Role of Ets-2 Phosphorylation in Inflammation, Development and Cancer

ROLE OF ETS-2 PHOSPHORYLATION IN INFLAMMATION, DEVELOPMENT AND CANCER

DISSERTATION

Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Guo Wei, M.S.

* * * * *

The Ohio State University

2003

Dissertation Committee: Approved by:

Dr. Michael C. Ostrowski, Adviser

Dr. Lee F. Johnson ______Adviser Dr. Gustavo Leone Department of Molecular Genetics Dr. Michael Weinstein

ABSTRACT

Ets family transcription factors are regulated by signal transduction pathways.

Phosphorylation of the Ets-1 and Ets-2 at a single conserved threonine residue (T38 and

T72, respectively) by ras-MAPK pathways leads to their activation and persistent target genes expression. The phosphorylation of Ets-2 affected its activity, as well as its protein partnership. Ets-2 interacted with Brg-1 or BS69 co-repressors in a phosphorylation dependent manner, and repressed target gene expression.

Ets-2 knockout mice are embryonic lethal due to an extra-embryonic defect, whereas ets-2T72A/T72A mice are viable and fertile, with no obvious abnormality. Ets-2 is constitutively phosphorylated in macrophages derived from motheaten-viable (mev) mice, while its phosphorylation is tightly regulated in wt cells. The aberrant Ets-2 phosphorylation correlated with increased target gene expression and cell survival. To directly test the role of Ets-2 phosphorylation in inflammation, the ets-2T72A allele was introduced into mev mice. In contrast to ets2+/+, mev/mev mice, ets-2T72A/T72A, mev/mev mice were fertile, had increased life span and body weight, elevated macrophage apoptosis in the absence of CSF-1, but reduced inflammation and expression of inflammatory genes, including cytokines (TNFα), chemokines (MIP1α, β), extracellular matrix proteases (MMP9), cell adhesion molecule (integrin αM) in lungs and macrophages.

ii Both ets-1-/- mice and Ets-2 T72A/T72A mice are viable and fertile. To reveal Ets-2 function possibly masked by gene redundancy, we mated ets-2T72A/T72A mice with Ets-1 knockout mice. Ets-1-/-, Ets-2T72A/T72A mice died between embryonic day 11.5 to 14.5, with dramatic angiogenesis and cardiovascular defects. Compared to control embryos, the double mutant embryos expressed lower levels of Ets target genes, such as Ang1, Tie2,

MMP3, MMP9, and Fli-1, but elevated levels of VEGF.

Therefore, Ets-2 phosphorylation is important in immune response, angiogenesis and cancer. To further explore the in vivo function of Ets-2, an ets-2 conditional knockout allele was developed. This allele is useful to address the cell antonymous function of Ets-2 in inflammation, angiogenesis and tumorgenesis (in tumor cells or stromal cells, including fibroblasts, macrophages and vessel cells) and other human diseases. Understanding how

Ets-2 works these diseases may have important clinical implications, both for early diagnosis and developing of novel, effective therapy methods.

iii DEDICATION

This work is dedicated to my parents,

to my wife Jianping Guo, and to my daughter Hope and son Abraham

iv ACKNOWLEDGMENTS

I wish to thank my adviser, Dr. Michael C. Ostrowski, for his guidance, support,

advice, encouragement, patience and generosity throughout my graduate studies in OSU.

I would like to thank the members of my committee, Dr. Lee F. Johnson, Dr.

Gustavo Leone and Dr. Michael Weinstein, for their time, support, guidance and advice.

I would also like to thank Dr. Thomas Rosol, Dr. Donna Kusewitt, Dr. Natarajan

Muthusamy, Dr. Michael Robinson, Dr. Andrea Doseff, Dr. Nicanor Moldovan and Dr.

Christoph Plass (The Ohio State University), Dr. Robert Oshima (The Burnham Institute)

and Dave Hume (The University of Queensland) for their advice and help.

I would also like to acknowledge the members of the Ostrowski lab, Department

of Molecular Genetics for their friendship and stimulating discussions.

This Research was supported by a grant from the National Institute of Health.

I am grateful to my family and friends for their continuous understanding, encouragement and support.

v VITA

1993 ………………………………………B.S. Biology, Shandong University

1996 ……………….………………..…….M.S. Plant Physiology, Chinese Academy of

Sciences

1996-1998 ………………………………..Graduate Teaching Associate, Ohio University

1998-present …………………………….. Graduate Fellow, Teaching and Research

Associate, The Ohio State University

PUBLICATIONS

1. Wei, G., Schaffner, A. E., Baker, K. M., Mansky, K. C. and Ostrowski, M. C. (2003). "Ets-2 interacts with co-repressor BS69 to repress target gene expression." Anticancer Res 23(3A): 2173-8.

2. Baker, K. M.*, Wei, G.*, Schaffner, A. E. and Ostrowski, M. C. (2003). "Ets-2 and Components of Mammalian SWI/SNF Form a Repressor Complex That Negatively Regulates the BRCA1 Promoter." J. Biol. Chem. 278(20): 17876-17884. *Equal contribution.

3. Smith, J. L., Schaffner, A. E., Hofmeister, J. K., Hartman, M., Wei, G., Forsthoefel, D., Hume, D. A. and Ostrowski, M. C. (2000). Ets-2 Is a Target for an Akt (Protein Kinase B)/Jun N-Terminal Kinase Signaling Pathway in Macrophages of motheaten-viable Mutant Mice." Mol. Cell. Biol. 20(21): 8026-8034.

vi 4. Wei, G., Shan, X., Ding, Q., Liu, B. and Jing, Y.: Transgenic rice plants with Parasponia haemoglobin gene and its expression. In KA Malik, MS Mirza and JK Ladha (eds.), Nitrogen Fixation with Non-Legumes. Kluwer Academic Publishers, Dordrecht, Netherlands,1998, pp. 125-131.

5. Zhao, S., Su,Y., Wei, G.: High Efficient Transformation of Escherichia coli by Electroporation. In Scientific Research Progress in Shandong Province. Science and technology Press of China, Beijing, 1993 pp. 839-845.

FIELDS OF STUDY

Major Field: Molecular Genetics

vii TABLE OF CONTENTS

Page ABSTRACT...... ii DEDICATION...... iv ACKNOWLEDGMENTS ...... v VITA...... vi LIST OF FIGURES ...... xii LIST OF TABLES ...... xiv LIST OF ABBREVIATIONS ...... xv CHAPTER 1 INTRODUCTION...... 1 1.1. ETS FAMILY TRANSCRIPTION FACTORS...... 3 1.1.1. The Ets domain...... 6 1.1.2. The Pointed domain...... 11 1.2. BIOLOGICAL ROLES OF ETS PROTEINS...... 16 1.2.1 Ets factors in embryonic development...... 16 1.2.2. Ets proteins and cancer...... 22 1.2.3. Ets Target Genes...... 24 1.3. REGULATIONS OF ETS PROTEINS ACTIVITY AND SPECIFICITY...... 27 1.3.1 Tissue specific expression of Ets family members...... 27 1.3.2. DNA binding...... 35 1.3.3.Protein partners of Ets family members...... 37 1.3.4. Signal transduction and post-translational modification of Ets family members...... 39 1.4 ETS-2 AND RAS SIGNALING...... 41 1.4.1 Ras and Ras signaling...... 41 1.4.2 Persistent activation of Ets-1 and Ets-2 through phosphorylation by Ras- MAPK pathway...... 43 CHAPTER 2 MATERIALS AND METHODS...... 49 2.1 PLASMIDS AND DNA MANIPULATIONS...... 49 2.1.1 Plasmids...... 49 2.1.2. Plasmid miniprep and maxiprep...... 50 2.1.3. Colony lifts...... 51 2.1.4. DNA cloning...... 52 2.1.5. High molecular weight genomic DNA isolation and purification...... 54

viii 2.1.6. ES cell DNA extraction...... 54 2.1.7. Polymerase chain reaction (PCR) ...... 55 2.1.8. Southern blots...... 56 2.2. RECOMBINANT PROTEIN EXPRESSION AND PROTEIN INTERACTIONS...... 58 2.2.1. GST fusion protein expression and isolation...... 58 2.2.2. Phosphorylation of recombinant Ets-2 pointed domain...... 59 2.2.3. Pull Down assay...... 59 2.2.4. Co-immunoprecipitation...... 59 2.3. QUANTITATIVE REAL TIME PCR ...... 60 2.3.1. RNA extraction...... 60 2.3.2. Elimination of DNA contamination...... 61 2.3.3. Reverse transcription...... 62 2.3.4. Primers for real time PCR...... 62 2.3.5. Real time PCR...... 67 2.3.6. Real time PCR data analysis...... 67 2.4. CELL SURVIVAL ASSAYS...... 67 2.4.1. Nuclear morphology...... 67 2.4.2. Viability assay...... 68 2.4.3. DNA fragmentation...... 68 2.4.4. Caspase-3 assay...... 69 2.5. CELL LINE CULTURE AND TRANSFECTIONS...... 70 2.5.1. Raw264 cell transfection...... 70 2.5.2. Transient Transfections with calcium phosphate method...... 71 2.5.3. Generation of stable SW13 clones...... 73 2.5.4. Transfection of stable SW13 clones...... 73 2.5.5. Protein Assays...... 74 2.6. PRIMARY CELLS ISOLATION AND CULTURE...... 74 2.6.1. Preparation of L-cell conditional media as a source of CSF-1...... 74 2.6.2. Bone marrow derived macrophages...... 75 2.6.3. Peritoneal macrophages...... 75 2.6.4. Lung alveolar macrophages...... 76 2.6.5. Mouse embryonic fibroblasts (MEFs)...... 77 2.6.6. Mammary stromal and epithelial cells...... 78 2.7. MOUSE LINES AND GENOTYPING...... 79 2.7.1. Mouse lines...... 79 2.7.2. Tail DNA prep for genotyping...... 79 2.7.3. Genotyping primers and PCR conditions...... 80 2.8. GENE TARGETING...... 82 2.8.1. BAC library Screening...... 82 2.8.2. Conditional ets-2 targeting construct cloning...... 82 2.8.3. DNA recombination by Cre recombinase...... 83 2.8.4. ES cell transfection and selection...... 83 2.8.5. Conditional Pten targeting...... 85 2.8.5. Conditional Pten targeting...... 86 2.9. HISTOLOGY...... 86

ix 2.9.1. Cryosection...... 86 2.9.2. H & E staining of paraffin-embedded tissue...... 87 2.9.3. Wholemount staining of mammary gland...... 87 2.9.4. Immunohistochemistry with whole mouse embryos...... 88 2.10. BIOINFORMATICS...... 89 2.10.1. Databases...... 89 2.10.2. On-line bioinformatics tools...... 90 CHAPTER 3 ETS-2 AND INFLAMMATION ...... 92 3.1 INTRODUCTION ...... 92 3.1.1. Macrophages and Ets factors...... 92 3.1.2. CSF-1 signaling regulates Ets-2 phosphorylation...... 95 3.1.3. SHP1 and CSF-1 signaling...... 97 3.1.4. Phosphorylation of Ets-2 by PI3K/Akt pathway in macrophages...... 99 3.1.5. Ets-2 and macrophage survival and motheaten pathology...... 100 3.2 RESULT ...... 102 3.2.1. Ets-2 phosphorylation was required for the mev mice inflammation phenotype...... 102 3.2.1.1. Breeding strategies...... 102 3.2.1.2. Mutation of ets-2 increased mev mice life span, body weight and fertility...... 103 3.2.1.3. Mutation in ets-2 relieved inflammation in peripheral tissues of mev mice...... 105 3.2.1.4. Mutation in ets-2 relieved inflammation in lungs of mev mice...... 109 3.2.2. Ets-2 is important for macrophage survival but not for proliferation...... 113 3.2.4. Expression of many inflammatory genes was reduced in lungs with mutant ets-2...... 119 3.2.5. Ets-2 modulated lung alveolar macrophages gene expression...... 124 3.2.6. Peritoneal macrophages gene expression was regulated by Ets-2...... 126 3.2.7. Ets-2 is required for LPS induction of persistent TNFα expression...... 128 3.3 DISCUSSION ...... 130 CHAPTER 4 ETS-2 AND EMBRYO DEVELOPMENT ...... 136 4.1 INTRODUCTION ...... 136 4.1.1. Angiogenesis...... 137 4.1.2. Genes involved in angiogenesis...... 138 4.1.3 Ets factors and angiogenesis...... 142 4.2. RESULTS ...... 146 4.2.1. Ets1-/-, Ets-2 T72A/T72A mice were embryonic lethal and died between E11.5- E14.5...... 146 4.2.2. Ets1-/-, Ets-2 T72A/T72A mice had numerous dilated blood vessels...... 148 4.2.3. Ets1-/-, Ets-2 T72A/T72A mice had cardiovascular and angiogenesis defects.... 151 4.2.4. Gene expression in embryos was affected by Ets-1 and Ets-2 mutations..... 156 4.2.5. Ets-1 and Ets-2 mutations reduced the expression of matrix proteinases in embryo fibroblasts...... 161 4.3. DISCUSSION ...... 163 x CHAPTER 5 ETS-2 AS A TRANSCRIPTIONAL REPRESSOR...... 172 5.1 INTRODUCTION ...... 172 5.2 RESULTS ...... 176 5.3 DISCUSSION ...... 186 CHAPTER 6 ETS-2 CONDITIONAL KNOCKOUT MICE ...... 192 6.1 INTRODUCTION ...... 192 6.2 RESULTS ...... 195 6.3 DISCUSSION ...... 207 CHAPTER 7 DISCUSSION AND FUTURE DIRECTIONS ...... 209 7.1 PHOSPHORYLATION OF ETS-2 REGULATES PROTEIN PARTNERSHIP AND ACTIVITY. 210 7.2. ETS-2 IS IMPORTANT FOR INFLAMMATION AND OTHER IMMUNE DISEASE...... 212 7.3. ETS-2 AND CANCER...... 213 7.2.1. Ets-2 and tumor cells...... 213 7.3.2. Ets-2 and tumor microenvironment...... 214 7.2.3. Ets-2 in tumor angiogenesis and metastasis...... 218 7.2.5. Define the role of Ets-2 phosphorylation in tumorgenesis by tissue specific deletion...... 219 BIBLIOGRAPHY...... 223

xi LIST OF FIGURES

Page Figure 1.1. The Ets domain of Ets family proteins...... 7 Figure 1.2. The Pointed domain of Ets proteins...... 13 Figure 1.3. Ets proteins expression pattern...... 30 Figure 2.1. Generation of mice with PTEN conditional knockout allele...... 86 Figure 3.1 Mutation in ets-2 increased mev mice life span and body weight...... 104 Figure 3.2. Mutation in ets-2 relieved inflammation in the foot of mev mice...... 107 Figure 3.3. Mutation in ets-2 reduced macrophage accumulation in lungs of mev mice 110 Figure 3.4. Ets-2 was important for macrophage survival but not for proliferation...... 117 Figure 3.5. Mutation of ets-2 affected the expression of inflammatory genes in lungs. 122 Figure 3.6. Ets-2 regulates lung alveoli macrophages inflammatory gene expression…125 Figure 3.7. Inflammatory gene expression in mev mice peritoneal macrophages was reduced by ets-2 mutation…………………………………………………………127 Figure 3.8. Ets-2 is important for persistent expression of TNFα in BMMs upon LPS stimulation...... 129 Figure 4.1 Ets1-/-, Ets-2 T72A/T72A mice had abundant dilated blood vessels…………….149 Figure 4.2 Ets1-/-, Ets-2 T72A/T72A mice had dilated blood vessels, edema and hemorrhage...... 150 Figure 4.4. Ets1-/-, Ets-2 T72A/T72A embryos had defects in vessel branching and reduced vascular complexity...... 154 Figure 4.5. Ets-1 and Ets-2 mutations affected target gene expression in mutant embryos...... 158 Figure 4.6. Matrix proteinases expression was reduced in Ets1-/-, Ets-2 T72A/T72A mice embryonic fibroblasts...... 162 Figure 4.7. Model for the role of Ets-1 and Ets-2 in angiogenesis...... 167 Figure 5.1. Brg-1 and ets-2 repressed BRCA1 promoter reporter in SW13cells………178 Figure 5.2. Phosphorylation-dependent direct interaction between BS69 and Ets-2 in vitro...... 182 Figure 5.3. BS69 and ets-2 repressed uPA and BRCA1 reporter genes in SW13 cells.. 185 Figure 5.4 Ets-2 contains a consensus PXLXP motif...... 187 Figure 5.5. Model for ets-2 as a signaling-regulated repressor and activator...... 189 Figure 6.1. Illustration of ets-2 mRNA transcribed from different targeting alleles...... 196 Figure 6.2. Outline of ets-2 conditional targeting vector cloning...... 198 Figure 6.3. Ets-2 gene targeting illustration...... 199 Figure 6.4. ES clones having desired homologous recombination were generated...... 201 Figure 6.5. ES clones with neo cassette deletion were generated...... 204

xii Figure 6.6. Gene targeted mice harboring Ets-2 floxed and knockout alleles were generated...... 206 Figure 7.1. Constitutive Ets-2 phosphorylation in stromal cells from pre-invasive mice tumors...... 217

xiii LIST OF TABLES

Page Table 1.1. The human and mouse Ets proteins …………………………………….…..…5 Table 1.2. Phenotype of mice with disrupted ets genes...... 19 Table 1.3. Partial list of Ets target genes...... 26 Table 2.1 Real time PCR primers...... 63 Table 2.2. Genotyping primers and PCR conditions ………………………….………...81 Table 4.1. Ets1-/-, Ets-2 T72A/T72A mice were embryonic lethal...... 147 Table 4.2 Ets1-/-, Ets-2 T72A/T72A mice died from E11.5 to E14.5...... 147 Table 4.3. Some growth factors and their receptors expression in ets-1/2 double mutant mice embryos ………………………………………………..……..……157

xiv LIST OF ABBREVIATIONS

A alanine Ang1 angiopoietin-1 Ang2 angiopoietin 2 ATP adenosine triphosphate Bcl2 B-cell leukemia/lymphoma 2 Bclx Bcl2-like bFGF fibroblast growth factor 2 BMM bone marrow derived macrophages BRCA1 breast cancer 1 BSA Bovine Serum Albumin CBP CREB binding protein CDC2 cell division cycle 2 homolog A ChIP chromatin immunoprecipitation Cre cyclization recombination CSF1 colony stimulating factor 1 DMEM Dulbecco's Modified Eagle Medium DNA deoxyrobose nucleic acid dNTP Deoxyribonucleotide Triphosphate DTT Dithiothreitol E1AF E1A enhancer-binding protein EBS Ets binding site EC endothelial cell ECM extracellular matrix EDTA Ethylenediaminetetraacetic Acid Elf-1 E74-like factor 1 eNOS nitric oxide synthase 3, endothelial cell eotaxin small chemokine (C-C motif) ligand 11 ER71 Ets-related protein 71 ER81 ets variant gene 1 ERF Ets2 repressor factor ERG v-ets erythroblastosis virus E26 oncogene like ERK extracellular signal regulated kinase ERM ets variant gene 5 ES cell embryonic stem cell ESE-1 epithelium-specific Ets 1 ESE-2 epithelium-specific Ets 2

xv ESE3 epithelium-specific Ets 3 Ets1 v-ets erythroblastosis virus E26 oncogene homolog 1 Ets2 v-ets erythroblastosis virus E26 oncogene homolog 2 Fli-1 Friend leukemia virus integration 1 Flk-1 vascular endothelial growth factor receptor-2 Flt-1 fms-like tyrosine kinase 1 Floxed loxP-flanked fms colony stimulating factor 1 receptor GABPalpha GA binding protein transcription factor, alpha subunit 60kDa GST Glutathione-S-Transferase HAT histone acetyltransferase HBS HEPES-buffered saline Hcph hematopoietic cell phosphatase HDAC histone deacetylase HIF1a hypoxia inducible factor 1, alpha subunit HSV-tk herpes simplex virus thymidine kinase Il12 interleukin 12 alpha IL1a interleukin 1 alpha IL1b interleukin 1, beta Il6 interleukin 6 IPTG isopropyl-beta-D-thiogalactopyranoside JNK c-jun N-terminal kinase LB Luria broth LoxP locus of X-over of P1 MAPK mitogen activated protein kinase MCP-1 monocyte chemoattractant protein-1 Me motheaten Mev motheaten viable MEF myeloid elf-1-like factor MEFs murine embryonic fibroblasts MEK mitogen-activated protein kinase kinase MIP1a chemokine (C-C motif) ligand 3 MIP1b chemokine (C-C motif) ligand 4 MMP1 matrix metalloproteinase 1 MMP3 matrix metalloproteinase 3 MMP9 matrix metalloproteinase 9 MMP14 matrix metalloproteinase 14 (membrane-inserted) mRNA messenger ribose necleic acid NERF new Ets-related factor NET SRF accessory protein 2 PCR polymerase chain reaction PDEF prostate epithelium-specific Ets transcription factor PDGF-b platelet derived growth factor, B polypeptide PDGFRb platelet derived growth factor receptor, beta polypeptide PE1 ets variant gene 3

xvi PECAM platelet/endothelial cell adhesion molecule PI3K Phosphoinositide 3-kinase PMSF Phenylmethyl Sulphonyl Fluoride PU1 spleen focus forming virus (SFFV) proviral integration oncogene spi1 RANTES chemokine (C-C motif) ligand 5 RNA ribose nucleic acid RTK receptor tyrosine kinase SAP1 SRF accessory protein 1 SDS sodium dodecyl sulfate SimMets similar to mitogenic Ets transcriptional suppressor METS smMHC myosin heavy chain 11, smooth muscle SHP1 Src-homology 2-domain phosphatase-1 Spi-B spleen focus forming virus (SFFV) proviral integration oncogene spib Spi-C spleen focus forming virus (SFFV) proviral integration oncogene spic T threonine TCF ternary complex factor TEL ets variant gene 6 TEL2 ets variant gene 7 TGFb transforming growth factor, beta 1 Tie1 tyrosine kinase receptor 1 Tie2 endothelial-specific receptor tyrosine kinase Tk thymidine kinase TNFa tumor necrosis factor alpha uPA urokinase plasminogen activator VE-cadherin cadherin 5 VEGF vascular endothelial growth factor A

xvii

CHAPTER 1

INTRODUCTION

Characterized by a conserved Ets DNA binding domain, Ets family transcription factors regulate many important biological processes, including cell proliferation, differentiation, and apoptosis. These factors also contribute to human diseases, in particular to cancer. All of the Ets proteins recognize a purine rich GGAA/T sequence

(Ets binding site, EBS) in the promoter. The specificity of target gene regulation is achieved through several mechanisms within this large gene family: A). Tissue specific expression; B). Discrete binding affinity achieved through differences in primary sequence and structure of DNA binding domain; C). Protein partners, including other transcription factors (e.g. Jun, NFκB), co-activators (e.g. CBP, P300) or co-repressors, modulate Ets protein DNA binding and activity; D). Signal transduction and posttranslational modification.

Many Ets family proteins are downstream nuclear targets of signal transduction cascades. Specificity can be achieved through modification of specific family members by distinct signal transduction pathways (Yordy and Muise-Helmericks, 2000, Oikawa and Yamada, 2003). Our lab is interested in studying how Ets transcription factors, especially Ets-1 and Ets-2, are modulated by signal transduction pathways, and

specifically regulate target gene expression. Ets-1 and ets-2 are phosphorylated at a

conserved residue (Threonine 38 and Threonine 72, respectively) by a well-characterized ras-effector pathway. Phosphorylation of the Ets-1 and Ets-2 by ras-dependent pathways leads to persistent expression of target genes including urokinase plasminogen activator

(uPA) in cell culture (Yang et al., 1996, Fowles et al., 1998, Smith et al., 2000). Signal transduction pathway also modulates protein-protein interactions (Li et al., 2000, Verger and Duterque-Coquillaud, 2002). For example, phosphorylated Ets-2 and non- phosphorylated Ets-2 interact with discrete nuclear proteins (Baker et al., 2003). Because ets-2 is expressed ubiquitously throughout embryonic development and in adult tissue, while ets-1 expression is relatively restricted, this work primarily focuses on the study of ets-2.

Hypothesis

Phosphorylation of Ets-1 and Ets-2 by ras signal transduction pathway regulates their activity, protein partnerships and specification of downstream target genes. Disruption of this phosphorylation may lead to functional and physiological abnormalities.

Significance

Ets-2 is implicated to be involved in many diseases, such as cancer. This study discovered the novel roles of Ets-2 in inflammation by controlling macrophages survival and activities through regulating genes involved in cell survival (e.g. Bclx), inflammatory cytokines and chemokines (e.g. TNFα, Il-1α, Il-12, MCP1, MIP1α and MIP1β), cell

adhering molecules (e.g. integrinαM, β2) and extra cellular matrix proteinases (e.g. 2

MMP1, MMP9 and uPA). The embryo development studies provided the first in vivo evidence that Ets-1 and Ets-2 were important for angiogenesis and embryo development.

Target genes whose expression was affected by Ets-1/2 were defined. Furthermore, by biochemical and cellular studies, the repression function of unphosphorylated Ets-2 was revealed. The results from this study strongly indicated that the functions of Ets-2 in cancer were through controlling target gene expression not only in cancer cells, but also in cells in tumor microenvironment, including fibroblasts, macrophages and blood vessel cells, and may be essential for tumor angiogenesis and metastasis. The development of the conditional targeting allele provided key tools to further define the roles of Ets-2 in various diseases, especially in cancer and immune diseases, and may lead to development of novel strategies for treating patients with these diseases.

1.1. Ets family transcription factors.

The Ets family transcription factors are characterized by an evolutionarily conserved DNA-binding domain constituted by about 85 amino acids. They regulate expression of a variety of viral and cellular genes by binding to a purine-rich GGAA/T core sequence in cooperation with other transcriptional factors and co-factors. The first founding member of the ets (E26 transformation-specific sequence) family gene, v-ets, was originally identified as a gag-myb-ets fusion oncogene of the avian transforming retrovirus E26, which induces both erythroblastic and myeloblastic leukemia in chickens

(Leprince et al. 1983). The cellular homologue of v-ets, c-ets-1, was identified by using the viral ets sequence as a probe (Leprince et al. 1983). Many other family members from several different species were identified thereafter. To date, there are 27 characterized 3

human Ets proteins and one unknown function protein (LOC343059, named simMets in

this dissertation because its similarity to PE1, i.e. METS) predicted from human genome

database (Table 1.1). Except Tel2, all of those Ets proteins are present in mice, with high

homology to those in humans. For example, human and mouse Ets-2 overall share 92%

similarity and 89% identity, with higher homology in two conserved domains, the Ets

domain (100% identity) and the pointed domain (98% similarity and 96% identity). Many

ets proteins are also characterized in other organisms, including 10 Ets proteins in

Caenorhabditis elegans (Hart et al. 2000b) and 8 in Drosophila melanogaster (Hsu and

Schulz 2000). Most of mammalian Ets subfamilies are present in C. elegans and

Drosophila, except the Pu.1 subfamily, which is only present in vertebrates.

Most of the known ETS proteins have been shown to activate transcription.

However, several ETS proteins, such as YAN, ERF, NET and TEL, are shown to be

transcriptional repressors. In Drosophila, YAN antagonizes the activity of the Pointed-

P2, an activator of the ETS family. In mammalian cells, Bclx can be activated by Ets-2

(Smith et al. 2000), but repressed by TEL (Irvin et al. 2003). Some other ETS-domain proteins have been shown to repress transcription in a context specific manner. This issue is discussed further in Chapter 5.

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1.1.1. The Ets domain.

Ets family proteins can be divided into several subfamilies on the basis of their structural composition and their similarities in the DNA-binding Ets domains (Fig.1.1.

C). Most ets proteins have the Ets domains in their C-terminal regions. However, several

Ets family proteins like the ternary complex factor (TCF) subfamily have the Ets domains in their N-terminal regions (Fig. 1.1 A).

The Ets domain contains three α-helixes (α1-α3) and four-stranded β-sheets (β1-

β4) arranged in the order α1-β1-β2-α2-α3-β3-β4 forming a winged helix-turn-helix

(wHLH) structure (Fig. 1.1 A) (Donaldson et al. 1996; Kodandapani et al. 1996; Mo et al.

1998). The majority of the highly-conserved amino acids are found within the α-helices and β-strands, while the regions with less sequence conservation map to the loops or turns (Fig. 1.1 B). The HTH, composed of helices α1-α3, lies in the major groove of

DNA. All of the base-specific contacts are mediated by residues within α3 and the turn immediately preceding it. Particularly, the two absolutely conserved arginines in α3 provide base contacts to the two guanine (G) residues of the GGA core. A β-hairpin

“wing” and a loop connecting the recognition helix and its preceding helix make multiple

DNA phosphate backbone contacts to nucleotides flanking the GGA core (Donaldson et al. 1996; Kodandapani et al. 1996; Mo et al. 1998).

Figure 1.1. The Ets domain of Ets family proteins. A. Illustration of Ets family proteins domains. The red rectangle indicates the Ets domain. The green rectangle indicates the Pointed domain. B. Alignment of Ets domain from human Ets proteins. H: α-helix. S: β-sheet. C. Phylogeny relationship of human Ets proteins. The phylogeny tree was generated by average distance PID method, based on sequence conservation of Ets domain. The alignment and phylogeny tree were constructed by ClustalW (Thompson et al. 1994). Nomenclatures of subfamilies are based on Graves and Petersen, 1998, Laudet et al., 1999, except PDEF subfamily, which was discovered recently (Oettgen et al. 2000; Yamada 2000).

Continued on next page

Fig. 1.1 continued.

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Fig. 1.1 continued.

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Fig. 1.1 continued.

1.1.2. The Pointed domain.

Beside the conserved Ets domain, 11 out of 28 human Ets family proteins (Fig

1.2), including Ets-1, Ets-2 and TEL, and Drosophila Ets proteins pointed-P2 and Yan, have another evolutionarily-conserved domain, called the Pointed domain, by analogy to the Drosophila melanogaster Ets domain transcription factor Pointed-P2, at their N- terminal regions. This domain forms a helix–loop–helix (HLH) structure that is important for protein-protein interactions (Slupsky et al. 1998; Seidel and Graves 2002). This

domain is proposed to mediate protein-protein interactions and has important biology

roles. For example, in human leukemia, oligomerization of the Tel pointed domain is

implicated in the self-association of chimeric oncoproteins, which result from

chromosomal translocations of the Tel gene with segments of genes encoding several

tyrosine kinases or AML (Eguchi et al. 1999; Buijs et al. 2000; Million et al. 2002).

The pointed domain is a target for signal transduction (Yordy 2000). Ets-1, Ets-2 and Drosophila Pointed-P2 protein have a conserved MAPK phosphorylation site

(threonine 38 in Ets-1 and threonine 72 in Ets-2) at the N-terminal of the Pointed domain

and are thereby regulated by ras-dependent signaling (Yang et al. 1996; Fowles et al.

1998a) (discussed in detail in section 1.4). The pointed domain of Ets-1 and Ets-2 contain

an ERK2 docking site (LXLXXXF) (Fig 1.2 A). Three hydrophobic residues, which are

clustered on the surface of the pointed domain, are involved in docking (Seidel and

Graves 2002). The docking site is not conserved in other Ets factor, such as GABPα (Fig

1.2A). Mutation of the docking site in Ets-1 and Ets-2 prevents Ras pathway-mediated

enhancement of their trans-activation function (Seidel and Graves, 2002).

Ets-1 pointed domain forms a monomeric five alpha-helix bundle. The

architecture is distinct from that of any know DNA- or protein-binding module (Graves

and Petersen 1998a; Slupsky et al. 1998). The structure of the pointed domain indicates

several potential protein binding sites. Protein-protein interaction results from both

hydrophobic and electrostatic/hydrogen bonding interactions between interfaces

composed of complementary non-polar and charged/polar residues (Graves and Petersen

1998b; Slupsky et al. 1998). The postulated association surfaces of Ets-1 formed by the

hydrophobic and charged groups are not strictly conserved among pointed domains of other ets protein, thus providing the potential for specific interactions with other partners.

The MAPK site is in a highly flexible region of both the unphosphorylated and phosphorylated forms of Ets-1. Phosphorylation of Threonine 38 residue of Ets-1 does not change its structural or dynamic properties. It implies that binding of the phosphorylation site by potential partner transcription factors, perhaps in conjunction with the pointed domain, is coupled to the ordering of these residues (Graves and

Petersen 1998a; Slupsky et al. 1998). Therefore, the phosphorylation may provide another potential source for specificity and regulation. Ets-2 closely resembles Ets-1 in the pointed domain (Fig 1.2), suggesting that Ets-1 and Ets-2 have similar three- dimensional structure in these regions.

Figure 1.2. The Pointed domain of Ets proteins. A. Alignment of pointed domain from Ets proteins. The MAPK phosphorylation site was included to show the conservation of this site between mammalian Ets-1, Ets-2 and Drosophila Pointed P2 protein. The arrow indicates the threonine residue which can be phosphorylated by MAPK. The ERK docking site (Seidel and Graves 2002) is denoted with rectangle boxes. MuEts1: Mouse Ets-1; MuEts2: Mouse Ets-2; PointedP2: Drosophila Ets protein Pointed P2. B. Phylogeny relationship of human Ets proteins that have pointed domain. The phylogeny tree was based on sequence conservation of the pointed domain. The alignment and phylogeny tree were constructed by ClustalW (Thompson et al. 1994).

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Fig. 1.2 continued

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Fig. 1.1 continued

1.2. Biological roles of Ets proteins.

Ets factors play crucial roles in regulating a variety of cellular function including growth, proliferation, differentiation, apoptosis, migration and oncogenic transformation.

Major themes of Ets protein actions include roles in hematopoiesis, vasculogenesis and neuronal development. Disruption of these proteins often leads to embryonic lethality

(Table 1.2) or cancer. Furthermore, Ets binding site (EBS) were among the eight most important DNA motifs in minimal responsive synthetic promoters generated using random oligonucleotides (Edelman et al., 2000).

1.2.1 Ets factors in embryonic development.

ETS-domain proteins are expressed ubiquitously or tissue-specifically (see section

1.3.1) and contribute to a variety of embryonic development processes. Disruption or dysregulation of their expression often leads to embryonic lethality or developmental abnormality (Table 1.2). Fli-1 null embryos die at E11.5 with loss of vascular integrity leading to bleeding in the midbrain/forebrain boundary and in the hindbrain (Hart 2000a).

PU.1 knockout-mouse is absent of morphologically normal B cells and macrophages. It has disrupted granulopoiesis, and aberrant T-lymphopoiesis (Scott et al. 1994;

McKercher et al. 1996). Tel null mice are embryonic lethal and die between E10.5-11.5 with defective yolk sac angiogenesis and intro-embryonic apoptosis of mesenchymal and neural cells (Wang et al. 1997). Through mouse chimeras with TEL-/- ES cells, TEL was shown to be essential for the establishment of hematopoiesis of all lineages in the bone marrow (Wang et al. 1998a). Ets-1 knockout mice lack NK cells and may have short life 16

span, also have defects in B- and T-cell apoptosis (Muthusamy et al. 1995; Barton et al.

1998).

Ets-2 knockout mice are severely growth retarded early in development, resulting

in embryonic lethality and resorption by E8.5. These mutant embryos exhibit several

defects. The mutant embryos have small ectoplacental cone region, apparently caused by

a failure of trophoblasts migration. The mutant embryos have no amnion or chorion

membranes and hence have only one cavity. The trophoblastic tissue in the ectoplacental

cone failed to proliferate and the primary ectoderm begin to die by apoptosis at E7.5.

Compared to that in control embryos, trophoblasts cells from the mutant embryos express

much less matrix metalloproteinase-9 (MMP-9), which is a key molecule in extracellular

matrix remodeling, and PECAM-1, an endothelial marker also expressed in trophoblasts

cells directly connected to maternal endothelium (Yamamoto et al. 1998). Ets-2-/-

embryos were rescued by aggregation with tetraploid mouse embryos, which complement

the developmental defects by providing functional extraembryonic tissues. Rescued ets-2

deficient mice are viable and fertile but have wavy hair, curly whiskers, and abnormal

hair follicle shape and arrangement (Yamamoto et al. 1998). By gene targeting, the

threonine 72 residue in ets-2 was substituted to alanine in mice. Ets-2T72A/T72A mice are viable, fertile and develop normally. There are no hair abnormalities as observed in ets-2 knockout mice. However, combining the Ets-2T72A allele with a deletion mutant of ets-2 resulted in embryonic lethality at E11.5. Ets-2T72A/- embryos are significantly smaller and

retarded in development and the yolk sacs cannot develop blood vessels. Those

development defects was believed to arise secondarily from placenta defects (Man et al.

2003), because Ets-2T72A/- embryos have smaller, disorganized placentas lacking a 17

labyrinth region where maternal and embryonic vascular systems intermingle and exchange nutrients and oxygen (Man et al. 2003). The phenotypes of ets-2T72A/- embryos, ets-2-/- embryos and ets-2T72A/T72A mice imply that ets-2T72A is a hypomorphic allele and ets-2 dosage and phosphorylation may affect embryo development.

Human ets-2 is located in the minimal Down’s syndrome (trisomy 21) region in chromosome 21, and is over-expressed in Down’s syndrome. Transgenic mice overexpressing Ets-2 in particular organs develop abnormalities similar to those in trisomy 16 mice and in Down’s syndrome human patients, such as neurocranial, viscerocranial and cervical skeletal abnormalities, lymphocyte abnormalities and smaller thymus (Sumarsono et al. 1996a). Ets-2 is not crucial for macrophage differentiation from embryonic stem cells (Henkel et al. 1996). However, the expression of ets-2 is rapidly induced in a variety of myelomonocytic cell lines as they differentiate into macrophages.

Constitutive expression of ets-2 in the MID+ myeloblast leukemic cell line is sufficient to push these cells to a more differentiated state, including upregulation of TNFα (Aperlo et al. 1996).

Ets-1 and Ets-2 was also shown to be important for embryonic development in other animals. In Xenopus, both ets-1 and ets-2 genes are transcribed in regions of the embryo undergoing important morphogenetic modifications, especially in migrating cells and/or along their migration pathways (Meyer et al. 1997). Overexpression of Ets-2 causes posteriorized embryos and leads to the induction of mesoderm in ectodermal explants. Expression of a dominant-negative form of Ets-2 or injection of antisense oligonucleotides against Ets-2 inhibits the formation of the trunk and tail structure

(Kawachi et al. 2003). 18

Table 1.2. Phenotype of mice with disrupted ets genes. The major phenotypes of 12 Ets genes, which were reported to be knocked out, are summarized, based on the information provided by the reference papers, which are indicated in the table.

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Table 1.2. Continued.

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Table 1.2. continued.

1.2.2. Ets proteins and cancer.

Deregulated expression or formation of chimeric fusion proteins of Ets family due to proviral insertion or chromosomes translocation is associated with leukemia and specific types of solid tumors. For example, the human Ets genes, FLI1, TEL and ERG, are located at the translocation breakpoints of several leukemia and solid tumors, forming chimeric proteins believed to be responsible for tumorgenesis (Dittmer and Nordheim,

1998). Fli-1 was first identified from mouse Friend leukemia virus integration site and human T cell leukemia cell line (Ben-David et al. 1991; Prasad et al. 1992; Watson et al.

1992). Mouse Fli-1 was shown to be involved in 75% erythroleukemias induced by

Friend murine leukemia virus (Prasad et al. 1992). Transcriptional activation of Fli-1 by either chromosomal translocation or proviral insertion leads to Ewing’s sarcoma in humans (Delattre et al. 1992) and erythroleukemia in mice (Ben David et al, 1991). The

Tel gene is frequently rearranged in human leukemia of both myeloid and lymphoid origin. For example, the Tel/AML-1 fusion is common in childhood acute lymphoblastic leukemia, where this gene rearrangement account for 25-30% of cases (Shurtleff et al.

1995).

Over-expression of Ets genes has been correlated with a number of human neoplasia. For example, several studies have established the correlation between Ets-1 and Ets-2 protein activity and neoplastic transformation in vivo. Ets-1 and Ets-2 are overexpressed in several experimental and prostatic carcinomas and many cancer cell lines (Fig. 1.3). Ets-2 is overexpressed in prostate and breast cancer during cancer development (Sapi et al. 1998; Sementchenko et al. 1998). An acute non-lymphoblastic leukemia with a complex t(6;18;21) chromosomal translocation was associated with 20- 22

fold amplification of Ets-2 sequences and 3- to 4-fold elevation of ets-2 mRNA level

(Santoro et al. 1992). Ets-2 is an important mediator of cellular transformation.

Overexpression of ets-2 stimulates cells to proliferate and abolishes their serum requirement. The fibroblast cells transfected with ets-2 shows foci of densely growing, morphologically altered cells, either in low-serum or serum-free medium. The ets-2 transfected cells formed colonies in semisolid medium and induced tumors in nude mice, indicating that c-ets-2 can be a transforming gene when overexpressed in these cells (Seth et al. 1989). Similarly, ets-1 gene transforms NIH3T3 cells and the ets-1 transfected cells form colonies in soft agar and induce tumors in nude mice (Seth and Papas 1990). Stable expression of a dominant negative constructs of Ets-2 can block transformation by Ras or

Her2/neu (Foos et al. 1998; Sapi et al. 1998). Furthermore, mammary tumors are suppressed by lowering the dosage of ets-2 by half (Neznanov et al. 1999), and further restricted in ets-2T72A/T72 mice (Man et al. 2003).

In most of these tumors, ets gene expression levels correlate with tumor progression. For example, ets-1 and ets-2 expression level correlates with successive events of colon carcinogenesis. Ets-1 and ets-2 expression was directly linked to lymph node metastasis in adenocarcinoma (Ito et al. 2002b). Ets-2 expression is significantly higher in highly aggressive pancreatic adenocarcinomas. Pancreatic carcinoma cells ectopically expressing ets-2 cause the formation of large tumors when injected into athymic mice (Ito et al. 2002a). Thyroid cell neoplastic transformation is associated with a dramatic increase in Ets transcriptional activity. Expression of a dominant negative Ets construct induces apoptosis in thyroid carcinoma cells lines (de Nigris et al. 2001). The phosphorylated Ets-2 is important for the invasiveness of tumor cells. For instance, the 23

amount of phosphorylated Ets-2 correlates with metastatic ability in breast cancer cell lines (Baker et al. 2003).

Several Ets family proteins also participate in malignancy of tumor cells including invasion and metastasis by activating the transcription of several protease genes and angiogenesis-related genes. For example, the expression of genes encoding for enzymes involved in degradation of the extracellular matrix (ECM), such as MMP-1 (collagenase-

1), MMP-3 (stromelysin-1) and MMP-9 (gelatinase B), is regulated by Ets family proteins including Ets-1, Ets-2 and PEA3/E1AF (Yang et al. 1996; Westermarck and

Kahari 1999a; Trojanowska 2000b; Singh 2002). Generally, expression levels of Ets-1 correlate well with the grade of invasiveness and metastasis (Nakayama et al. 2001;

Behrens et al. 2001a; Behrens et al. 2001b; Behrens 2003) and therefore can be useful for predicting poor prognosis of the cancer patients.

Tumorgenesis is a complex process involving not only the tumor cell, but also stromal cells and immune cells. Ets factors also play important roles in microenvironment of tumor development. Ets-1 is expressed in endothelial cell and stromal fibroblasts during the onset of tumor stroma generation. It is significantly upregulated in the stroma of invasive ductal and lobular cancers. The upregulation of Ets-1 in stoma is correlated with increase level of Ets target genes, such as MMP1 and MMP9 (Behrens et al. 2001b).

1.2.3. Ets Target Genes.

All Ets transcription factors bind to unique GGAA/T DNA sequences (Ets

Binding Sites, EBS) (Graves and Petersen 1998a; Sementchenko 2000). Ets target genes have been identified by the presence of EBS in the promoter/enhancer, electrophoresis 24

mobility shift assays (EMSAs), DNA foot printing, transient transfections, knockout or

transgenic models, and more recently, chromatin immunoprecipitation (ChIP)

(Sementchenko 2000). Over 200 Ets target genes have been identified to date, and the

number of genes shown to be regulated via EBS is constantly increasing. Collectively, functional Ets sites have been characterized in viral genes and cellular genes encoding transcription factors, transforming and tumor-associated products, proteinases, cell cycle and apoptosis regulators, signaling molecules, receptors and other cell surface molecules, ligands and tissue specific products (Graves and Petersen 1998a; Sementchenko 2000;

Oikawa 2003). Through the regulation of the expression of these genes, Ets proteins control cell growth, proliferation, differentiation, apoptosis, transformation, and are important for development, hematopoiesis, tissue remodeling, angiogenesis and tumor metastasis. Table 1.3 shows a partial list of Ets target genes listed according to their functions.

Growth factors, a-FGF, b-FGF, GM-CSF, IL-1α, IL-1β, IL-12, cytokines and Interferon-γ, MIP-1α, PDGF, RANTES, TF, TGFα , chemokines: TGFβ, TNFα

Receptors: Flt, Flk, HER2/neu, IL-2R β, MSR, Met/HGFR, TCR α, TCR β, Tie1, Tie2 Matrix proteinases: MMP1, MMP3, MMP9, MMP14, uPA Transcription factor: c-fos, Fli-1, Hoxb-3, JunB, N-myc2, c-myb, p53, TBP, TFEC Cell adhering Integrin αM, Integrin αV, Integrin β2, Integrin β3, VE molecules: cadherin Cell cycle: Cyclin D1, CDC2, mdm2, p21CIP Cell viability: Bcl-2, Bclx Viral genes HIV LTR, HTLV-1 LTR Others BRCA1, eNOS

Table 1.3. Partial list of Ets target genes. The list is mainly adopted from (Sementchenko 2000)

1.3. Regulations of Ets proteins activity and specificity.

Although some overlap in the biological function of different Ets proteins may exist, the emergence of a family of closely related transcription factors suggests that individual Ets members may have evolved unique roles, manifested through the control of specific target genes. The important roles of the Ets family of transcription factors in various biological and pathological processes necessitate the identification of downstream cellular target genes of specific Ets proteins, and the mechanisms defining

Ets protein specificities. There are several mechanisms that define the specificities among different family members. These proteins are controlled by a complex series of inter and intramolecular interactions, and signaling pathways impose on these proteins to further regulate their action. These mechanisms are not mutually exclusive. In fact, they may cooperatively define the specificity.

1.3.1 Tissue specific expression of Ets family members.

Some Ets family proteins are expressed ubiquitously, while others are preferentially expressed in a tissue-specific manner: they are only expressed in specific cell lineages and are involved in their development and differentiation by increasing the enhancer or promoter activities of the genes specific for the cell lineages, such as genes encoding growth factor receptors and integrin families (Fig. 1.3).

Ets proteins are expressed ubiquitously or specifically in different tissues in different stages of embryo and neonatal development. Fli-1 is preferentially expressed in hematopoietic lineages and endothelial cells. It is expressed in the blood islands of the extra embryonic visceral yolk sac at murine E8.5. Later in gestation, it is expressed in the 27

developing vasculature and the liver (Prasad et al. 1992; Melet et al. 1996). Pu.1 is highly expressed in most hematopoietic lineages, with highest levels in the erythroid, monocytic, granulocitic and B-lymphoid lineages (Hromas et al. 1993). The Tel gene is widely expressed throughout embryonic development and in the adult (Maroulakou 2000).

Ets-1 first appears in blood islands of yolk sac and then in several organs including blood vessels, developing brain and bone, and mesodermal cells in organs undergoing morphogenetic processes during mammalian development. At early embryo development, ets-1 expression is clearly observed during a narrow developmental stage in the developing nervous system, including the presumptive hindbrain regions, the neural tube, as well as neural crest and the first and second branchial arches. Ets1 mRNA is also found in the endothelial cells of villous trophoblast and in the extravillous trophoblastic cells invading the uterine vessels (Luton et al. 1997). In later fetal stages, Ets-1 is limited in lymphoid cells, vascular endothelial cells and organs that are undergoing branching morphogenesis (e.g., lung) but is dramatically reduced in other organs such as the stomach and intestine (Maroulakou et al. 1994a). In neonatal development, Ets-1 is expressed only in the lymphoid organs and brain. In adult tissues, Ets-1 is abundantly expressed in B and T lymphocytes but is not abundant in myelomonocytic lineages

(Ghysdael et al. 1986; Bhat et al. 1987; Kola et al. 1993).

Ets-2 is ubiquitously expressed throughout embryonic and neonatal development.

In extraembryonic tissues, ets-2 is expressed highly in trophoblasts, and expressed at a relatively lower level in the extraembryonic ectoderm and trophectoderm (Yamamoto et al., 1998). In embryos, ets-2 is highly expressed in developing bone, tooth buds,

epithelial layers of the gut, nasal sinus and uterus, several regions of the developing brain, developing lung, gut and skin (Maroulakou et al. 1994a).

Recently microarray experiments have generated a lot of data regarding ets protein expression in various human tissues and cell types (Fig. 1.3). Some ets proteins, such as Elk1, Elf1, NERF and ERM, are highly expressed in most tissues and cell types studied. ets-2 is highly expressed in many tissues and cells, including thymus, lung, trachea, skin, skeletal muscle, kidney, liver, spleen, small intestine, colon, bladder prostate, uterus, monocytes, bronchial/Tracheal epithelial cells and neonate epidermal keratinocytes, and moderately expressed in other tissues and cells. Some ets proteins are highly expressed only in a subset of tissues and cells. For example, ets-1 is expressed abundantly in thymus and blood. ESE1 is highly expressed trachea, breast, pancreas and colon. ESE2 is primarily expressed in salivary gland, lung, trachea and breast. PDEF is highly expressed in salivary gland, trachea, breast, colon, and prostate. It is worth noting that many of the microarray data are based on the expression of ets factors in whole tissues that consists many cell types. The expression of a certain protein in a certain type of cells within a tissue might be much higher.

Figure 1.3. Ets proteins expression pattern. A. Ets proteins expression in developing mouse. Each Ets protein is indicated by different colors. This figure is adopted from Table 1 in Maroulakou IG and Bowe DB, 2000. B. Ets proteins expression levels in normal human tissue. C. Ets proteins expression levels in cancer cells. B and C are adopted from published microarray data compiled by Timothy Ravasi, The University of Queensland, Brisbane, Australia. The coloring of the number is arbitrary, but basically the levels, from high to low, is indicated by red, brown, purple, gray, dark blue or light blue, respectively.

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Fig. 1.3. Continued.

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Fig. 1.3. Continued.

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Fig. 1.3. Continued.

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Fig. 1.3. Continued.

1.3.2. DNA binding.

Non-conserved residues within the Ets domain

The structure studies with Ets domain of several proteins suggested that DNA

contacts from conserved and nonconserved protein residues coupled with sequence-

dependent DNA structural properties appear to contribute to individual “innate” Ets

protein specificity (Grave and Petersen, 1998, Verger and Duterque-Coquillaud, 2002). A single amino-acid substitution (K388 of Ets-1 to T54 of Elf-1) within the Ets domain switches Ets-1 DNA binding specificity to that of related Ets family transcription factor

Elf1 and E74 (Bosselut et al. 1993). Two amino acids (A231 and N236) in the Ets domain of PU.1 determine that PU.1 cannot recognize GGAT sequence (Pio et al. 1996).

Residues flanking the Ets domain.

The Ets domain flanking sequences is also important to determine the preferential binding of individual Ets proteins (Szymczyna and Arrowsmith, 2000). TCF subfamily

Ets protein SAP-1 binds to the c-fos promoter with high affinity, however, the DNA binding domain of Elk-1, a member from the same subfamily, binds poorly to the c-fos promoter (Shore et al., 1996). Nearly one-third of the interactions between the protein recognition helix and the DNA are different between the SAP-1 and Elk-1 DNA complexes (Mo et al., 1998, Mo et al., 2000). The differential DNA binding properties of

Ets proteins are mediated by non-conserved residues distal to the DNA binding surface that function to orient conserved residues in the DNA recognition helix for protein- specific DNA contacts. Alteration of a single amino acid in this region changes their specificity (Mo et al, 2000). 35

Autoinhibition of the Ets domain.

DNA binding is a dynamic process that is tightly regulated. Several ETS-domain proteins are autoinhibited in the absence of appropriate stimuli or partner proteins.

Conversely, such inhibition is lost in the presence of appropriate triggers. The Ets-1 autoinhibitory elements were discovered when proteolysis of Ets-1 or deletion of regions flanking the Ets domain enhanced DNA binding activity (Jonsen et al., 1996, Skalicky et al., 1996, Graves and Petersen, 1998). Ets-1 and activated Ets-1 polypeptides differ in

DNA-binding affinity as much as 23-fold. Two regions flanking the minimal DNA- binding domain mediate the inhibition of DNA binding. Both regions regulate affinity by enhancing dissociation of the protein-DNA complex. Truncation of either the N-terminal inhibitory region (residue 280-330) or the C-terminal inhibitory region (residue 416-440) fully activates DNA binding (Jonsen et al., 1996). An intramolecular conformational switch forms the basis of an autoinhibitory mechanism. The autoinhibitory elements are also found in other Ets proteins, such as Ets-2, Elk-1 and PEA3. Contrary to the conserved Ets DNA binding domain, those elements are not conserved in these Ets proteins. Therefore, those autoinhibitory elements, regulated by signal pathways and partners, could specify gene expression in a defined environment.

Autoinhibition of Ets proteins is counteracted by a protein partnership and reinforced by phosphorylation (discussed later in this Chapter). Direct contacts between a partner and inhibitory sequence can alleviate autoinhibition and provide cooperative energy. The first inhibitory helix HI-1 could be stabilized in a different conformation, allowing the Ets domain to adopt a high affinity DNA-binding form (Garvie et al. 2002). 36

AML1 enhances Ets-1 binding affinity for DNA by counteracting the autoinhibitory mechanism. AML1 could contact HI-1, stabilize it and break its intramolecular interactions. This removes the inhibitory effect of HI-1 on the remainder of the protein and result in high affinity binding (Garvie et al. 2002).

1.3.3.Protein partners of Ets family members.

Combinatorial control is a characteristic property of Ets family members. Many

Ets family proteins modulate gene expression through protein-protein interactions with other cellular partners. Protein-protein interactions regulates DNA binding, subcellular localization, target gene selection and transcriptional activity of Ets proteins (Li et al.

2000; Verger and Duterque-Coquillaud 2002).

Ets family proteins regulate gene expression by functional interaction with other transcription factors and co-factors on composite DNA-binding sites. Specific domains of

Ets proteins interact with many protein motifs such as bHLH, bZipper and Paired domain. Interaction between Ets and other key transcriptional factors such as AP-1,

AML1, LEF-1, Sp1, c-Myb, Pax-5, NF-κB, Stat-5 and Maf-B, regulates the expression of cell-type specific genes (Li et al. 2000; Verger and Duterque-Coquillaud 2002). Such interactions coordinate cellular processes in response to diverse signals including cytokines, growth factors, antigen and cellular stresses (Graves and Petersen 1998b; Li et al. 2000; Verger and Duterque-Coquillaud 2002). One well-known example is interaction of Ets family with Jun family proteins on the DNA sequences called the Ras-responsive element (RRE) consisting of an Ets binding site and an AP-1 (Fos/Jun) site. The RRE was originally identified on the polyomavirus enhancer and is located on the sequence in 37

various cellular genes which are responsive to Ras/MAPK signaling (Imler et al. 1988).

For example, RRE is found in the promoters of matrix metalloproteinase 1(MMP1) and

urokinase plasminogen activator (uPA) genes, which are responsible for organ

remodeling and tumor invasion. The promoters of these genes are synergistically

activated by Ets-1 and AP-1 with the co-activator CBP/p300. Ets-1 and Ets-2 recruit

transcription adapter proteins p300 and CBP (cAMP-responsive element-binding protein)

during the transcriptional activation of the human MMP-3 promoter, which contains

palindromic Ets-binding sites. Although MMP3 promoter contains palindromic Ets-

binding sites, other Ets family transcription factors failed to cooperate with p300/CBP in

stimulating the expression of MMP-3 (Jayaraman et al. 1999).

Ets proteins can also obtain “acquired” DNA-binding specificity mechanism

through interaction with partners. When other transcription factors binds to adjacent sites,

an uninterrupted protein-DNA interface extends across both recognition elements,

thereby increases the specificity and affinity of DNA binding. For example, Elk-1 binds

to c-fos promoter poorly by itself. However, when Elk-1 is recruited by SRF, DNA

binding of SRF nearby may promote the reorientation of the appropriate Elk-1 residues to

facilitate its sequence-specific binding to the c-fos promoter (Sharrocks and Shore 1995).

Similarly, Ets-1 or Elk-1 needs to be recruited by Pax-5 to recognize efficiently the mb-1 promoter (Fitzsimmons et al. 1996; Fitzsimmons et al. 2001).

Ets proteins bind to coactivators and corepressors that modulate their transcriptional regulatory properties (Graves and Petersen 1998b; Li et al. 2000; Verger and Duterque-Coquillaud 2002). A major mode of action of ETS-domain transcription

factors is to recruit histone acetyl transferases or deacetylases to activate or repress transcription. This interaction is discussed further in Chapter 5.

1.3.4. Signal transduction and post-translational modification of Ets family members.

Many Ets proteins are downstream targets of signal transduction cascades. This post-translational modification of the proteins often changes their DNA binding, transcriptional activities, association with cellular partners (other transcription factors or co-activators), subcellular localization and/or protein stabilities (Yordy and Muise-

Helmericks 2000; Sharrocks 2001). Many Ets family proteins are phosphorylated in response to growth factors and cellular stress, through the activation of signal transduction pathways, such as the Ras/MAPK pathway (Wasylyk et al. 1998; Yordy and

Muise-Helmericks 2000).

TCF/elk-1 ets family transcription factors are phosphorylated, thereby activated, by the ras/MAPK pathway at multiple serine and threonine residues in the C-terminal activation domain. Activation of those ets proteins activates expression of many immediate-early genes, such as c-fos (Whitmarsh 1995). Ras also induces persistent gene expression through regulating the activities of Ets-1 and Ets-2 through the Pointed domain that contains a single MAPK phosphorylation site (threonine 38 in Ets-1 and threonine 72 in Ets-2) (discussed in detail later in this chapter). Mutation of the phosphate-acceptor threonine within this site in either protein abolishes responsiveness to activated Ras (Yang et al. 1996; Fowles et al. 1998a).

The regulation of Ets protein by ras signal transduction pathways is conserved through evolution. For instance, the Drosophila pointed gene codes for two Ets transcription factor, pointed P1 and P2. Pointed P1 is a constitutive activator of transcription. Pointed P2 protein is a nuclear target of Sevenless receptor tyrosine kinase-

Ras-Raf-MAPK signal transduction cascade (Brunner et al. 1994; O'Neill et al. 1994). A single consensus MAPK phosphorylation site (homology to T38 of Ets-1 and T72 of Ets-

2) in pointed P2 is phosphorylated by MAPK in vitro. Mutation of this site abrogates

MAPK responsiveness in vivo (Brunner et al. 1994; O'Neill et al. 1994). The Ras-MAPK pathway does not only regulate transcription activators, but it also regulates Ets family transcription repressors. Yan is another Drosophila Ets protein. It is a negative regulator of photoreceptor determination and acts as an antagonist of Ras1 (Lai and Rubin 1992;

O'Neill et al. 1994). Activation of the Ras-MAPK signal transduction cascade leads to the phosphorylation of Yan, which is then exported from the nucleus into the cytoplasm where it is degraded (O'Neill et al. 1994; Rebay and Rubin 1995; Gabay et al. 1996).

Therefore, Ras-MAPK controls neural development through phosphorylation of two antagonizing Ets transcription factors, pointed P2 and Yan; manipulates the balance of pointed P2 and Yan that is necessary for proper Drosophila eye development.

Signal transduction pathways can also repress an Ets transcriptional activator. At least 4 serines, which are at N-terminal of the Ets domain near Ets-1 autoinhibitory helices HI-1 and HI-2, are phosphorylated by Calcium induced signal pathway (Rabault and Ghysdael 1994). This phosphorylation stabilizes an inhibitory conformation and greatly inhibits the Ets-1 DNA binding ability (Cowley and Graves 2000).

Besides phosphorylation, Ets factors can undergo other forms of posttranslational

modifications. For example, ER81 is acetylated by two coactivators/acetyltransferases,

p300 and p300- and CBP-associated factor (P/CAF) in vitro and in vivo. p300 acetylates

two lysine residues (K33 and K116) within the ER81 N-terminal transactivation domain,

while P/CAF targets only K116. Acetylation of ER81 enhances its DNA binding activity,

stability and also its ability to trans-activate target gene expression. HER2/Neu

overexpression stimulates the ability of p300 to acetylate ER81, likely by inducing

phosphorylation of p300 through the Ras-MAPK pathway (Goel and Janknecht 2003).

1.4 Ets-2 and Ras signaling.

1.4.1 Ras and Ras signaling.

Ras proteins are now well recognized for their essential function in transducing

extracellular signals and regulating cell growth, survival, and differentiation. Mutations in codon 12, 13, or 61 of one of the three ras genes convert these genes into active oncogenes. Ras gene mutations can be found in a variety of tumor types, although the incidence varies greatly. Approximately a third of human tumors have activating mutations in one of the three ras genes. The highest incidences are found in adenocarcinomas of the pancreas (90%), the colon (50%), and the lung (30%); in thyroid tumors (50%); and in myeloid leukemia (30%) (Bos 1989; Adjei 2001). Most human tumors are of epithelial origin, and these tumors gradually lose their epithelial character in a process called the epithelial-mesenchymal transition. Epithelial cells transformed by oncogenic Ras acquire a mesenchymal phenotype that is associated with a decrease in cell-cell adhesion and an increase in focal adhesions and stress fibers resulting from 41

alterations in the organization of the actin cytoskeleton and adhesive interactions (Bar-

Sagi and Hall 2000a; Bar-Sagi 2001).

Ras belongs to a group of proteins called Small GTP-binding proteins (Macara

1996; Bar-Sagi and Hall 2000a). These proteins are monomeric G proteins with molecular masses of 20-30 kDa. GTPases of the Ras superfamily act as molecular switches to control a wide range of essential biochemical pathways in all eukaryotic cells.

Like all GTPases, they exist in an inactive (GDP-bound) and an active (GTP-bound) conformation. An upstream signal stimulates the dissociation of GDP from the GDP- bound form, which is followed by the binding of GTP, eventually leading to the conformational change of the downstream effector-binding region so that it interacts with the downstream effectors. The rate-limiting step of GDP/GTP exchange reaction is the dissociation of GDP from the GDP bound form. Guanine nucleotide exchange factors

(GEFs) catalyze the release of GDP. An intrinsic GTPase activity, catalyzed further by

GTPase activating proteins (GAPs), completes the cycle and the GTPase returns to its inactive, GDP bound state (Macara 1996; Bar-Sagi and Hall 2000b).

Active GTP-bound Ras interacts with several effector proteins: among the best characterized are the Raf kinases, phosphatidylinositol 3-kinase (PI3K), RalGEFs and

NORE/MST1 (Joneson 1997; Adjei 2001). Ras activates Raf-1 by recruiting it in a complex with 14-3-3 to the plasma membrane. Activated Raf activates mitogen-activated protein kinase (MAPK) kinase (MEK)1 and MEK2 by serine phosphorylation. Activated

MEK, in turn, phosphorylates and activates ERK/MAPK. Ras also binds directly to the p110 subunit of PI3K and upregulates lipid kinase activity. K-ras is the more potent

activator of Raf-1, and H-ras is the more potent activator of PI3K in vivo (Joneson 1997;

Adjei 2001).

Mitogen-activated protein kinases (MAPKs) are widely expressed serine- threonine kinases that mediate important regulatory signals in the cell (Seger and Krebs

1995). Three major groups of MAPK exist: the extracellular signal-regulated kinase (Erk) family, the c-Jun N-terminal kinase (JNK) family, and the p38 MAPK family. MAPK participates in the generation of various cellular responses, including gene transcription, induction of cell death or maintenance of cell survival, malignant transformation, and regulation of cell-cycle progression. MAPK is activated by a MAPK kinase (MAPKK), which is a ‘dual-specific’ kinase that phosphorylates at both Ser/Thr and Tyr sites, targeting a Thr-X-Tyr motif on the MAPK (where X is glutamate, proline or glycine for the ERK, JNK and p38 modules, respectively) (Seger and Krebs 1995; Cowan and Storey

2003).

1.4.2 Persistent activation of Ets-1 and Ets-2 through phosphorylation by Ras-

MAPK pathway.

Ets-1, Ets-2 and Drosophila pointed P2 share one conserved MAPK site at the N- terminal of their pointed domain. The integrity of this site is crucial for superactivation of

Ets target genes by Ets-1, Ets-2 and Ras pathway. Mutation of a single conserved threonine residue (Ets-1 T38, or Ets-2 T72) to alanine abrogates the ability of Ets-1 or

Ets-2 to superactivate report gene expression. Phospho-amino acid analysis, mutation of

T72 to alanine, and Western blotting using a polyclonal antibody specifically recognizing

phosphorylated Ets-2 threonine 72, revealed that Ras induced normally absent threonine specific phosphorylation (Yang et al. 1996), (Fowles et al. 1998a).

Extracellular stimuli like CSF-1 stimulation results in the stable, persistent expression of specific genes, for example, uPA, in mature macrophages or in fibroblasts ectopically express the CSF-1 receptor, c-fms. The uPA gene has an RRE (Ras response elements, composed of Ets binding site and AP1 site) located 2-3 kb from the transcription initiation site(Yang et al. 1996). Ras/MAPK kinase pathway activates Elk-1 ets family transcription factors (Wasylyk 1998). These events occur early after growth factor stimulation and result in expression on immediate-early genes such as c-fos, but the phosphorylation of Elk-1 is transient. Therefore, the phosphorylation of elk-1 cannot account for persistent expression of Ets target genes (Fowel et al., 1998). In contrast,

Threonine 72 of Ets-2 is persistent phosphorylated in a ras-dependent fashion in response to CSF-1/c-fms signaling. In transient transfections, Ets-1 and Ets-2 stimulates uPA expression, and synergistically superactivate uPA expression with Ras. In contrast, Fli-1,

Elf-1 and PEA-3 can only moderately activate uPA expression, and they does not display superactivation in combination with Ras (Yang et al. 1996). Therefore, Ets-2 and Ets-1 appear to be the primary Ets factors that transduce persistent ras/MAPK signal.

Erk-1 and Erk-2 are the major kinases that phosphorylate Ets-2 in fibroblast cells.

In-gel kinase assay using recombinant pointed domain of Ets-2 suggested that p42 and p44 MAPK are the major renaturable kinases responsible for phosphorylation of threonine 72 of Ets-2 following activation of Raf in NIH 3T3 cells (McCarthy et al, 1997,

Fowles et al, 1998). Purified recombinant p42 and p44 MAPK phosphorylate purified recombinant protein corresponding to Ets-2 pointed domain. In the same condition, a 44

recombinant Ets-2 protein containing the Ala 72 substitution (T72A) was not

phosphorylated by MAPK. MAPK activity correlates with Ets-2 phosphorylation. MAPK

is activated from minutes to hours in ∆Raf-1:ER NIH3T3 cell following addition of

estrogen. Prior to ∆Raf-1:ER activation, there was low basal level of threonine 72-

phosphorylated Ets-2 that was significantly increased within 30 minutes and was

maintained for at least 24 hours following ∆Raf-1:ER activation (McCarthy et al, 1997).

The ERK inhibitor PD98059 inhibits MAPK activation and greatly reduces Ets-2

phosphorylation (Ghosh et al, 2003). The phosphorylation of Ets-2 is followed by Ets-2

target genes expression. For example, Heparin-binding epidermal growth factor (HB-

EGF) gene was shown to be an Ets-2 target gene. Its expression correlates with Ets-2

phosphorylation state (McCarthy et al, 1997). uPA and Bclx mRNA level also increased

significantly after Ets-2 phosphorylation (Fowles et al. 1998a; Smith et al. 2000).

Ets-2 is also phosphorylated at the same threonine residue by other signaling

pathways. In wild type bone marrow derived macrophages (BMMs), ERKs are activated

transiently and persistently by CSF-1 stimulation and followed by phosphorylation of

Ets-2. However, in an animal model if acute inflammation, motheaten viable (mev) mice

(discussed in Chapter 3), ERKs are not responsive to CSF-1 stimulation in macrophages.

ERKs are not activated transiently, or persistently, by addition of CSF-1 to BMMs

(Krautwald et al. 1996; Smith et al. 2000). However, Ets-2 is still phosphorylated, even in the absence of exogenous CSF-1. Ets-2 target genes, uPA and SR, are also expressed at high level in mev BMMs in the absence of CSF-1 (Smith et al, 2000). PI3K level are higher in mev macrophages than in wild-type cells (Roach et al, 1998). When LY294002, a drug specifically inhibit PI3K activity, is added to the mev cells, Ets-2 phosphorylation 45

is greatly reduced. Akt is an important downstream target of PI3K pathway, and also has higher activity in mev macrophages. Akt immunoprecipitates are able to phosphorylate the T72 form of Ets-2, but not the A72 form, indicating that Akt-associated kinase phosphorylated the same T72 residue of Ets-2 as the ERKs. Since Ets-2 lacks the Akt consensus phosphorylation motif, a kinase associated with Akt, like JNK p54, could be the kinase responsible for phosphorylation of Ets-2 (Smith et al, 2000).

The above sections indicated that Ets-2 phosphorylation by Ras pathway correlated with macrophage survival and mev mice inflammation phenotype, and also correlate with tumor development. However, there was no direct in vivo evidence demonstrating the role of Ets-2 in inflammation. Although Ets-2 was shown to be important for placenta development, the function of Ets-2 in embryo development was not revealed. Furthermore, the early embryonic lethality of ets-2 knockout mice hampered the study of Ets-2 in later development stages and in diseases models, such as cancer. The phosphorylated form of Ets-2 was shown to activate target gene expression.

However, the function of the unphosphorylated form of Ets-2 was not well defined.

This study used genetic, biochemical and cellular approaches to understand the roles of Ets-2 phosphorylation in vivo and in vitro. Ets-2 was an important regulator of inflammation in an inflammation model, mev mice. Inflammation phenotypes of mev mice were corrected, at least partially, in mice with half dosage of ets-2 or with homozygous ets-2T72A allele. Ets-2 mutation affected macrophage survival, and the expression of genes encoding inflammatory cytokines and chemokines (such as TNFα,

IL-1α, IL-12, MCP1, MIP1α and MIP1β), cell adhering molecules (e.g. integrin αM, 46

β2), extra cellular matrix proteinases (e.g. MMP1, MMP9 and uPA) and survival factors

(e.g. Bclx). Ets-2 was not only shown to be important for pathological processes; it was

demonstrated to be critical in normal physiological processes, such as in embryo

development. To reveal the roles of Ets-2 in development possibly masked by gene

redundancy, ets-2T72A/T72A mice was studies in the ets-1-/- background. Ets-1-/-, ets-

2T72A/T72A mice were embryonic lethal with severe angiogenesis and cardiovascular

defects, indicating that Ets-1 and Ets-2 were essential for angiogenesis and embryo

development. Ets-1/2 mutations caused reduced expression of some Ets target genes,

such as Ang1, Tie2, Fli-1, MMP1, MMP9, VE-cadherin, BRCA1, CDC2, Cyclin D1 and

HIF1α, but led to overexpression of VEGF. The phenotype similarities between Ets-1-/-,

ets-2T72A/T72A mice and Tie2 or Ang1 knockout mice and the downregulation of Tie2 and

Ang1 in the ets-1/2 double mutant mice indicated that there was close genetic relationships between ets-1/2 and Tie2 and Ang1, and Ets-1/2 might directly regulate

Tie2 or Ang1 expression. Moreover, Ets-1-/-, ets-2T72A/T72A mice had less macrophages,

and had reduced extracellular matrix proteases expression in fibroblasts. This study

indicates the roles of Ets-2 in tumorgenesis are controlling target genes expression not

only in cancer cells, but also in cells in tumor microenvironment, including fibroblasts,

macrophages and blood vessel cells, and may be essential for tumor angiogenesis and

metastasis. An ets-2 conditional knockout allele was developed, and will be very useful

to study the function of Ets-2 by cell type specific deletion of ets-2. Finally, our

biochemical and cellular assays discovered that the unphosphorylated form of Ets-2 was

not idle bystander in gene regulation. It interacted with Brg-1 or BS69 corepressor

complex, and repressed target gene expression in a chromatin dependent fashion. Further 47

studies with Ets-2 in immune disease or cancer may further define the role of Ets-2 in these diseases and development of novel strategies for early diagnosis and treatment for patients with these diseases.

CHAPTER 2

MATERIALS AND METHODS

2.1 Plasmids and DNA manipulations.

2.1.1 Plasmids.

The vector used for expression of ets-2, uPA-luciferase, were previously described (Fowels, et al., 1998, Yang et al., 1996). The luciferase report of BRCA1 was kindly provided by Ellen Soloman (Guy’s hospital, Lodon). pREP4-luciferase were a kindly gift from Dr. Liu (National Heart, Lung, and Blood Institute, National Institutes of

Health, Bethesda, MD ). BS69 and NCoR expression vectors were kindly provided by

Dr. Bernard (The Netherlands Cancer Institute, Amsterdam, The Netherlands). The expression vectors for Brg-1 and Brg-1 K798R were previously described (Fryer and

Archer, 1998). The triple-lox cloning vectors, pLoxL, pLoxC and pLoxR, were provided by Dr. Leone (The Ohio State University). The PGK-cre vector was previously described

(Abuin and Bradley, 1996). pLoxP vector was kindly provided by Dr. Weinstein (The

Ohio State University).

2.1.2. Plasmid miniprep and maxiprep.

Plasmid miniprep:

The day before miniprep, a single isolated bacterial colony was pick up by a sterile toothpick and transferred to a 15 ml sterile tube with 3-5 ml LB media supplemented with 100ug/ml Ampicillin or other appropriate antibiotics. The cells were grown overnight in a shaker at 225-300 rpm at 37°C. 1.5ml of bacteria culture were transferred to a mini-centrifuge tube and centrifuged in a microfuge at top speed for 1 min. The supernatants were discarded. (Optional: another 1.5ml from the same tube were added and centrifuged). After the supernatant were decanted, the tube was left upside down on paper towel briefly. 0.3 ml P1 solution (15mM Tris, pH 8.0, 10 mM EDTA,100 ug/ml RNase A) was added into each tube. The pellet was vortexted to resuspended. 0.3 ml of P2 solution (0.2N NaOH, 1% SDS) was added. The tube was gently inverted several times to mix the contents and sit at room temperature for about 5 min. The appearance of the suspension should change from very turbid to almost translucent. 0.3 ml cold P3 solution (3M KOAc, pH 5.5) was added to each tube and the tube was gently inverted several times to mix the contents. A thick white precipitate of protein and E. coli genomic DNA would form. The tube was incubated on ice for 20-30 min before centrifuged in a microfuge at top speed for 5 min. 0.8ml of supernatant was transferred using a P1000 pipette to the 1.5 ml eppendorf tube that contains 0.56 ml isopropanol. The tube was inverted a few times to mix the contents and then placed at room temperature for 10 min before centrifuge in a microfuge at top speed for 5 min. The pellet was washed with 1 ml of 70% ethanol and spined in a microfuge for 2 min before air-dried and resuspended in 50 µl TE. For BAC plasmid, narrow bore pipettes tips were not used to 50

mechanically resuspend DNA sample; rather, the solution was allowed to sit in the tube

with occasional tapping of the bottom of the tube at 37oC. For isolation of plasmid for

sequencing, the Plasmid mini kit (Qiagen) was used according to manufacture’s

instructions.

Plasmid maxi prep was using plasmid maxi kit (Qiagen) according to manufacture’s

instructions. For BAC DNA, 1-4 litters of bacteria culture were used. After the P3 step,

the supernatant was transferred to a fresh centrifuge tube. DNA was precipitated with

0.67 volume of isopropanol. After centrifugation, the pelleted DNA was washed with

70% ethanol once, air-dried and resuspended in appropriate amount of TE. All the BAC

DNA was combined and then equilibrated with QBT and loaded on one maxi-prep column (Qiagen), and purified according to manufacture’s instructions. To elute BAC

DNA, solution QF was warmed up to 50oC before elution.

2.1.3. Colony lifts.

The transformants colonies were picked-up and grided-out in duplicate dishes.

The dishes were incubated at 37oC overnight. 4 3MM paper squares (large enough for 3

85mm nitrocellulose circles) were placed on spread out saran-wrap. 1%SDS/5mM EDTA solution was poured on the first 3MM square paper, lysis solution (0.5M NaOH, 1.5M

NaCl) on the next, neutralization solution (1M Tris-Cl, pH 8.0, 1.5M NaCl) on the next and 2X SSC (0.3M NaCl, 30mM Na citrate, pH 7.0) on the last, to saturate the 3MM

paper, without leaving pools of liquid on top on the paper. A dry nitrocellulose filter

(85mm circle) was placed onto each plate to be lifted and taped down enough so that the 51

filter was wet all over. 5 minutes later, the nitrocellulose filters (most of cells of each

colony should adhere to the filter) was lifted and placed colony-side up onto the 1%

SDS/5mM EDTA 3MM paper. 5 minutes later, the filter was transferred to the lysis

solution 3MM paper and incubated for 5 min. The filter was transferred to the

neutralization solution 3MM paper and incubated for 5 min. The filter was transferred to

the 2X SSC 3MM paper and incubated for 5 min. The filter was placed colony-side up

onto dry 3MM paper and left air-dry for 15 min. After being baked in a oven for 2 hr at

80oC, the filter was used in hybridization, as in Southern blots.

2.1.4. DNA cloning.

Restriction digests.

Restriction digests of DNA were conducted with commercially available endonucleases (NEB, Fermentas and Roche). The digests were performed using the appropriate buffers and under conditions recommended by the manufacturer in a total volume of 20-100µl, depending on the amount of DNA. In case of a sequential digest, the vector was digested with the first enzyme for 2 hours, ethanol precipitated, and digested with the second enzyme. The products of restriction digestion were analyzed by agarose electrophoresis along with undigested controls and DNA size markers. For ligation or making probes, the appropriate DNA fragments were cut out from the gel and purified using a QIAquick Gel extraction kit (Qiagen) according to manufacture’s instruction.

Alkaline phosphatase treatment of vector.

Linearized vector DNA was subjected to alkaline phosphatase treatment prior to

ligation, to avoid self-ligation of “sticky ends”. The vector DNA was digested with the

appropriate restriction enzyme or enzymes. 1-3 µl (1unit/µl) of Calf Intestine Alkaline

Phosphatase (Roche) was added to the DNA sample and incubated at 37oC for one hour.

The restricted DNA fragments were loaded on an agarose gel, and purified by QIAquick

Gel Extraction Kit (Qiagen), according to manufacturer’s directions. The DNA was

eluted in 30µl of water.

Generation of blunt end by Klenow filling-in 5’ overhangs.

After restriction digest, dNTP was added to the buffer to make a final

concentration of 66µM for each dNTP. 1 unit Klenow enzyme (NEB) per microgram

DNA was added and incubated at room temperature for 15 minutes. EDTA was added to

the solution to make a final concentration of 10mM to stop the reaction. The enzyme was

heat inactivated at 75oC for 20 minutes.

Ligation.

Linearized vectors (after alkaline phosphotase treatment) were ligated to inserts

containing compatible sticky or blunt ends using a molar ratio of 1:3. The reaction was

performed in a 20 µl reaction in the presence of 1-6 units of T4 DNA ligase (NEB or

Fermentas) using the buffer supplied by the manufacturer, at room temperature for sticky

end ligations for 30 minutes to 3 hours, or at 4oC or 16oC overnight for blunt end ligations.

2.1.5. High molecular weight genomic DNA isolation and purification.

Mouse genomic DNA was isolated from 0.5-1cm long tail tip clips. Tails were digested overnight at 55oC in 0.7ml TE-SDS (100mM Tris-Cl; pH 8.0, 50mM EDTA,

100mM NaCl and 0.5% SDS) and 1mg/ml of fresh proteinase K. The tail extract was

extracted once with Tris-Cl buffer-saturated phenol (pH 8.0). The aqueous phase was

transferred to a fresh tube and further extracted with phenol-chloroform-isoamyl alcohol

(24:24:1) and finally with chloroform-isoamyl alcohol. The aqueous phase was

transferred to a fresh tube and the genomic DNA was precipitated in the presence of

1/10th the volume of 3M NaAc (pH 5.2) and 2 volume of 100% ethanol. The DNA

precipitates were visible and easy to be spooled by pipette tips and transferred to a fresh

tube with 1ml of 70% ethanol. After centrifugation, the DNA pellet was allowed to air-

dried and resuspended in 50-100ul of TE at 37oC overnight. The DNA was used for

Southern Blot or PCR.

2.1.6. ES cell DNA extraction.

ES cells were growing in 96, 24, or 12 well dishes for the desired amount of time.

The media was aspirated off and 200ul or 400ul of lysis buffer (0.5% SDS, 100mM

NaCl, 10mM EDTA, 20mM Tris-Cl, pH 7.6, 100ug/ml proteinase K) was added to each

well, and set at room temperature for 5 minutes, swirled to lyse and detach cells. The

lysate was transferred to an eppendorf tube and incubate at 50oC for 2 hours, and cooled 54

at room temperature. 125 or 250µl of saturated (6M) NaCl was added to each tube. The

tube was vortexted vigorously for about one minute and incubated on ice for 10 minutes.

The samples were centrifuged in a microcentrifuge at top speed for 10 minutes.

Supernatant was transferred to a fresh tube, and 2 volumes of ethanol were added. The

sample was centrifuged in a microcentrifuge at top speed for 2 minutes. DNA was

washed once with 70% ethanol, air-dried and resuspended in 50ul of TE (10mM Tris-Cl,

pH 8.0, 1mM EDTA) at 37oC for 1 hour to overnight. The DNA can be used in PCR or

Southern blots.

2.1.7. Polymerase chain reaction (PCR)

PCR amplification from plasmid DNA was performed by using a reaction mixture containing 1-10 pg of plasmid DNA, or 1ng of BAC DNA, 0.2µM each of forward and reverse primers, 1.5µM MgCl2, 20µM deoxy-nucleotide triphosphates (dNTPs) (Roche),

1X PCR buffer and 1 unit of Tag DNA polymerase (Invitrogen) in a total volume of 25-

50µl. PCR was performed using the following thermo cycling conditions: an initial denaturation at 95oC for 2 minutes followed by 20 - 35 cycles of denaturation at 94oC for

30 seconds, annealing at appropriate temperature for 30 seconds and extension at 72oC

for appropriate time ( 1kb amplification product per minute, minimum 1 minute) and a

final extension step at 72oC for 10 minutes. Reaction products were analyzed by

electrophoresis or purified using QIAquick PCR-purification kit (Qiagen). For PCR from

genomic DNA, please refer to the genotyping section.

2.1.8. Southern blots.

Random Prime Labeling of DNA Probes

1-2 µg of restriction digested plasmid DNA, or a PCR product, was run on a 1%

agarose gel. The DNA fragments were isolated using QIAquick gel extraction kit

(Qiagen). The DNA was eluted from the QIAquick column in 30µl of distilled water.

25ng of DNA was labeled in a 20µl reaction containing 50µCi of 32P labeled dATP using

a random primed DNA labeling kit (Roche) according to manufacture’s instructions. The

labeling reaction was performed for 10 minutes at 37°C. The random primed probes were purified using the QIAquick Nucleotide Removal Kit (Qiagen).

Gel blotting.

After appropriate enzyme digestion, the DNA samples were electrophoresed in an agarose gel with ethidium bromide (0.5µg/ml of gel volume) at 1-2 volts/cm overnight.

The gel was photographed with a fluorescent ruler under UV light for later reference. The gel was incubated in the depurination solution (0.25 M HCl) with gentle shaking. The gel was rinsed twice with water and incubated in denaturing solution (0.5 M NaOH, 1.5 M

NaCl) for 30 minutes. After rinsed with water briefly, the gel was transferred to neutralization solution (0.5 M Tris-Cl, pH 8.0, 1.5 M NaCl) for 15 minutes, and switched to fresh neutralization solution for 30 minutes. 40 pieces of 3M paper (same size and shape as the gel) were prepared. 16 pieces of paper was soaked in 10X SSC and plated on a glass plate. The gel was flipped over with the well side facing down, labeled with a notch at the upper left corner, and placed on the wet paper. Air bubbles were squeezed

out. A piece of nitrocellulose membrane (slightly bigger than the gel) was wet in water

and placed on top of the gel. All air bubbles were gently squeezed out. 16 pieces of dry

3M paper was placed on top of the nitrocellulose membrane. 1-2 inches of dry paper

towers were placed on top of the dry 3M papers. The whole paper/gel/glass plate

complex was flipped over and placed on a second glass plate. The top glass plate was

removed and 8 pieces of wet 3M paper was placed on top. Four pieces of parafilm was

prepared and placed at the sides of the gel to prevent direct contact of the wet paper and

the dry paper. The glass plate was put back on. The whole blotting complex was wrapped

with Saran wrap. Some weight (500g-1kg) was put on top of the glass plate. The DNA

was allowed to transfer overnight. The nitrocellulose membrane was baked at 80oC for

two hours and ready for hybridization.

Hybridization.

The membrane was incubated in 20 ml pre-hybridization solution (50%

Formamide, 5X SSC, 1X Dennhart solution, 0.1% SDS, 100µg/ml denatured CT DNA)

for 2 hrs at 42oC. The pre-hybridization solution was removed and the membrane was incubated with 10-20 ml hybridization solution (pre-hybridization solution plus denatured probe (1-2 million counts per ml, denatured by boiling for 5 minutes and placed in salted ice water) at 42oC for 1-2 days. The membrane was washed twice with 2X SSC, 0.1%

SDS at RT for 5min, and 30 min with 0.2X SSC, 0.1% SDS at 65oC. The membrane was

then placed in an autoradiography cassette with an X-ray film. The film was developed

several hours (for BAC and plasmid) later, or one day to a week later (for genomic

DNA). 57

2.2. Recombinant protein expression and protein interactions.

2.2.1. GST fusion protein expression and isolation.

The GST (Glutathione-S-Transferase) -BS69 (corresponding to BS69 amino acids

403-522, including the MYND domain) was expressed and purified using GST beads

(Amersham-Parmacia Biotech) according to manufacture’s instructions. Briefly, the

plasmid was transformed into BL21 competent cells. The next day, a colony was picked

up and grew in 5 ml of LB at 37oC overnight, and placed in 250ml LB. The bacteria were allowed to grow at 37 oC till OD600 was 0.6. 50ul 1M IPTG was added to the 250 ml culture. The cells grew at 30 oC for 2-3 hours and were harvested by centrifugation at

4000 rpm in a Sorvall S6-3 at 4 oC for 10 minutes, resuspended in 10 ml ice cold buffer

(100 mM NaCl, 20 mM Tris-Cl, pH8.0, 1mM EDTA), and transferred to a pre-chilled 15

ml conical centrifuge tubes. The cells were centrifuged in a pre-cooled tabletop at 3000

rpm for 10 min at 4 oC, and resuspended in 2 ml of ice cold complete buffer (100mM

NaCl, 20mM Tris-Cl, pH 8.0, 1mM EDTA, 0.5% NP40, and proteinase inhibitors:

10µg/ml aprotinin, 10µg/ml leupeptin, 10µg/ml antipain, and 100 µg/ml PMSF). The sample was aliquoted in microcentrifuge tubes, and sonicated 2X for 10 seconds with a sonicator. Sonicates were centrifuged and the supernatant was incubated with

Glutathione-Sepharose beads (Amershan-Pharmacia Biotech) for 2-6 hours at 4 oC. The

beads were washed 4 times with PBS and boiled in 2X SDS buffer. Commassie-staining and/or Western blots with GST antibody (Santa Cruz Biochemicals) was used to determine the quality and quantity of the recombinant protein.

2.2.2. Phosphorylation of recombinant Ets-2 pointed domain.

Recombinant Ets-2 protein corresponding to the pointed homology region (amino

acids 60-167) was prepared as described (Fowles et al., 1998, McCarthy et al., 1997).

1µg recombinant protein was phosphorylated in a reaction that contained kinase buffer

(30mM Hepes, pH 7.2; 20mM MgCl2; 2mM DTT), 100uM ATP, 10 µCi 32P-γATP (500

Ci/mmole, Dupont/NEN Boston, MA) and 5ul activated recombinant MAPK p42

(Upstate Biotechnology, Lake Plaid, NY) in a 30ul reaction.

2.2.3. Pull Down assay.

1 µg of GST-BS69 fusion protein was immobilized using glutathione beads

(Amersham Biosciences). The beads were incubated with 1 µg of the recombinant ets-2

pointed domain that was unphosphorylated, or phosphorylated by recombinant MAPK

p42. Beads and Ets-2 protein were incubated in lysis buffer for 16 h at 4 °C. Beads were

washed and the material bound analyzed by Western blotting using 6X his tag antibody

(Santa Cruz Biochemicals) for Ets-2, or by autoradiography and phosphorimaging for

32P-labeled protein.

2.2.4. Co-immunoprecipitation.

2.5-3 X 106 COS-7 cells transfected with ets-2, BS69, and/or NCoR were washed

2X with ice-cold PBS. Cold IP buffer (50 mM Tris, pH7.4, 150 mM NaCl, 3mM MgCl2,

1% NP-40, 0.5% deoxycholate) containing protease inhibitors (10µg/ml aprotinin,

10µg/ml leupeptin, 10µg/ml antipain, and 100 µg/ml PMSF) and phosphatase inhibitors 59

(1mM EGTA, 10mM NaF, 1mM tetrasodium pyrophosphate, 0.1 mM β-

glycerophosphate, 1mM NaVO3) was added to the dish (500 ul/100mm dish) and the

cells were scraped off, transferred into an eppendorf tube and pipetted ~10 times up and

down, and lysed in a tube on the rotating wheel for 20-30 min at 4 oC. The sample was

centrifuged for 20 min at 20-30,000 rpm. The supernatant was pre-clear with 20ul of

protein G Gamma Bind Plus Sepharose beads (Amershan-Pharmacia Biotech,

Piscataway, NJ) for ~45 min and centrifuged. The supernatant was mixed with (2-4 µg)

appropriate antibody (HA or FLAG antibody, Santa Cruz Biochemicals ) and 20ul of a

50% slurry of beads, and incubated overnight at 4oC (rotated on a wheel). The beads were

washed 4X with IP buffer, 1X with wash buffer (50 mM Tris, pH7.6, 50 mM NaCl,

6mM MgCl2, 1% NP-40, 0.5% deoxycholate). 30-40 µl of SDS sample buffer was added to the beads and boiled for 5-7 min, spinned for 20 min at 20,000 rpm, and the proteins were analyzed by Western blots.

2.3. Quantitative real time PCR

2.3.1. RNA extraction.

Fresh tissues were placed immediately into at least 10 volume of RNAlater reagent (Ambion #7020, or Qiagen #76104) in a 1.5ml tube, and stored at 4oC or -80 oC according to manufacture’s instructions. The tissue was transferred into an RNase-free blue microfuge tube (Fisher) with 500ul of Trizol (Invitrogen, Cat# 15596-026). Tissue samples were homogenize in 1 ml of Trizol Reagent per 50-100 mg of tissue using a

Fisher homogenization rod (better with a electric drill). The sample volume should not exceed 10% of the volume of Trizol Reagent used for homogenization. To isolate RNA 60

from cultured cells, 1-2ml of Trizol reagent was directly added to the dish to lyse the

cells, and transferred to a fresh RNase-free tube. RNA was extracted according to

manufacture’s instructions. At the end of the procedure, the RNA pellet was briefly air-

dried for 5-10 minutes. RNA was dissolved in appropriate volume (30-100µl) of RNase-

free water by passing the solution a few times through a pipette tip, and incubating for 10

minutes at 55 to 60°C. RNA was quantified by OD260, and the purity of the nucleic acid

was determined by the ratio of OD260 to OD280 (should be greater than 1.8). RNA could

be used immediately or stored at -80°C. For lung alveolar macrophages, RNA was extracted with RNeasy mini kit column (Qiagen) according to manufacture’s instruction.

DNA contaminants was removed by on-column DNase I digestion.

2.3.2. Elimination of DNA contamination.

10ul Roche 5X cDNA synthesis buffer (1st strand) (250 mM Tris-HCl, 40 mM

MgCl2, 150 mM KCl, 5 mM dithiothreitol, pH 8.5 (20°C) was added to the RNA sample.

DNase I was added at 1u/1ug nucleic acid. RNase-free water was added to bring the

volume to 100µl. The sample was incubated at RT for 15 minutes. Then RNA was purified using RNeasy mini kit (Qiagen) according to manufacture’s instruction. The

RNA was eluted with 30-60µl of RNase-free water. RNA was quantified by checking

OD260, and the purity was determined by the ratio of OD260 to OD280 (should be greater than 1.8, normally around 2.0).

2.3.3. Reverse transcription.

1-5 µg of RNA were transferred to a 200ul RNase-free tube. RNase-free water

was added to make to 9 µl. 2 µl of 0.04OD/µl random hexmer primers (Roche) were

added to the RNA sample. The RNA-primer mixture was denatured at 70oC for 10 min

before chilled on ice. Other RT reagents were added to the tube: 4 µl RT buffer, 2 µl

10mM dNTP, 1 µl 100mM DTT, 1 µl RNase inhibitor (Roche), and 1ul Superscript III

reverse transcriptase (Invitrogen). RT reaction was carried out in a thermocycler at 25 oC

for 10 minutes to allow the primers to anneal with RNA, 50oC for one hour for reverse

transcription, and 99oC for 5 minutes to inactive the enzyme. The samples were dilute to

100ul to 200ul. 2-4 µl of each cDNA sample was used in each Real-Time PCR reaction.

2.3.4. Primers for real time PCR.

Primers were picked by Primer 3 software (Rozen S and Skaletsky HJ, 2000, http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) with the following

additional criteria: The primers should be 18-25 nucleotides long, and the Tm should be

about 58 to 60oC; The primers should have no more than 2 Gs or Cs among the last five nucleotides at the 3’ end, preferably an A or T at the very 3’ end; The G/C content should be about 45 to 60%; The amplified PCR product should be 100 to 250 bases (Qiagen recommends 100-150 bps); For multi-exon genes, primers should be in different exons separated by at least one long intron; or one or both primers span an intron/exon junction, with 3’ end 4-5 nucleotide located in a different exon from the rest part of the primer. The primers for Real-Time PCR were listed in Table 2.1.

Table 2.1 Real time PCR primers. The primers sequence and basic information about the genes are listed.

Continued on next page 63

Table 2.1. Continued.

Continued on next page

Table 2.1. Continued.

Continued on next page 65

Table 2.1. Continued.

2.3.5. Real time PCR.

Each Real time PCR reaction mixture had 7-9ul Nuclease-free water, 300nM of

each primer, 12.5 µl SYBR Green super mix (Bio-Rad) and 2-4 µl of diluted cDNA

samples. The 25 µl volume reactions were carried out using the Icycler iQ Real-Time

Detection system (BioRad) according to manufacture’s instructions.

2.3.6. Real time PCR data analysis.

The threshold was determined by iCycler PCR base line subtracted curve fit method. The threshold setting was base on PCR standard curve from known fold dilutions of cDNA samples. Then the threshold of a certain gene was adjusted by that of a reference gene (ribosomal protein L4 gene, 18s rRNA, or lysozyme). The raw mRNA copy number was calculated as: ½(adjusted threshold). The relative copy number was obtained

by dividing each raw mRNA copy number with a certain sample raw mRNA copy

number. The melting curve was checked for each reaction. Data from reactions with

multiple melting curves were discarded.

2.4. Cell survival assays.

2.4.1. Nuclear morphology.

The cells were fixed in 3.7% paraformaldehyde for one hour to overnight. The

cells were washed once with PBS. The bisbenzimide stock (10uM) was diluted 500-1000

folds in PBS plus 0.45% NP40. Appropriate volume (200µl to 1ml, just need to cover the cells) of diluted bisbenzimide solution was added to cells. After staining for 5-10 67

minutes, the cells were washed twice with PBS. The samples were observed or imaged in

a fluorescent microscope with a DAPI filter. Viable cell nuclei were recognized by

relatively even diffused staining, while apoptotic cells nuclei were characterized by

condensed sharp bright dots (Figure 3.1A).

2.4.2. Viability assay.

The viability assay was carried out using the LIVE/DEAD®

Viability/Cytotoxicity Kit (Cat# L3224, Molecular Probes, Eugene, Oregon), according

to manufacture’s instructions. The cells growing in a 6-well dish was washed twice with

PBS. 1ml PBS solution with 1 µM calcein AM and 4 µM EthD-1 was added to the cells

and incubated at room temperature for 20 minutes. The cells were then fixed in 4%

paraformaldehyde for 30 minutes to one hours, and imaged in a reverted fluorescent

microscope.

2.4.3. DNA fragmentation.

DNA fragmentation method in this work was adopted from (Ishida et al. 1992)

with modifications. 1X107 mouse BMMs, growing in RPMI media with 0.1% heat inactivated calf serum and no CSF-1, in a square dish were rinsed with PBS. The media with floating cells and the rinsing PBS were collected in a 50ml tube. 1ml of non- enzymatic cell dissociation buffer (Sigma) was added to the plate. 5 minutes later, 10ml of RPMI (with 5% heat inactivated calf serum) were added to the plate and the cells were

transferred to the 50 ml tube with floating cells. Cells were centrifuged and washed once with cold PBS. The cell pellet was lysed in 600 µl of a buffer (10 mM Tris-Cl, 10 mM 68

EDTA and 0.2% Triton X-100, pH 7.5) for 10 min on ice. The lysate was centrifuged for

10 min at 4oC in an Eppendorf tube. The supernatant (containing RNA and fragmented

DNA, but not intact chromatin) was extracted first with phenol (Tris saturated), phenol:

chloroform:isoamyl alcohol (25:24:1) and then chloroform:isoamyl alcohol (24:1). The

aqueous phase is made to 300 mM NaAc. Apoptotic DNA was precipitated with 2

volumes of ethanol. The pellet was washed once with 70% ethanol, air-dried and

dissolved in 20 µl of TE plus 0.6 mg/ml RNase A. RNA was eliminated by digesting with

RNase A at 37oC for one hour. The apoptotic DNA was mixed with SyBr Green

(Molecular Probes, final concentration 1X), and electrophoresed in 1.2% agarose gel. The gel was visualized under UV light in a gel imager (Bio-Rad).

2.4.4. Caspase-3 assay.

3X106 mouse BMMs, growing in RPMI media with 0.1% heat inactivated calf serum, with or without 50ng/ml CSF-1 in a square dish, were rinsed with PBS. The media with floating cells and the rinsing PBS were collected in a 50ml tube. 1ml of non- enzymatic cell dissociation buffer (Sigma) were added to the plate. 5 minutes later, 10ml of RPMI (with 5% heat inactivated calf serum) were added to the plate to collect the cells to the 50 ml tube with floating cells. Cells were collected by centrifugation and washed with KPM buffer (50 mM KCl, 50 mM PIPES, 10 mM EGTA, 1.92 mM MgCl2, pH 7.0,

1 mM DTT, 0.1 mM PMSF, 10 µg/ml of cytochalasin B and 2 µg/ml of protease inhibitors: chymostatin, pepstatin, leupeptin, antipain). Cells were snap-frozen in liquid nitrogen and and kept at –80oC. Cells were lysed by 5 cycles of freeze (in liquid

nitrogen)-thawing (in a water bath at ambient temperature). Extracts were then 69

centrifuged for 20 min at 14,000 x g in a microcentrifuge. 10ul of supernatants were used

in caspase-3 activity assay. The presence of active caspase-3 was determined by the

aminotrifluoromethylcoumarin assay (afc), as previously described (Fahy et al. 1999;

Doseff et al. 2003; Zeigler et al. 2003). Briefly, lysates were incubated with DEVD-afc to determine the presence of active caspase-3 in a cyto-buffer (10% glycerol, 50 mM Pipes, pH 7.0, 1 mM EDTA) containing 1 mM DTT and 20 µM tetrapeptide substrate (Enzyme

Systems Products, Livermore, CA). Release of free afc was determined using a Cytofluor

4000 fluorimeter (Perseptive Company, Framingham, MA. Filters: excitation; 400 nm, emission; 508 nm).

2.5. Cell line culture and transfections.

2.5.1. Raw264 cell transfection.

RAW264 cells were grown in RPMI containing heat inactivated (1 hour at 55oC)

3% new-born Calf Serum (Hyclone), Penn/Strep, L-Glutamine (2mM) in square dishes

(Lab Tek/Nunc #4021 square petri dishes (Fisher# 08-757-10K)) in a humidified

o incubator at 37 C and 7% CO2. A 10cc syringe with an 18 G needle was used to remove

the cells and disperse for passage. For passaging, cells were plated at 4X106 cells per dish with 12 ml media and passaged every day. The cell number should increase to 2-3 fold after 24 hours. If cell number increased to more than 3 fold, they were not useful for transfections. One day before transfection, cells were plated at 4X106 cells per dish and harvested within 24 hrs. Cells were collected by centrifugation (1000 rpm in a Sorvall

RT6000D benchtop) and resuspended in normal growth media at a concentration of 25 million cells/ml. 5 million cells (0.2 ml) were mixed in a sterile tube with 5 µg of plasmid 70

DNA purified from Qiagen columns. The DNA and cells were gently mixed and allowed

to stand at room temperature for at least 5 min. The DNA/cell mixture was transferred to

pre-chilled 4mm gap electroporation cuvettes and electroporated using a Bio-Rad Gene

Pulser equipped with a capacitance extender (0.26 kv, 980 uF, time constant was usually

around 40-60). Cuvettes were immediately placed on ice. Electroporated cells were

transferred to a 6-well tissue culture dish containing 3 ml of normal culture media as

quickly as possible. The cuvettes were washed with media from the wells 2-3 times to

transfer all material. About 50% of the cells were normally lysed or fused and the

mixture should be slightly viscous at this point. Pasteur pipettes were used to transfer the material and wash the cuvettes. Cells were grown overnight at standard conditions, and harvested 16-24 hrs following electroporation. In some cases, certain promoters were more active 36-48 hr following transfection. During harvesting, media was removed, cells were washed twice with PBS, and then were lysed on the dish by adding 0.1 ml of

1X cell culture lysis reagent (Promega, catalog # E153A). Cells were scraped, and the lysed mixture was transferred to a microfuge tube. The samples were used to measure luciferase activity immediately or stored in –20oC. Before measuring luciferase activity,

the cell lysis samples were centrifuged for 1 min at 10-12K rpm. 10µl of supernatant was

used in each assay.

2.5.2. Transient Transfections with calcium phosphate method.

Cells were plated at 4X105 per 60mm dish or 2 X105 per well of a 6-well dish 20-

24 hours before transfection. A total of 10µg of DNA was used per 60mm dish and 5µg

DNA was used per well of a 6-well dish. An expression vector for Renilla luciferase 71

(pRL-CMV, Promega, SW13 cells) was included as an internal control for transfection

efficiency (1ng /DNA precipitate). The DNA plasmids to be transfected were brought up

to a final volume of 375 µl or 187.5 µl of ddH2O to which 125µl or 62.6 µl of CaCl2 was

added. After mixing, the DNA/ CaCl2 solution was added drop by drop (normally 30-60

seconds to finish adding the solution) to an eppendorf tube containing 500µl of 2X HBS

(28nM NaCl, 50mM HEPES, 1.5mM NA2HPO4, pH 7.05-7.15). The 2X HBS was

bubbled gently with a Pasteur pipette while adding the DNA/CaCl2 solution. The

mixture was incubated at room temperature for 30 minutes to allow precipitates

containing DNA to form. The precipitates were mixed gently. 500µl or 250ul of the

precipitates was added drop-wise to the 60mm dish or 1 well of a 6-well dish,

respectively. The plates were rocked gently to distribute the precipitates evenly over the

cells. 16 hours after the transfected cells had been incubated at 37°C, the precipitates

were removed by washing twice with sterile Tris-buffered saline (1X TBS; 50mM Tris-

HCL pH 7.4, 150mM NaCl) and then fresh media was added. The cells were incubated

for an additional 24-48 hours at 37°C before harvesting to measure luciferase activity.

Cells were harvested by washing twice with PBS and then lysed in dish with 1X cell culture lysis reagent (Promega). 150µl of lysis buffer was used per 60mm dish and 100µl was used per well of a 6-well dish. Cells in lysis buffer were scraped from the dishes with a spatula and the cells were transferred to an eppendorf tube. The samples were used to measure luciferase activity immediately or stored in –20oC. Before measuring

luciferease activity, the cell lysis samples were centrifuged for 1 min at 10-12K rpm.

10µl of supernatant was mixed with 100µl of luciferase reagent (20mM Tricine; 0.1mM

EDTA; 33.3mM DTT; 2.67mM MgSO4; 1.07mM Magnesium carbonate; 0.27mM

Coenzyme A; 0.47mM luciferin, 0.53mM ATP, pH 7.8) injected by the Bio-Lumat luminometer. Relative luciferase activity is equal to (raw luciferase activity)/(raw activity of the internal control) X (the protein concentration of the extract). Fold-repression is the ratio of relative luciferase activity for the BRCA1-luciferase reporter alone (with empty expression vectors) to the activity in the presence of ets-2, Brg-1, or the combination of both ets-2 and Brg-1.

2.5.3. Generation of stable SW13 clones.

Human carcinoma SW13 cells were obtained from American Type Culture

Collection (Bethesda, MD) and cultured in DMEM plus 5% bovine calf serum. Stable

SW13 cells expressing luciferase under BRCA1 promoter was generated by the following: SW13 cells in a 100mm dish were co-transfect by 3µg of BRCA1-luciferase plasmid and 1µg pLoxC (neomycin-resistance vector) plasmid DNA using

LipofectAMINE Plus (Invitrogen) according to manufacture’s instructions. 48 hours after transfection, cells were selected with G418 (400ug/ml, Invitrogen) and media was changed every 3 days. 14 days after selection, the colony cells were pooled and cultured with 100-200µg/ml G418.

2.5.4. Transfection of stable SW13 clones.

The SW13 cells with stable integration of BRCA1-luciferase were transfected using LipofectAMINE Plus (Invitrogen) by 0.5ug puroBABE, a puromycin-resistant expression vector, and the plasmids (Ets-2, Brg1 and Brg1 mutant). 36 h after 73

transfection, cells were selected with 4 µg/ml puromycin and subsequently harvested 36 h after the initial selection. Luciferase activity was adjusted by cell lysate protein concentration.

2.5.5. Protein Assays.

The Bradford method was used to determine the protein concentration of cellular extracts. 200µl of 1X Bio-Rad reagent was added to samples (diluted in ddH2O) in a 96 well-microtiter plate. Absorbance at 600nm was determined using an ELISA plate reader

(model 2250, Bio-Rad). Sample readings were compared against a standard curve generated simultaneously using a known amount (0, 1, 2, 4, 6, 8, 10, and 12 mg) of BSA in order to determine the protein concentration (mg/ml) of each sample.

2.6. Primary cells isolation and culture.

All the mice used in the study were housed in Keck or Heart & Lung Institute animal facilities in the Ohio State University, according to NIH guidelines. All the mice were sacrificed according to animal resources standard operating procedures.

2.6.1. Preparation of L-cell conditional media as a source of CSF-1.

L-cell conditioning media was prepared as described (Stanley 1985) with modifications. L-cells (CCL-1, ATCC) were amplified in DMEM media with 10% fetal bovine serum (FBS). 5X106 cells were seeded in each 175 cm2 tissue culture flask. After

the cells reached confluency, the cells were washed with PBS twice and grew in 100 ml

serum-free DMEM. The cell morphology was monitored daily to avoid harvesting media 74

with massive dead cells. 2-3 weeks later, the conditioned media was harvested and

centrifuged. The supernatant was filtered through 0.2µm filter, aliquoted, and stored at –

80oC.

2.6.2. Bone marrow derived macrophages.

Bone marrows cells from femur and tibia bones from each mouse were cultured for 3 days in 2 square dishes, each containing 10-12 ml RPMI media plus 5% heat inactivated calf serum and 50ng/ml of recombinant CSF-1 or 10% L-cell media. Floating cells were aspirated off and cells continued to grow in RPMI media plus 5% heat inactivated calf serum and 50ng/ml of recombinant CSF-1 or 10% L-cell media for 2 more days. Cells were washed twice with PBS. 1ml of non-enzymatic cell dissociation buffer (Sigma) was added to the cells and the cells were incubated for 5 min in a 37oC incubator. 10 ml RPMI with 5% heat inactivated calf serum was added to the dish.

BMMs were collected, counted, centrifuged and resuspended in appropriate solutions.

2.6.3. Peritoneal macrophages.

The collection of thioglycolate elicited peritoneal macrophages was described previously (Conrad 1981). Briefly, 2.9% Brewer’s thioglycolate medium (Sigma) was autoclaved and stored for 3 months or longer till the color of the solution was all brown. 1 ml of thioglycolate medium was injected aseptically via the interperitoneal route, using a

1 ml insulin syringe. 4-5 days later, the mouse was sacrificed by CO2. The animal fur was

dampened with 70% ethanol. Using scissors, a small incision was made in the animal

skin at the abdomen. The skin was pealed around the body to expose abdomen and 75

thoracic cage. 10 ml cold PBS plus 10 units/ml heparin was injected into the peritoneum

with a 10 ml syringe and a 27 G needle. For smaller size animals (e.g. mev mice), less

volume of solution (5-7 ml) was injected. The needle was removed from the body. The

abdomen was massaged and rotated for about 1 minute. A syringe with an 18 G needle

was used to aspirate the lavage fluid. The lavage fluid was centrifuged, and the cells were

plated in 6-well dishes (non-tissue culture treated). 30 minutes later, the unattached cells

were washed off and the macrophages were harvested for real time PCR analysis.

2.6.4. Lung alveolar macrophages.

The collection of murine alveolar macrophages was carried out with a

protocol described before (McGee and Myrvik 1981) with modifications. Mice

were brought to laboratory in animal housing cages and placed in a fume hood.

The mouse was killed by CO2, following animal resources standard operating procedures, immediately prior to lavage. The animal fur was dampened with 70% ethanol. Using scissors, a small incision was made in the animal skin at the abdomen. The skin was pealed upwards to expose thoracic cage and neck. The salivary glands were removed. Other tissues were also dissected from the neck to expose trachea. A small incision was made in the trachea by a 21G needle. A 21G needle was put halfway into a 4-5 cm Intramedic polyethylene tubing (Clay

Adams, #7421, PE90, I.D. 0.86mm, O.D. 1.27mm). The lavage tube was passed into trachea. Depend on the size of the mice, 0.4ml-1ml of sterile cold PBS was load into a 1cc syringe. The syringe was placed in end of the 21-gauge needle

connected to the trachea by the tubing, and PBS solution was injected into the lung. It should be visible that the lung was inflated. The lavage was aspirated by pulling barrel of syringe. The syringe was removed from the needle/tube, and the recovered lavage fluid was injected into a 15ml falcon tube on ice. The lavage procedure was repeated 5-10 times per animal. The pooled lavage fluid was centifuged (1000 rpm in a Sorvall RT6000D benchtop centrifuge) and lung alveolar cells were resuspended in 1ml RPMI media with 0.1% heat inactivated calf serum, and transferred to a well of a 24-well dish (non-tissue culture treated).

6 hours later, floating cells were washed off and attached macrophages were used to extract RNA.

2.6.5. Mouse embryonic fibroblasts (MEFs).

Pregnant mouse (E12.5-E13.5) was sacrificed, and dipped in a beaker with 70% ethanol. Using scissors and forceps, an incision was made on abdominal skin. The skin was peeled off and the peritoneum was washed with 70% ethanol. The abdominal cavity was opened and the uterus was taken to a 50 ml conical tube containing sterile PBS. The uterus was transferred to another tube with sterile PBS in a tissue culture hood. The uterus was transferred to a 100 mm dish with about 10 ml sterile PBS. The embryos were removed from the uterus and placed into individual 60 mm dishes containing PBS.

Placenta and yolk sac was removed from embryo and yolk sac, and the yolk sac was used for genotyping. “Red” tissues (liver, spleen, heart etc.) were removed, and the rest of the embryo was transferred into one well of a 6-well dish containing 5 ml trypsin, minced with scissors and scalpels, and then incubated in a incubator for 15 minutes. The

digested embryo was pipetted up and down several times with a 5 ml pipette and

transferred to a tube with 10 ml DMEM medium with 10% FBS. After centrifugation,

cells were plated in DMEM medium with 10% FBS in a 60 mm dish (for E12.5) or a 100

mm dish (for E13.5). The next day, cells were switched to fresh medium. When the cells

were almost confluent, they were passaged. When those passage 1 cells were almost

confluent, they were passaged again. The next day, RNA was extracted from those

passage 2 cells, and subjected to real time PCR analysis.

2.6.6. Mammary stromal and epithelial cells.

The mouse was sacrificed and dipped in 70% ethanol prior to dissection. The

mouse was fastened with pins in the paws on a dissection board. A gentle cut along the

midline and along the top of the legs was made. The mammary glands were dissected

using a scalpel and a cotton swab, minced thoroughly with scissors or a scalpel, and

placed in collagenase solution (2.43 mg/ml DMEM/F12, 7.48 mg/ml 199 medium, 2

mg/ml NaHCO3, 0.15% collagenase, 160 units/ml hyaluronidase, 1X Pen/Str, 1µg/ml

hydrocortisone, 10µg/ml insulin, 10% heat inactivated FBS) for one hour in an incubator.

The digested glands were centrifuged and resuspended in 10 ml of DMEM/F-12 (Atlanta

Biologicals) high Calcium medium (10% FBS, 1.05 mM calcium, 10ug/ml insulin, 0.02

µg/ml EGF, 0.5 µg/ml hydrocortisone, 0.1 µg/ml cholertoxin, Pen/Str, 1.2 g Na

Bicarbonate). The organs (epithelial cells) were allowed to come to the bottom of the tube by gravity 12-15 minutes for 3-5 times. The supernatant was enriched for stromal cells. The cells (pelleted cells and supernatant separately) were centrifuged and resuspended in high calcium medium with 10% FBS and grow in the same media in a 60 78

mm dish (For stromal cells) or a 100 mm dish (for epithelial cells). When the epithelial cells were at 50% confluency, low calcium medium (same as high calcium medium except CaCl2 concentration was 0.04 mM) was used. 10% conditioning media from epithelial cells might help stromal cells to grow. The cells were passaged and harvested for Western blot analysis.

2.7. Mouse lines and genotyping.

2.7.1. Mouse lines.

The mice with ets-2T72A mutation were kindly provided by Dr. Oshima (The Burnham

Institute, La Jolla, California). The ets-1 knockout mice were provided by Dr.

Muthusamy (Children’s hospital, Columbus, OH). EIIA-cre mice were kindly provided by Dr. Weinstein (The Ohio State University). Mox-2-cre mice were provided by Dr.

Leone (The Ohio State University). Motheaten-viable mice, C3(1)SV40-T antigen transgenic mice, PyMT mice and FVB/N wt control mice were purchased from Jax lab.

All the mice used in the study were housed in Keck or Heart & Lung Institute animal facilities in the Ohio State University, according to NIH guidelines.

2.7.2. Tail DNA prep for genotyping.

A 0.5-1cm long mice tail was digest each tail in 200ul of tail lysis buffer (50 mM

KCl, 10mM Tris-HCl (pH8.3), 0.1 mg/ml gelatin, 0.45% NP40, 0.45% Tween20, 1mg/ml proteinase K), digested at 55oC overnight or until tail degrades completely. Vortex was required if a short incubation time is needed. After being boiled for 12-15 minutes to

inactivate Proteinase K, and cooled down on ice, 2ul of each tail DNA sample was used in a 25-30µl PCR reaction.

2.7.3. Genotyping primers and PCR conditions.

The PCR programs for genotyping were: 1cycle of 95 oC , 2 minutes; 35 cycles of: 94 oC, 45 seconds, appropriate annealing temperature as listed in Table 2.2, 45 seconds, 72 oC, 1 minute; 1 cycle of 72 oC, 10 minutes. The PCR products were run in a

1.5-2% agarose gel.

annealing temperature in PCR conditions.

81 Table 2.2. Genotyping primers sequence and

2.8. Gene targeting.

2.8.1. BAC library Screening.

Plasmid DNA corresponding to mouse Ets-2 exon3-5 were purified, labeled with

P32 (See Southern blot section), and used to screen the mouse RPCI-22 129 BAC library.

The filter contains 129 BAC library was prehybridized in prehybridization solution (0.5

M Phosphate, 7% SDS) at 65°C for 1-3 hours. The prehybridization solution was removed and the filter was hybridized with the denatured probe in 20ml of prehybridization solution at 65°C overnight. The filter went through low stringency wash

(20ml of 0.2M phosphate, 0.1% SDS, 10min at 65°C). The sealed wet filter in plastic bag was exposed to a film in cassette, overnight at room temperature, to get a nice grid. The filter went through high stringency wash (20ml of 0.02M phosphate, 0.1%SDS, 30min at

65°C). The sealed wet filter in plastic bag was exposed in film cassette with 2 screens overnight at –70°C. The ID number of positive clones was identified by the hybridization signal pattern and position.

2.8.2. Conditional ets-2 targeting construct cloning.

The cloning strategy was decided through mapping the restriction enzyme site in

Ets-2 gene by Southern blots of genomic DNA and BAC DNA, and sequencing. The vectors were the Triple lox system, a gift from Dr. Gustavo Leone, Ohio State

University). The cloning process was illustrated in Fig 6.3. The 4.3 kb DNA fragment between EcoR I site in intron 2, and Bam HI sit in intron 5, was flanked with loxP site. A

1 kb fragment immediate upstream of the EcoR I site was generated by PCR and used as the short homologous recombination arm. The 8.5 kb fragment between BamH I site in 82

intron 5 and EcoR I site at the 3' end of the gene was used as the long recombination arm.

The final construct is illustrated in Figure 6.5. The functionality of loxP site was

confirmed by in vitro treatment of purified Cre enzyme (see below) followed by PCR

using primers shown in Fig 6.5.. The 21kb targeting construct was linearized by Not I

before transfection.

2.8.3. DNA recombination by Cre recombinase.

0.5-1 µg of Not I linearized targeting vector was put in a tube with 3µl of Cre

recombinase buffer, appropriate amount of water to make the final volume to 30µl, and 2 units of Cre recombinase (Novagen), and incubated at 37oC for one hour. The enzyme

was heat inactivated at 70oC for 5 minutes. 10ng of Cre treated DNA was used in PCR

reactions to confirm the correct localization and integrity of loxP sites.

2.8.4. ES cell transfection and selection.

ES cells (from Dr. Weinstein, The Ohio State University) were growing with mitomysin D treated mouse embryonic fibroblast (MEF) feeder cells in ES medium

(DMEM (Invitrogen) plus 15% heat inactivated FBS, Penicillin/Strepmycin, non- essential amino-acid, LIF, beta-mercaptol-ethanol). On the day of transfection, 2X107 ES

cells were lifted by trypsin, and resuspended in 0.5ml ES media. 50ug of Not I linearized

targeting construct was added to the ES cells. Transfection was achieved by

electroporation at 25uF, 500 volt in 0.4mm cuvettes. After electroporation, the cells were

transferred to 4 100mm dishes with feeder cells. 2 days later, G418 was added to select

transfected cells. 7-10 days later, colonies were picked and screened by PCR. Primers 83

Ets2I2P5 and PGKrev2 (selecting 5' homologous recombination) were used in the initial screening. Primers loxNB and Ets-2E6 (selecting 3' homologous recombination) were used for second round of screening (Figure 6.5).

9X106 ES-LE 18 clone cells were electroporated with 9 µg PGK-cre (Abuin and

Bradley, 1996), and were plated in 2 100mm plates. 2 days later, the cells were plated at the density of 106, 105 and 104 cells per plate. 2 days later, 2X10-6 M Gancyclovior was used to select clones that had neo and tk genes excised. 7-10 day later, clones were picked and screened for excision of neo and tk by PCR with primers Ets2I2P10/I2P9 and

Ets2I5P2/I5P3. Positive ES cells were injected into blastocysts in Children’s Hospital,

Columbus, Ohio, to generate chimera mice with correct allele.

. g in ES clone screenin

Table 2.3. Primers used

2.8.5. Conditional Pten targeting.

Figure 2.1. Generation of mice with PTEN conditional knockout allele. Two loxP sites were introduced to two Hpa I sites (indicated as H) in intron 3 and intron 5, respectively. This caused PTEN exon 4 and exon 5 flanked by two loxP sites. Exon 5 of PTEN encode the lipid phosphatase active site. Tissue specific expression of cre removes PTEN exon 4 and exon 5 and removes the PTEN lipid phosphatase activity (Cristofano et al, 1998, Suzuki et al, 1998).

2.9. Histology.

2.9.1. Cryosection.

Embryos or other tissues were fixed in 3.7% paraformaldehyde at 4oC for 10

minutes to 2 hours. Tissues were placed in cryomolds (Tissue-Tek), filled with OCT

Embedding medium (Tissue-Tek) and placed on dry ice. After frozen, the samples were

stored at -80 oC. The tissues were sectioned with a cryostat at 4-8 microns, and stored at -

80 oC for immunohistochemistry or enzyme assays.

2.9.2. H & E staining of paraffin-embedded tissue.

The slides were incubated at 60 °C for 40 min, and hydrated through Xylene,

100%, 95%, 70% ethanol and deionized water, and dipped in Hematoxylin for 30 -45 seconds at RT. Slides were rinsed with tap water, and went through though 70% and 95% ethanol, then immersed in Eosin for 30 seconds at RT. The slides were dehydrated through 95%, 100%, 100% ethanol and Xylene, and mounted using Permount, and covered with coverslips.

2.9.3. Wholemount staining of mammary gland.

Murine mammary glands were dissected and spread on glass slides, and fixed in

Carnoy's fixative (Carnoy's solution I: 1 part glacial acetic acid, 3 parts 95 % or absolute ethanol or Carnoy's Fix(II): 6 parts 100 % ethanol, 3 parts chloroform, 1 part glacial acetic acid) for 2 to 4 hours at RT. The slides were washed in 70 % ethanol for 15 min, and gradually changed to distilled water. The slides were rinsed in distilled water for 5 min, and stained in carmine alum (2mg carmine, 5mg aluminum potassium sulfate) overnight. The slides were then washed and rehydrated through 70 %, 95%, 100% ethanol (15 min each step), cleared in xylene and mounted with Permount. After photographic documentation the tissue could be embedded in paraffin for sectioning and conventional histological staining.

2.9.4. Immunohistochemistry with whole mouse embryos.

This protocol was adopted from Dr. Sato’s lab protocol (The university of Texas

Southwestern medical center, Dallas, Texas) with modifications. Embryos were collect in ice cold PBS and extraembryonic membranes were dissected away. For embryos older than E9.0, a sharp incision was introduced along the dorsal midline of the hindbrain using a pulled injection needle or scissors. The embryos were rinsed in ice cold PBS for 10 min, and fix in 4% paraformaldehyde/PBS overnight at 4°C. The embryos were rinsed in

PBS at room temperature (5 min, 3 times). The embryos were dehydrated through

25%MeOH, 50%MeOH, 75%MeOH, 100%MeOH, 100%MeOH (15 min each step) at room temperature. The embryos were incubated in 5%H2O2 in MeOH for 4-5 hours at room temperature to bleach the embryos and block endogenous peroxidase. The embryos were rinsed with 100%MeOH twice. At this point, the embryos could be processed immediately or stored in MeOH at -20°C for at least a few weeks. The embryos were rehydrated through 75%MeOH, 50%MeOH, 25%MeOH, PBS, PBS (15 min each step) at room temperature. The embryos were incubated in PBSMT (3% instant skim milk powder, 0.1% Triton X-100, in PBS, made fresh each time) (1 hour, 2 times) at room temperature. The embryos were incubated in diluted primary antibody (10 µg/ml final concentration, 50X for PECAM antibody (BD Pharmigen, Clone MEC 13.3)) in PBSMT at 4°C overnight. Only the minimum volume (0.2 ml) was used. The embryos were washed in 1ml of PBSMT (1 hour X 5) at 4°C, then incubate with the diluted secondary antibody in PBSMT at 4°C overnight. For HRP coupled goat anti-rat IgG (mouse absorbed, from K&P), 1/100 dilution was used. The embryos were washed in 1ml of

PBSMT (1 hour X 5) at 4°C, and washed in PBT (0.2%BSA, 0.1% Triton X-100 in PBS. 88

Made fresh each time) at room temperature for 20 minutes. The developing solution was

made as follows: 10mg tablet of DAB (Sigma) was dissolved in 10ml of PBT by gently

shaking in a 15ml centrifuge tube on an orbital shaker by laying down the tube. After

DAB was dissolved, 0.17g NiCl2 was added and dissolved by gently shaking on the orbital shaker. 3 parts of this concentrated solution was mixed with 7 parts of PBT (i.e. final developing solution concentration is 0.3mg/ml DAB, 0.5% NiCl2 in PBT). The embryos were incubated in this developing solution for 20 min at room temperature.

H2O2 was added to 0.03% final concentration (i.e. 1µL of 30% H2O2 for 1ml of developing solution) and immediately mixed gently. It took about 5-10 min for the color to develop. The embryos were rinsed in PBT (5 min, X 2) and then PBS (5 min, X 2) at room temperature. The embryos were fixed in 2%paraformaldehyde/ 0.1%glutaraldehyde

/ PBS at 4°C overnight. The embryos were rinse in PBS (5min. X 3) at RT. The embryos were equilibrated in 50% glycerol at room temperature for 1 hour and followed by 70% glycerol for one hour. The pictures were taken in 70% glycerol.

2.10. Bioinformatics.

2.10.1. Databases.

Genomic, mRNA and protein sequences were obtained from Celera

(http://www.celeradiscoverysystem.com/index.cfm), NCBI

(http://www.ncbi.nlm.nih.gov/) and EMI (http://www.ebi.ac.uk/index.html).

Other database used were as follows:

Mouse Genome Informatics (MGI): http://www.informatics.jax.org/

Cre Transgenic Database: http://www.mshri.on.ca/nagy/cre.htm 89

Transgenic/Targeted Mutation Database: http://tbase.jax.org/

Tet mouse database: http://www.zmg.uni-mainz.de/tetmouse/

Mouse Atlas and Gene Expression Database: http://genex.hgu.mrc.ac.uk/

Mouse Tumor Biology Database: http://tumor.informatics.jax.org/FMPro?-

db=TumorInstance&-format=mtdp.html&-view

The Tumor Gene Family Databases: http://www.tumor-gene.org/tgdf.html

Atlas of Genetics and Cytogenetics in Oncology and Haematology: http://www.infobiogen.fr/services/chromcancer/

Genome databases for farm and other animals: http://www.thearkdb.org/

The Biomolecular Interaction Network Database (BIND): http://binddb.org/

Nucleic Acid Research databases: http://www3.oup.co.uk/nar/database/a/

Protein families, domains and functional sites: http://www.ebi.ac.uk/interpro/

Real-time PCR primer and probe sequences:

http://medgen31.ugent.be/primerdatabase/index.php

Stanford Microarray Database: http://genome-www5.stanford.edu//

2.10.2. On-line bioinformatics tools.

Similarity search were performed by using BLAST

(http://www.ncbi.nlm.nih.gov/BLAST/ or http://us.expasy.org/tools/blast/ ).

Alignment of a pair of DNA or protein sequences were performed by BLAST 2

(http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html ).

Alignment of multiple DNA or protein sequences were performed by ClustalW

(http://www.ebi.ac.uk/clustalw/index.html#), BCM Search Launcher 90

(http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) or Multiple Sequence

Alignment (http://genome.cs.mtu.edu/map/map.html ).

Assemble of multiple sequences were performed by Cap (http://bio.ifom-

firc.it/ASSEMBLY/assemble.html).

Alignment of mRNA sequence and genomic sequence was performed by Spidey

(http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey/).

Restriction site analysis of DNA was performed by Webcutter

(http://www.firstmarket.com/cutter/cut2.html).

Translation of DNA or mRNA sequence into protein sequence was performed by the Translate Tool (http://ca.expasy.org/tools/dna.html).

PCR primer design was performed by Primer 3 (http://www-

genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi), or ePrimer3

(http://bioinfo.pbi.nrc.ca:8090/cgi-bin/emboss.pl?_action=input&_app=eprimer3).

3-D protein structure prediction was performed by CE (http://cl.sdsc.edu/ce.html),

EDPSSM (http://www.sbg.bio.ic.ac.uk/~3dpssm/), or Swiss Model

(http://www.expasy.org/swissmod/SWISS-MODEL.html).

CHAPTER 3

ETS-2 AND INFLAMMATION

3.1 Introduction

Ets factors activity is regulated by extracellular stimuli through various signal transduction pathways (Chapter 1). For example, Ets-2 is phosphorylated upon macrophage colony-stimulating factor (CSF-1) binding to c-fms and the subsequent activation of ras signaling, and correlates with the expression of many CSF-1 target genes, such as Bclx and uPA. However, it was not clear to what extent that Ets-2 contributes to inflammation, and what genes were regulated by Ets-2 in an inflammation process. This Chapter used a genetic approach to address this question and examined the roles of Ets-2 in an inflammation animal model (motheaten viable, i.e. mev mice) in vivo.

3.1.1. Macrophages and Ets factors.

The macrophage is the major differentiated cell of the mononuclear phagocyte system, which comprises bone marrow monoblasts and promonocytes, peripheral blood monocytes, and tissue macrophages (Murphy 1996; Gordon 1999). Macrophages are widely distributed throughout the body, and are important in many physiological and pathological processes, such as hematopoiesis, homeostasis, destruction of microorganisms, disposal of damaged or senescent cells, rheumatoid arthritis, wound 92

healing, tissue repair and remodeling, angiogenesis, tumorgenesis and autoimmunity

(Gordon 1999; Ross and Auger 2002). For example, alveolar macrophages are involved in local defense against a variety of pathogenic and particulate entrance via the airway and play an early role in inflammation and control of infection (Murphy 1996; Gordon

1999; Ross and Auger 2002).

Macrophages have important functions in immune responses: 1) Phagocytosis and destruction of microorganisms; 2) Antigen processing and presentation; 3) Secretion of enzymes, such as lysozyme, which mediates digestion of the bacteria cell wall; 4)

Secretion of extracellular matrix proteases (MMP1, MMP3, MMP9, uPA); 5) Secretion of cytokines, such as TNFα, Il-1α, Il-1β, Il-6 and Il-12; 6) Secretion of chemokines, such as MCP1, MIP1 and MIP1, RANTES and eotaxin; 7) Secretion of other molecules, such as enzyme inhibitors, complement components, reactive oxygen intermediates and coagulation factors (Gordon 1999; Hamilton 2002; Ross and Auger 2002).

TNFα, Interleukin-1α (Il-1α) and Il-1β have multiple local and systemic host defense functions. TNFα, one of the earliest cytokines produced by activated macrophage-monocytes, occupies a pivotal role in the pathogenesis of inflammation, septic shock and tissue injury. Biochemically characterized in 1984, TNFα has since been found to be a pleiotropic agent produced mostly by activated macrophages and monocytes. On stimulation by a variety of stimuli, the transcription and translation of the

TNFα gene are rapidly upregulated, enabling the release of large quantities of soluble

TNFα. The variability of TNFα production between individuals appears to be critical in determining susceptibility to diseases such as multiple sclerosis, rheumatoid arthritis, and

infections with parasites, bacteria, or viruses (Wang and Tracey, 1999). Interleukin-6 (Il-

6) is a multifunctional cytokine involved in the induction of B-cell terminal differentiation, induction of acute phase protein synthesis in hepatocytes, stimulation of hematopoietic progenitors, and activation of T cells and thymocytes (Wang and Tracey,

1999). Interleukin-12 (Il-12) acts as a proinflammatory cytokines, activating natural killer cells. Through its ability to induce interferon-γ production, Il12 enhances the phagocytic and bactericidal activity of macrophages and monocytes and their ability to release proinflammatory cytokines, including Il-12 itself (Trinchieri, 1999).

During early stages of infection or tissue injury, leukocytes emigrate outward into tissues and move toward the agent stimulating the inflammation response. This process of directional locomotion, called chemotaxis, is partially mediated by members of the chemokine superfamily members. MCP-1 was the first CC chemokine to be characterized. It attracts monocytes, T lymphocytes and basophils. MIP1α and MIP1β are chemoattractant and hematopoietic regulators. RANTES is a powerful chemoattractant for basophils and eosinophils. It also recruits monocytes and helper T cells. Eotaxin attracts eosinophils and basophils (Ross and Auger 2002).

It has been reported that Ets factors regulate the expression of many of those genes. Ets-1 and Ets-2 bind to TNFα promoter in vitro and in vivo as shown by DNase I foot printing and chromatin immunoprecipitation (ChIP). Mutation of –84 EBS impairs

LPS and mycobacteria induced TNFα expression (Tsai et al. 2000; Barthel et al. 2003).

There are two conserved EBSs in Il-1α promoter. Mutation of the two sites greatly decreased Il-1α expression (Kawaguchi et al. 2003). Similarly, there are Ets binding sites

located in the promoters of Il-1β (Kominato et al. 1995), Il-12 p40 (Ma et al. 1997; Gri et al. 1998), MIP1α (Grove and Plumb 1993), integrin αM (Bottinger et al. 1994) and integrin β2 (Hickstein et al. 1992), and those sites are required Ets proteins binding and gene expression. The Ets regulation of extracellular matrix proteases, such as MMP1,

MMP3, MMP9 and uPA, are well documented, as discussed in several review papers

(Crawford and Matrisian 1996; Westermarck and Kahari 1999b; Trojanowska 2000a;

Cox and O'Byrne 2001; Singh et al. 2002).

3.1.2. CSF-1 signaling regulates Ets-2 phosphorylation.

CSF-1 and its cognate receptor tyrosine kinase c-fms control the proliferation, differentiation, and survival of cells of the mononuclear phagocyte cell lineage by activating multiple pathways (Hume et al., 1997, Sweet and Hume, 2003). The importance of CSF-1 signaling was demonstrated by a classic mouse mutation, osteopetrotic (op). The op mutation results from an in-frame stop codon in the csf-1 gene that precludes translation of a functional growth factor (Yoshida et al. 1990). The op/op mice have drastically reduced numbers of circulating monocytes and have decreased numbers of some classes of tissue macrophages and of osteoclasts and thus exhibit the op phenotype (Wiktor-Jedrzejczak et al. 1990; Yoshida et al. 1990). The c-fms proto- oncogene encodes a tyrosine kinase receptor for csf-1 and is usually expressed in monocytic phagocytes and in placental trophoblasts. The phenotype of the fms knockout mice resembles the phenotype of op mice, including the osteopetrotic, hematopoietic, tissue macrophage, and reproductive phenotypes (Dai et al. 2002). C-fms mediates its pleiotropic effects through the coupling of its ligand-activated tyrosine kinase to multiple 95

intracellular effector proteins, the combined actions of which determine the magnitude

and specificity of the biological response. Upon CSF-1 binding, c-fms dimerizes and

autophosphorylates, and a number of cytosolic and membrane proteins become transiently phosphorylated on tyrosine residue. Those proteins include the 85-kDa subunit of phosphatidylinositol-3 kinase (PI3K), Src family kinases, Shc, SHIP and SHP-

1 (Pierce et al. 1990; Insogna et al. 1997; Koths 1997; Rohrschneider et al. 1997; Stanley et al. 1997; Fowles et al. 1998b; Kelley et al. 1999; Liu et al. 2001b). As with other tyrosine kinase receptors, ligand binding leads to subsequent activation of signal transduction pathways, such as Ras, PI3K, Src, Fyn, Yes and other signaling pathways

(Hamilton 1997; Stanley et al. 1997; Aziz et al. 1999). Ras activation is required for the proliferative response to CSF-1. v-raf could induce CSF-1 independent macrophage proliferation (Aziz et al., 1999). One effect of these signaling events in macrophages is the stable, persistent expression of specific genes, such as the urokinase plasminogen activator (uPA) gene, the macrophage scavenger receptor A (MSR) gene, and the Bclx gene (Smith et al., 2000). Ets-2 regulates these three CSF-1 target genes.

The Ras pathway is activated upon CSF-1 binding to c-fms (Aziz et al. 1999). As discussed in Chapter 1, Ets-2 is activated by ras-dependent phosphorylation of threonine residue 72, and CSF-1-c-fms signaling leads to persistent phosphorylation of this site

(McCarthy et al. 1997; Fowles et al. 1998a). Erk1/2 is the major renaturable kinase that is able to phosphorylate Ets-2 in fibroblast cells. However, there appear to be cell type specific regulation of Ets2 phosphorylation. In macrophages, the ras induced phosphorylation of Ets-2 is only partially inhibited by PD98059, even though Erk activity

is not detected after drug application. In gel kinase assay using macrophage extracts 96

suggest that there are additional kinase in macrophages that can phosphorylate threonine

72 of Ets-2 (McCarthy et al. 1997; Fowles et al. 1998a).

3.1.3. SHP1 and CSF-1 signaling.

Interestingly, it has been reported that CSF-1 does not activate MEK-1 and Erks

in primary macrophages obtained from mice for the motheaten-viable (mev) mice

(Krautwald et al. 1996; Smith et al. 2000), but Ets-2 is still phosphorylated in those cells

(Smith et al. 2000).

Mev and motheaten (me) are resulted from recessive mutations in hematopoietic

cell phosphatase (hcph). Those mutations cause one of the most deleterious abnormalities

in hematopoiesis and dysregulation of the immune system (Shultz and Sidman, 1987). To

be concise, “me mice” (referring to me/me mice) and “mev mice” (referring to mev/mev mice with wild-type ets-2, unless indicated) are used in this work. The me mutation was first reported in C57BL/6J mouse strain (Green and Shultz, 1975). Later, the mev mutation was found in the same strain. Me mice can be recognized by the second postnatal day by the patchy absence or thinness of hair that gave the mice a motheaten appearance. Mev mice can be recognized at 4-5 days of age. In both me and mev mice, neutrophils and macrophages accumulate in the dermis and penetrate as far as the panniculus pigment, giving the mice their characteristic “motheaten” appearance. The depletion of the thymus cortical starts at about 4 weeks of age. Thymus involution progresses in mev mice until 16 weeks of age when nothing grossly recognizable as a thymus can be found. Splenomegaly in me and mev mice results from increased

erythropoiesis and myelopoiesis and is accompanied by a depletion of lymphoid cells 97

from the periarteriolar regions. The bone marrow shows decreased erythropoiesis and increased myelopoiesis. Me and mev mice are sterile and have short life span (3 and 9

weeks, respectively) (Green and Shultz 1975; Shultz et al. 1984). The me and mev mice

suffer a functional severe combined immunodeficiency of both B and T cells (Sidman et

al. 1978a; 1978b), and a virtual absence of natural killer (NK)-cell activity (Clark et al.

1981). Although the motheaten mutations greatly affect T and B cell signaling (Zhang et

al, 2000), mice absent of B and T cells still develop the motheaten phenotypes, indicating

that B and T cells are not required for mev phenotype (Scribner et al, 1987, Yu et al,

1996).

A major immunopathological abnormality in me and mev mice is the overgrowth

of macrophages and granulocytes in lungs, skin and extremities. The accumulation of

macrophages and neutrophils in lungs results in a fatal pneumonitis (Green and Shultz

1975; Shultz et al. 1984), while inflammation in the extremities is associated with

arthritis in the joints (Kovarik et al, 1994). Spleen macrophages from me mice

exponentially grow in culture in the absence of exogenous growth factors, while

macrophages from control mice could not grow at the same condition (McCoy et al.

1982). This unusual proliferative capacity probably results from the spontaneous

production of colony-stimulating activity of these cells (McCoy et al. 1984). Mev mice

show classic symptoms of osteoporosis due to an increased number and activity of

osteoclasts in the bone marrow (Aoki et al. 1999; Umeda et al. 1999).

Hcph gene encodes Src-homology 2-domain phosphatase-1 (SHP1) (Plutzky et al.

1992; Yi et al. 1992). The me mutation is caused by the deletion of a cytidine residue in

first SH2 domain, generating a novel splice donor site and frame shift, resulting in a null 98

mutation. The mev mutation is a thymidine-to-adenine transversion that destroys a splice

donor site. The activation of cryptic splice sites produces an in-frame insertion or deletion

in the phosphatase catalytic domain. Abnormal SHP-1 protein in mev hematopoietic cells

retains about 20% of the wild-type activity (Shultz et al. 1993).

SHP-1 apparently plays a central role in cell signaling events that regulate macrophage-dependent inflammatory responses. SHP-1 forms complexes with several cell surface receptors: c-kit (Kozlowski et al. 1998), the Il-3 receptor beta subunit, erythropoietin receptor, interferon-α receptor complex, CD22, BIT and PIR-B and p62DOK (Krautwald et al. 1996; Timms et al. 1998; Berg et al. 1999). SHP-1 may be a

negative regulator of CSF-1 signaling (Chen et al. 1996). SHP-1 is phosphorylated on

tyrosine following CSF-1 stimulation of macrophages, but does not directly bind to

ligand-activated c-fms (Yi and Ihle 1993). CSF-1 treatment of primary macrophages

obtained from me mice is reported to result in c-fms hyperphosphorylation, increased phosphorylation of signaling molecules known to be downstream of c-fms, and an increased rate of macrophage proliferation (Chen et al. 1996). However, another group reported that CSF-1 mitogenic signaling is unaffected in macrophages obtained from me or mev mice, but that granulocyte-macrophage (GM)-CSF mitogenic signaling is hyper- activated in such macrophages (Jiao et al. 1997).

3.1.4. Phosphorylation of Ets-2 by PI3K/Akt pathway in macrophages.

As mentioned earlier, CSF-1 does not activate MEK-1 and Erks in primary mev macrophages (Krautwald et al. 1996), implying a positive role for SHP-1 in CSF-1 activation of MEK-1 and Erks . The CSF-1 and ets-2 target genes coding for Bclx, 99

urokinase plasminogen activator, and scavenger receptor are also expressed at high levels independent of CSF-1 in mev cells. PI3K activity is several fold higher in mev macrophages compared to wild type macrophages (Roach et al. 1998; Smith et al. 2000).

A major effector of PI3K pathway, Akt (protein kinase B), is constitutively active in mev macrophages, and an Akt immunoprecipitate catalyzes phosphorylation of Ets-2 at threonine 72, as demonstrated by the phospho-specific Ets-2 antibody. The p54 isoform of c-jun N-terminal kinase-stress-activated kinase (JNK- SAPK) coimmunoprecipitates with Akt from mev macrophages. Treatment of mev cells with the specific PI3K inhibitor

LY294002 decreases cell survival, Akt and JNK kinase activities, ets-2 phosphorylation, and Bclx mRNA expression (Smith et al. 2000). Therefore, Ets-2 is a target for PI3K-

Akt-JNK action, and the JNK p54 isoform is likely an Ets-2 kinase in macrophages.

3.1.5. Ets-2 and macrophage survival and motheaten pathology.

Constitutive ets-2 activity may contribute to the pathology of mev mice by increasing expression of genes like the Bclx gene that promote macrophage survival.

There are several lines of evidence support this hypothesis. Expression of a dominant- negative ets-2 protein in macrophages in transgenic mice results in accelerated apoptosis following CSF-1 deprivation (Jin et al. 1995). In the macrophage cell line BAC1.2F5, overexpression of ets-2 promotes survival of cells in the absence of CSF-1 (Sevilla et al.

1999). Phosphorylation of ets-2 correlates with the expression of antiapoptotic Bclx gene.

In transient transfection assays, the promoter for the mouse Bclx gene is superactivated over 90-fold by the combination of ets-2 and a constitutive active form of Akt.

100

Phosphorylation of ets-2 and activation of target gene expression correlate with increased

mev macrophage survival (Smith et al. 2000).

The above results indicate that constitutive ets-2 activity may contribute to the

pathology of mev mice by regulating expression of genes that promote cell survival in

macrophages. Lowering the dosage of ets-2 or blocking Ets-2 phosphorylation may lead

to decreased macrophage survival and rescued motheaten phenotype. We addressed this

hypothesis through genetics approaches by introducing the ets-2 knockout allele or the

T72A allele into mev mice. Mev mice with half dosage of ets-2, or homozygous ets-2T72A

mutation had increased life span (30% or 100% in ets-2+/-, mev/mev or ets-2T72A/T72A, mev/mev mice, respectively) and body weight, and reduced inflammation in peripheral tissues (such as skin and feet) and lungs compared to mev mice with wt ets-2. The

relieved inflammation was not only due to increased macrophage apoptosis, but also

because of reduced expression of inflammatory genes, such as inflammatory cytokines

and chemokines (such as TNFα, Il-1α, Il-12, MCP1, MIP1α and MIP1β), cell adhering

molecules (e.g. integrin αM, β2) and extra cellular matrix proteinases (e.g. MMP1,

MMP9 and uPA). Furthermore, we found out that Ets-2 phosphorylation was not crucial

for acute phase of TNFα induction upon LPS stimulation in macrophages, but played

important roles in sustain persistent TNFα expression. Those findings demonstrated the

role of Ets-2 phosphorylation in immune responses, and implicated that Ets-2 may be

important for some human immune diseases.

101

3.2 Result

3.2.1. Ets-2 phosphorylation was required for the mev mice inflammation phenotype.

3.2.1.1. Breeding strategies.

To address the role of Ets-2 phosphorylation in macrophage survival and motheaten phenotype, mev/+ mice were mated with mutant mice either harboring a ets-2

deletion allele (ets-2+/-) (Yamamoto et al. 1998), or mice with a point mutation at the Ets-

2 phosphorylation site (ets-2T72A/T72A) (Man et al. 2003). The T72A mutation of Ets-2 has been tested in transfections and other experiments and shows similar property to unphosphorylated Ets-2 (Yang et al, 1996, Fowles et al, 1998). A study with mice embryonic development indicates that the ets-2T72A allele is a hypomorphic allele (Man et al. 2003).

Our first approach was to generate mutant mice breeders in an inbred background.

The ets-2+/- and ets-2T72A/+ mutant mice were in FVB/N strain, while the mev mice were in C57Bl/6 strain. To minimize strain background influence, the ets-2+/+, mev/+ mice and

ets-2+/-, mev/+ mice were backcrossed 9 generations to FVB/N background. The FVB/N

mev mice showed a similar phenotype as those in C57Bl/6 background: motheaten

appearance, inflammation in feet and lung, life span, etc (see later parts of this chapter).

After crossed to FVB/N background for 5 generations, ets2+/+, mev/+ mice were mated with ets2T72A/+ mice. The resulted ets2T72A/+, mev/+ mice were further mated to FVB/N

strain for 2 more generations. Then brother-sister mating of ets2T72A/+, mev/+ mice

yielded ets2T72A/T72A, mev/+ mice, which were used to generate ets2T72A/T72A, mev/mev

mice. 102

3.2.1.2. Mutation of ets-2 increased mev mice life span, body weight and fertility.

The heterozygous ets-2 knockout allele or homozygous ets-2T72A mutation did not affect embryonic and neonatal development of mev mice (data not shown). As reported before, mev mice had a short longevity. Their mean life span was about 61 days. In contrast, the introduction of mutated Ets-2 significantly increased the mev mice life span

(Figure 3.3 A). The mean life span of ets2T72A/T72A, mev/mev mice was about 83 days, while that of ets2T72A/T72A, mev/mev mice was 123 days, about doubled that of the ets2+/+, mev/mev mice. Ets2+/+, mev/mev mice were smaller in size. At 90 days of age, their body weight was just a little more than half of the control mice (Figure 3.3 B). However, homozygous Ets2T72A mutation almost restored the body weight of mev mice to that of wild type level (Figure 3.3B).

103

Figure 3.1 Mutation in ets-2 increased mev mice life span and body weight. A. Cumulative survival percentage of mev mice with wt or mutant ets-2. Data were based on 35 Ets2+/+, mev/mev mice, 43 ets2+/-, mev/mev mice and 37 ets2T72A/T72A, mev/mev mice. B. Mice body weight. The data were average weight of 6 mice from each genotype at 3 months of age. Error bars indicate standard derivation.

104

Mev mice are infertile. The infertility of male mev mice is due to infiltration of

inflammatory cells in testes and low levels of testosterone (Shultz et al, 1984). Low levels of testosterone is not the major reason responsible for sterility because although hormone treatment rescued spermatogenesis, those treated male mice still failed to pregnant normal females. In contrast, both male and female ets2T72A/T72A, mev/mev mice were

fertile. Especially the males were good breeders. They could actively mate with females

for several months. Therefore, those mice were used as breeders to produce more

ets2T72A/T72A, mev/mev mice. Female ets2T72A/T72A, mev/mev mice were also fertile. But for

some unknown reasons, they stopped breeding after giving one or two litters. Most of the

ets2+/-, mev/mev mice were not fertile. However, 3 out of 40 male ets2+/-, mev/mev mice were fertile. All the female ets2+/-, mev/mev mice were infertile.

3.2.1.3. Mutation in ets-2 relieved inflammation in peripheral tissues of mev mice.

Inflammation is evident throughout the body in mev mice, especially in the foot,

tail and ear (Figure 3.3A). This inflammation is absent in most ets2T72A/T72A, mev/mev

mice (Figure 3.3 B.) and partially reduced in ets2+/-, mev/mev mice (data not shown). In

the ets2+/+, mev/mev mice, the epidermis was markedly thickened, and there were

numerous intraepidermal pustules, some of which had ruptured to the skin surface

(Figures 3.3 C). In many areas, suppuration extended from the epidermis deep into the

dermis. Large areas of the dermis were heavily infiltrated by mixture of inflammatory

cells. There was evidence of extensive remodeling of the cortices of the long bones

(Figures 3.3 E). This remodeling appeared to be due both to bone resorption and new 105

bone formation and resulted in marked skeletal deformity. In marked contrast, the epidermis of the ets2T72A/T72A, mev/mev mouse was only 2-3 cell layers thick, which is the thickness for normal mice (Figure 3.3 D). The dermis did not contain unusually large numbers of inflammatory cells. Cortical surfaces of long bones were smooth and cortices were of uniform thickness (Figure 3.3 F). Therefore, the skin, bones, and joints of the ets2T72A/T72A, mev/mev mice were basically indistinguishable from those of normal mice.

106

Figure 3.2. Mutation in ets-2 relieved inflammation in the foot of mev mice. A-B. Representative feet from a motheaten-viable mice (A) and a ets2T72A/T72A, mev/mev mouse (B). The inflammation was evident in the motheaten-viable mouse (A), but not in ets2T72A/T72A, mev/mev mouse (B). C-F. Sections of feet from motheaten-viable mice (C, E) and the ets2T72A/T72A, mev/mev mice (D, F). The epidermis of the ets2T72A/T72A, mev/mev mouse was thin, no pustules were evident, and the dermis was free of inflammatory cells (D), while the skin of the motheaten mouse had a markedly thickened epidermis with intraepidermal pustules and the dermis contained numerous mixed inflammatory cells (C). In the doubly mutant mouse, cortices of long bones were uniform in thickness with smooth contours (F); in motheaten mice, there had been extensive remodeling of bone (E).

Continued on next page 107

Fig.3.2. Continued.

108

3.2.1.4. Mutation in ets-2 relieved inflammation in lungs of mev mice.

Alveolar macrophages are natural components of the normal lung. They are

involved in local defense against a variety of pathogenic and particulate and play an early

role in inflammation and control of infection. However, dysregulation of alveolar

macrophages may cause inflammation, fibrosis and other lung injuries. The primary

lethal cause of mev mice is due to lung injury, characterized by interstitial pneumonitis

accompanied by accumulations of alveolar macrophages and other immune cells in the

lower respiratory tract. There were large numbers of macrophages present in the alveoli

of ets2+/+, mev/mev mice lungs (Figure 3.4 C, D), as reported before (Shultz et al. 1984).

In contrast, there were few macrophages present in lungs from ets2T72A/T72A, mev/mev

mice (Fig 3.4 E, F), similar to the number observed in normal wt lungs (Fig 3.4 A, B).

There were still slightly increased number of macrophages in lungs from ets2+/-, mev/mev

mice compared to that in wt lungs, but the number of macrophages was less than that in

lungs from ets2+/+, mev/mev mice. To quantify the accumulation of macrophages in lung,

RNA was extracted from fresh whole lungs from 3 months old animals. Real-time

quantitative PCR with two macrophage markers, c-fms and lysozyme, revealed that

ets2+/+, mev/mev mice have more than 2-fold higher level of c-fms and lysozyme

expression in the lung than that in wt lungs. In contract, there was much reduced

expression of c-fms and lysozyme in lungs from ets2T72A/T72A, mev/mev mice and ets2+/-, mev/mev mice compared to ets2+/+, mev/mev mice. The expression level of the two

macrophage markers in ets2T72A/T72A, mev/mev mice was comparable to that in Wt mice

(Fig 3.4 G).

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Figure 3.3. Mutation in ets-2 reduced macrophage accumulation in lungs of mev mice. Representative sections of lungs from a wt (A, B) mouse, a ets2+/+, mev/mev mouse (C,D), a ets2T72A/T72A, mev/mev mouse (E, F) and a ets2+/-, mev/mev mouse. B, D, F and H are the same section as A, C, E and G, respectively, but at a higher magnification. I. The quantification of macrophages by the expression of macrophage markers. Total RNA was isolated from fresh dissected lungs, reverse transcribed, and then subject to real time PCR analysis. The expression level of each gene was standardized with the level of ribosomal protein L4. The data were average of 2 experiments of 2 mice from each genotype groups. Error bars indicate standard derivation of measurements.

Continued on next page 110

Fig.3.3. Continued

Continued on next page 111

Fig.3.3. Continued

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3.2.2. Ets-2 is important for macrophage survival but not for proliferation.

CSF-1 is important for macrophage survival. One of the mechanisms for CSF-1

promoting cell survival is to regulate the expression of pro-survival genes, like Bclx.

CSF-1 does so by activating Ras signaling. The Ras signaling leads to phosphorylation of

Ets-2, which activates the expression of target genes, such as Bclx. The phosphorylation status of Ets-2 correlated with CSF-1 dependent macrophage survival. To test the role of

Ets-2 phosphorylation in macrophage survival, mev mice bone marrow derived macrophages with normal or half dosage of ets-2, or homozygous for the ets-2 phosphorylation mutation were used to measure the CSF-1 dependent cell survival.

The viability assay was carried out using the LIVE/DEAD®

Viability/Cytotoxicity Kit, which provides a two-color fluorescence cell viability assay that is based on the simultaneous determination of live and dead cells with two probes that measure two recognized parameters of cell viability — intracellular esterase activity and plasma membrane integrity. Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant calcein AM to the intensely fluorescent calcein.

The polyanionic dye calcein is well retained within live cells, producing an intense uniform green fluorescence in live cells (ex/em ~495 nm/~515 nm). EthD-1 enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (ex/em

~495 nm/~635 nm). EthD-1 is excluded by the intact plasma membrane of live cells.

Bone marrow derived macrophages (BMMs) were derived from Wt, ets2+/+,

mev/mev, ets2+/-, mev/mev and ets2T72A/T72A, mev/mev mice as described in Chapter 2. The 113

BMM cells were counted and 1X106 cells were plated in each well of 6-well dish (non-

tissue culture treated), in RPMI with 0.1% heat inactivated calf serum and no CSF-1. At

indicated time, the cells were stained with LIVE/DEAD® Viability/Cytotoxicity Kit,

fixed and imaged with a fluorescent microscope. The cell number was obtained by

analyzing the images with ImageJ software. Wt cells were sensitive to CSF-1 withdrawal.

After 36 hours of CSF-1 withdrawal, about 50 percent of cells were not viable. Ets-2+/+,

mev/mev cell were not sensitive to CSF-1 withdrawal. There were more than 90% of

viable cell at 36 hours. In contrast, cells from ets-2T72A/T72A, mev/mev mice and ets2+/-, mev/mev mice were sensitive to CSF-1 withdrawal, although at a less extent than that in wt cells. There were only about 70% of viable cells at 36 hour (Fig3.5 A).

A biochemical hallmark of apoptosis is degradation of DNA by endogenous

DNases, which cut within the internucleosomal regions to generate double-stranded DNA fragments of 180–200 base pairs. The cleavage of DNA may not be complete, thus the

DNA fragments are detectable as a ladder pattern upon electrophoresis of isolated DNA.

Internucleosomal fragmentation has been correlated with other well-characterized apoptotic morphologic methods in a wide variety of cell types and treatment (Tian et al.

1991; Sarih et al. 1993; Heusel et al. 1994; Liu et al. 1997; Kuida et al. 1998).

To perform the DNA laddering analysis, 1X107 BMM cells were plated in each

square dish without CSF-1. 30 hours later, DNA from apoptotic cells were harvested,

stained with SYBR Green, and run on an agarose gel. Wt cells gave a strong DNA ladder.

There was only barely detectable DNA ladder in ets2+/+, mev/mev cell. In contrast, there

was significant amount of fragmented DNA apparent in ets2T72A/T72A, mev/mev cell lane

(Fig3.5 B) and in ets2+/-, mev/mev cells (data not shown). 114

Efficient removal of cells that are damaged, malignant, or otherwise compromised

occurs by selectively triggering latent “self-destruction” machinery present in each cell.

Apoptosis can be triggered rapidly (death within 2-8 hours) by activation of extracellular

cell death receptors, or slowly (death within 8 hours to 2 days) by removing the survival

signals that normally sustain cells. The latter form of apoptosis appears to be primarily

dependent on the regulation of mitochondrial integrity and/or function through

modulation of Bcl-2 family members. Loss of mitochondrial integrity leads to the release

of agents such as cytochrome c and apoptosis-inducing factor that can lead to caspase-

dependent or caspase-independent cell death (Tian et al. 1991; Heusel et al. 1994; Liu et

al. 1997; Kuida et al. 1998). Caspases are a family of cysteine proteases that cleave target

proteins at specific aspartate residues (Alnemri et al. 1996). The roles of caspases in

apoptosis first became evident when a cell death-related gene, ced-3, which is essential

for apoptosis in Caenorhabditis elegans, was found to be homologous to the mammalian

caspases (Tian et al. 1991; Yuan 1995). It is now clear that caspases are essential effector molecules for carrying out apoptosis in eukaryotic cells (Wang and Lenardo 2000).

Caspase-3 is one of the key executioners of apoptosis, being responsible either partially or totally for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly(ADP ribose) polymerase (PARP). During the execution phase of apoptosis, caspase-

3 is responsible either wholly or in part for the proteolysis of a large number of substrates, each of which contains a common Asp- X-X-Asp (DXXD) motif (Cohen

1997; Wang and Lenardo 2000). Therefore, caspase-3 activity can be measured by incubating cell extract with synthetic peptide that contains this motif (e.g. aminotrifluoromethylcoumarin assay), and the cleavage (release of free afc) can be 115

determined by measuring fluorescence, as previously described (Fahy et al. 1999; Doseff

et al. 2003; Zeigler et al. 2003).

To perform Caspase-3 assay, 3X106 BMM cells were plated in square dishes in

RPMI medium, with or without CSF-1. All the cells were harvested 24 hours later for

caspase-3 assay. In the presence of CSF-1, there was similar basal level of caspase-3

activity in cells from all 4 genotypes. Therefore, the caspase activity in the absence of

CSF-1 was standardized with caspase activity in the presence of CSF-1 for each genotype

to obtain CSF-1 dependent results. In the absence of CSF-1, there were high levels of

caspase-3 activity in wt cells. In contrast, ets2+/+, mev/mev cells did not have much

caspase-3 activity. However, there was significantly more caspase activity in cells from

ets2T72A/T72A, mev/mev mice and ets2+/-, mev/mev mice (Fig 3.5 C).

To determine whether Ets-2 phosphorylation affects macrophage proliferation, 1

X 105 BMMs from mev mice with wt or mutated Ets-2 were grown in RPMI with 5% heat inactivated serum, with 10% of L-cell conditional medium as a source of CSF-1. The cells were counted at indicated time. There was no major difference in cell proliferation rate for mev macrophages with wt or mutant Ets-2 (Fig3.5. D).

Therefore, the results of BMM cell viability after CSF-1 withdrawal were consistent from three different assays. Wt cells were very dependent on CSF-1 for survival, while ets2+/+, mev/mev cells showed CSF-1 independent cell survival. In

contrast, mev/mev cells with mutated Ets-2 demonstrated moderate yet consistent increase

in cell apoptosis. Therefore, Ets-2 phosphorylation plays an important, but not an

exclusive role, in regulating macrophage survival. However, the phosphorylation of ets-2

had no effect on macrophage proliferation. 116

Figure 3.4. Ets-2 was important for macrophage survival but not for proliferation. A. Viability assay. 1X106 BMM cells were plated in each well of 6-well dish (non-tissue culture treated), in RPMI with 0.1% heat inactivated calf serum and no CSF-1. At indicated time, the cells were stained with LIVE/DEAD® Viability/Cytotoxicity Kit (Molecular Probes) according to manufacture’s instruction. After staining, the cells were fixed in 3.7% paraformaldehyde and imaged with a fluorescent microscope. The cell number was obtained by analyzing the images with ImageJ software. At least 2000 cells were counted at each time point for each genotype. B. DNA fragmentation assay. 1X107 BMM cells were plated in each square dish without CSF-1 for 30 hours. DNA from apoptotic cells were harvested and run on a 1.2% agarose gel. Lane 1: Wt cells; Lane 2: +/+ T72A/T72A ets2 , mev/mev cells; Lane 3: ets2 , mev/mev cells. C. Caspase-3 assay. 3X106 mouse BMMs were plated in square dishes in RPMI medium with 0.1% heat inactivated calf serum, with or without CSF-1. The cells were harvested 24 hours later for caspase-3 assay. For each genotype, the caspase activity in the absence of CSF-1 was standardized 117

with caspase activity in the presence of CSF-1 to obtain CSF-1 dependent results. D. Proliferation assay. 1X105 cells were plated in each well of a 6-well dish (non-tissue culture treated) supplied with RPMI with 5% heat inactivated serum, with 10% of L-cell conditional medium as a source of CSF-1. The cells were fixed at indicated time, stained with 10uM bis-benzimide and imaged with a fluorescent microscope. At least 1000 cells at each time point for each genotype were counted with ImageJ software. The data for viability assay (A), Caspase-3 assay (C) and proliferation assay (D) results were average results of cells from 3 independent mice from each genotype. Error bars represent standard derivation. A represent result of 3 DNA fragmentation experiments was shown in (B). F, FVB/N wt mice; M: mev mice; AM: ets2T72A/T72A, mev/mev mice; EM: ets2+/-, mev/mev mice.

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3.2.4. Expression of many inflammatory genes was reduced in lungs with mutant ets-2.

As discussed earlier, macrophages produce many important molecules in immune response, and the expression of some of those molecules are regulated by Ets factors. To further examine the role of Ets-2 in macrophage inflammatory responses, RNA were extracted from fresh whole lung and quantitative real time PCR technique was used to examine the expression of several immune cytokine, chemokine, extracellular protease, integrins and other genes.

TNFα is mainly secreted by macrophages and is a key proinflammatory cytokine, and has been shown to be important for mev pathology. TNFα levels was significantly increased in mev serum and lungs compared to control mice. Administration of anti-

TNFα antibody to mev bone marrow recipient mice decreases the severity of acute lung injury (Thrall et al, 1997). In our real time PCR assay, TNFα was expressed more than

10-fold higher in mev lungs than in wt lungs. ets2T72A/T72A, mev/mev mice had similar levels of TNFα in lungs as in wt mice (Fig3.5 A). Il-1α and Il-12 were also expressed at higher levels in mev mice lungs than in ets2T72A/T72A, mev/mev mice. However, there was no significant difference in expression of Il-1β and Il-6 in mev mice with wt or mutant ets-2 (Fig3.5 A). There was no major difference of the levels of TNFα and Il-1α in lungs of ets2+/+, mev/mev mice or ets2+/-, mev/mev mice, but there was much less Il-12 in lungs from ets2+/-, mev/mev mice than in lungs from ets2+/+, mev/mev mice (Fig. 3.5 A).

Chemokines attract immune cells to sites of inflammation (Ross and Auger 2002).

Eotaxin, MCP-1, MIP1α and MIP1β were all expressed significantly higher in ets2+/+,

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mev/mev mice than in wt, ets2T72A/T72A, mev/mev or ets2+/-, mev/mev mice (Fig3.5 B).

Especially MIP1α and MIP1β were expressed at significantly higher level (more than 40

fold and about 15 fold, respectively) in ets2+/+, mev/mev mice than in wt mice. MIP1α

and MIP1β transcripts levels were elevated in ets2T72A/T72A, mev/mev mice and ets2+/-,

mev/mev mice compared to wt mice, but they were significantly lower than those in ets2+/+, mev/mev mice (Fig3.5 B). RANTES mRNA level were comparable in all four

genotypes (Fig3.5 B).

Macrophages secret uPA and matrix metalloproteinases (MMPs), such as MMP-

1, MMP-3, and MMP9. Those proteins are important for the break down of the

extracellular matrix to facilitate cell migration, and change cell microenvironment. MMP-

1, MMP-3, MMP-9 and uPA are identified as Ets target genes in various cell types.

MMPs and uPA expression were examined in lungs. MMP1, MMP9 and uPA were

expressed significantly higher in lungs from ets2+/+, mev/mev mice than in wt lungs. The

expression of these genes in lungs from ets2T72A/T72A, mev/mev mice or ets2+/-, mev/mev

mice were similar to that in wt lungs. The expression of MMP3, however, was not higher

in lungs from ets2+/+, mev/mev mice than in lungs from ets2T72A/T72A, mev/mev mice

(Fig3.5 C).

Integrins are important for cells adhering to other cells and to extracellular matrix, and regulate intra-cellular signaling events. Macrophages from mev mice adhere and spread to a greater extent than normal macrophages through integrin αMβ2 mediated contacts (Roach et al, 1998). To explore the role of Ets-2 in controlling those integrin expression, mRNA level of these genes were examined. There was about 15 fold increase

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in integrin αM expression in lungs from ets2+/+, mev/mev mice, compared to lungs from wt mice. The mutation of Ets-2 reduced the expression of integrin αM more 2 (ets2+/-, mev/mev) to 4 fold (ets2+/+, mev/mev mice). There was also a significant elevation of integrin β2 in ets2+/+, mev/mev mice lungs than in lungs from mice with other three genotypes.

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Figure 3.5. Mutation of ets-2 affected the expression of inflammatory genes in lungs. Total RNA was isolated from fresh dissected lungs, reverse transcribed, and then subject to real time PCR analysis. The expression level of each gene was standardized with the level of ribosomal protein L4. The expression of cytokines (A), chemokines (B), extracellular proteases (C), integrins and Bclx (D). The data were average of 2 experiments with lungs from 2 independent mice from each genotype groups. Error bars indicate standard derivation.

Continued on next page

122

Fig.3.5. Continued.

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3.2.5. Ets-2 modulated lung alveolar macrophages gene expression.

The gene expression profile in the lung showed that many inflammatory genes

were overexpressed in the lung. However, the lung is constituted of many cell types. To

monitor the macrophage specific gene expression, the lung alveoli cells were collected by lavage and plated in a 24 well dish (non-tissue culture treated). 6 hours later, total RNA was harvested from attached cells (alveolar macrophages), reverse transcribed and subject to real time PCR analysis. mRNA level for each gene was adjusted by the level of lysozyme, a macrophage marker (Fig. 3.6). There were much more TNFα (26 fold), Il-1α

(14 fold), MIP1α (49 fold), MIP1β (19 fold), MMP1 (7 fold), MMP9 (36 fold) and uPA

(4 fold) expressed in alveolar macrophages from ets2+/+, mev/mev mice than cells from in

ets2T72A/T72A, mev/mev mice. There were 7 fold higher levers of integrin αM expression in

ets2+/+, mev/mev cells than in ets2T72A/T72A, mev/mev cells. However, ets2T72A/T72A, mev/mev cells still expressed about 5 fold higher amount of integrin αM than in wt cells.

The expression of Bclx was also higher (about 7 fold) in ets2+/+, mev/mev cells than in ets2T72A/T72A, mev/mev cells. The mRNA level of Bclx gene was even lower (2 fold) in ets2T72A/T72A, mev/mev macrophages than in wt cells.

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A fo the standard te ca extract total RN i rs ind 24-well dishes (non-tissue macrophage marker. The data Lung alveoli cells (including ophages were used to re immediately plated in for each genotype. Error ba e mRNA level of lysozyme, a phages inflammatory gene expression. centrifugation, those cells we were washed away and the attached macr on was standardized with th macrophages from two animals ts of t f i t

125 Figure 3.6. Ets-2 regulates lung alveoli macro macrophages) were collected by lavage. After culture treated). 6 hours later, floating cells real time PCR analysis. All gene expressi are average results of 2 measuremen di

3.2.6. Peritoneal macrophages gene expression was regulated by Ets-2.

To see whether other types of macrophages behave similar to alveolar

macrophages, we isolated thioglycolate elicited peritoneal macrophages and monitored

gene expression in those cells (Fig. 3.7). There were much more TNFα (6 fold), Il-1α (2

fold), Il-12 (3 fold), MIP1α (3 fold), MIP1β (3 fold), MMP1 (3 fold), MMP9 (6 fold),

uPA (3 fold), integrin αM (3 fold) and Bclx (3 fold) expressed in ets2+/+, mev/mev

peritoneal macrophages than in ets2T72A/T72A, mev/mev cells. The expression of these

genes was comparable in ets2T72A/T72A, mev/mev and wt cells, except MMP1 and uPA,

which were expressed higher (3 fold) in ets2T72A/T72A, mev/mev cells than in wt cells (Fig.

3.7). The relative gene expression level among different genotypes in peritoneal macrophages might be different from that in lung alveoli macrophages. The difference might indicate the nature and environment of those macrophages.

126

es later, floating olate injection. After R analysis. All gene lture treated). 30 minut 4 days after thioglyc ge marker. The data are average results of dard deviation of measurements. used to extract RNA for real time PC mice peritoneal macrophages was reduced by ets-2 mutation. hages were mev plated in 6-well dishes (non-tissue cu e. Error bars indicate the stan y l yp diate enot g ttached macrop ophages) were collected by lavage and ach gene expression in for e e ized with the mRNA level of lysozyme, a macropha lls were imme d shed away and the a from 3 mic n, those ce a s e tio g ha p ifuga Figure 3.7. Inflammatory macro Peritoneal cells (including macr centr cells were w expression was standar

127

3.2.7. Ets-2 is required for LPS induction of persistent TNFα expression.

Lipopolysaccharide (LPS), a major component of the outer membrane of Gram- negative bacteria, is known to induce in the host a variety of pathological and physiologic responses, including fever, coagulant activity, septic shock, and death. LPS stimulates macrophages to produce a wide diversity of inducible gene products needed (For example, TNFα) for immediate host defense and priming of an appropriate acquired immune response (Ravasi et al. 2002). To examine the role of Ets-2 in TNFα expression after LPS treatment, macrophages derived from bone marrow were treated with 100ng/ml

LPS for 0 -18 hours. Total RNA was harvested from those cells, and TNFα mRNA level

was determined by real time PCR. There was no significant difference in the acute phase

(15 and 30 minutes) of TNFα production in response to LPS stimulation in cells from all

three genotypes (Fig. 3.8,). However, there was significantly higher (about 3 fold)

expression of TNFα in ets2+/+, mev/mev macrophages than in ets2T72A/T72A, mev/mev cells at 18 hours after LPS stimulation. The levels of TNFα in ets2T72A/T72A, mev/mev cells

were similar to that in wt macrophages. It implicated that Ets-2 was responsible for LPS

induced persistent TNFα expression in mev macrophages.

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Figure 3.8. Ets-2 is important for persistent expression of TNFα in BMMs upon LPS stimulation. 2X106 BMM cells were plated in a 6-well dish (non-tissue culture treated) with RPMI media supplied with 5% heat inactivated calf serum. At indicated time after applying 100 ng/ml of salmonella R595 LPS (Sigma-Aldrich, St. Louis, MO), cells were harvested and total RNA was extracted. After reverse transcription, TNFα mRNA level was determined by real time PCR and adjusted by ribosomal protein L4 mRNA level. The data were average results of 2 measurements of macrophages from two independent animals for each genotype. Error bars indicate the standard deviation of measurements.

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3.3 Discussion

Ets-2 involvements in inflammation and motheaten pathology.

Mev mutant mice are small in size, sterile, accumulate large numbers of

macrophages and other immune cells in the peripheral tissues, including skin, spleen and

lung, and subsequently succumb to an interstitial pneumonia (Green and Shultz 1975;

Shultz et al. 1984). Most of those phenotypes were corrected, or partially corrected by homozygous ets-2T72A mutation, or reduction the dosage of ets-2. ets2T72A/T72A, mev/mev

mice were bigger, fertile and had much less inflammation (Fig. 3.1, 3.2 and 3.3).

Therefore, Ets-2, the signaling pathways regulate Ets-2 phosphorylation and Ets-2 target genes are crucial to the motheaten pathology.

Ets-2 may be involved directly in controlling fertility by regulating macrophage- elicited inflammation in reproductive organs, or indirectly by affecting other phenotypes.

There could be many factors affecting fertility. Compare to the sterility of ets2+/+,

mev/mev mice, the fertility of ets2T72A/T72A, mev/mev mice could be due to several reasons:

1) Restored spermatogenesis. Ets2+/+, mev/mev mice have defects in spermatogenesis. The fertility of Ets2T72A/T72A, mev/mev mice indicated that they produced mature sperms. 2)

No or less inflammation in sex organs. Ets2+/+, mev/mev mice have inflammation in many

parts of the body, including the penis. The inflammation in the genitals might hamper the

mating ability of those mice. 3) Ets2+/+, mev/mev mice are smaller in size, and very slim.

It is hard to find fat tissues inside the body. In contrast, the ets2T72A/T72A, mev/mev mice

were bigger, stronger, chubbier and lived longer. Those factors might affect their

physical, hormonal and mental sex behavior. In fact, ovaries from ets2+/+, mev/mev mice

aged 10-14 weeks contained normal-appearing and variably sized oocytes. 130

Transplantation of ets2+/+, mev/mev mice ovaries into histocompatible ovari-ectomized

normal hosts yield live offspring genetically derived from donor ovaries (Shultz et al,

1984). Therefore, Ets-2 phosphorylation might not play direct role in oocyte

development, but affect inflammation and other factors affecting fertility.

Ets-2 is important for bone development. In developing embryos, ets-2 is

expressed in the cartilage primordia in regions of vertebrae, skull, ribs and extremities

where cartilage is being actively formed and later replaced with bone (Maroulakou et al.

1994b). Ets-2 overexpression in transgenic mice causes osteomalacia and dome-shaped

skulls which are defects seen commonly in trisomy-16 mouse (down's syndrome) where

the gene dosage of Ets2 is increased (Sumarsono et al. 1996b). At least two cell types are

crucial for bone development and maintenance: osteoblast and osteoclast (Karsenty

1999). Ets-2 is expressed in both cell types (Raouf and Seth 2000). ets2+/+, shp1mev/mev mice have decrease bone density, appeared due to active remodeling and bone resorption by elevated osteoclasts activity (Fig. 3.2). In contrast, ets2T72A/T72A, shp1mev/mev mice

bones were much stronger and there was less remodeling (Fig 3.2). Both osteoclasts and

macrophages belong to monocytic phagocyte linage and share many characteristics. Ets-2

regulates many genes (eg. extracellular proteases, integrins) involved in macrophage

action. Those genes are also important for osteoclasts activity. Therefore, Ets-2 might

directly involved in regulating osteoclasts activity, and responsible for the mev bone

phenotype. Alternatively, Ets-2 may also regulate osteoblasts gene expression, and

modulates osteoclast activity indirectly. Further experiments with osteoblasts and osteoclasts from those mutant mice may uncover the role of Ets-2 in bone development and bone disease. 131

Although macrophages appear to be the key player in mev phenotype, mev mice have abnormalities in other immune cells, such as T cells, B cells and neutrophils. To get an unambiguous understanding of the role of ets-2 in macrophages and mev phenotype, ets-2 conditional knockout mice (Chapter 6) can be used to generate mev mice with tissue specific deletion of ets-2 in macrophages. Those mice may have more inflammation than mev mice with ets-2 mutation in every cell types, and then it means Ets-2 function in other cell types was also required for motheaten phenotype. However, since the T72A mutation is a hypomorphic allele but not a null allele, deletion of ets-2 in macrophages may lead to even more dramatic reduction of inflammatory gene expression, and restriction of inflammation in mev mice.

Ets-2 was important for macrophage survival.

To maximize advantages and reduce costs associated with life as an interdependent community of cells, multicellular organisms have evolved common mechanisms to regulate the life and death of their individual cells. Critical to the health and survival of a multicellular organism is its ability to selectively sustain advantageous cells and selectively eliminate cells that threaten the survival of its cellular community.

Excessive cell survival can lead to disorders such as cancer and autoimmunity, and insufficient cell survival can lead to tissue degenerative and developmental disorders.

Thus, regulated survival of individual cells evolved in a manner consistent with organismal and population survival priorities (Ballif and Blenis, 2001).

Ets-2 was important for macrophage survival (Fig 3.4). The status of Ets-2 phosphorylation correlates with cell survival and pro-survival gene expression (Smith et 132

al. 2000).Cell survival studies with BMM cells indicated that ets-2 is important, yet not

exclusively responsible for CSF-1 dependent BMM cell survival. By real time PCR

analysis, there was about 50% more Bclx mRNA level in mev BMMs with wt ets-2 than

in mev cells with mutant ets-2 after 18 hours CSF-1 withdrawal (data not shown).

However, there was much higher level of Bclx mRNA (4-7 fold) in fresh isolated alveoli

and peritoneal macrophages from ets2+/+, mev/mev mice than in the same type of cells from ets2T72A/T72A, mev/mev mice. Therefore, there might be a larger difference in cell

survival of mev mature tissue macrophages with wt or mutant ets-2 in vivo. Ets-2 might

be involved in cell autonomous, as well as cell non-autonomous regulation of

macrophage cell survival.

Ets-2 regulated macrophage gene expression.

Ets-2 regulates the expression of many genes in macrophages. Cytokines and

chemokines secreted by macrophages could recruit other immune cells (for example,

neutrophils, T lymph cells) to the inflammation site, e.g., lung, and further strength the

inflammation. Extracellular proteinases (MMPs and uPA) and integrins are important for

macrophages to migrate to inflammation sites. Macrophages from mev mice with normal

ets-2 were active in transcribing several cytokines (TNFα and Il1α), chemokines

(eotaxin, MCP-1, MIP1α and MIP1β), extracellular proteases (MMP1, MMP9 and uPA),

integrins (integrin αM and integrin β2) and other genes. The expression of most of these

genes was reduced in ets2T72A/T72A, mev/mev mice. The expression levels of some genes even went down to a level comparable to that in wt mice. However, it is yet unknown

133

whether Ets-2 directly regulate those genes expression, or indirectly. Chromatin

immunoprecipitation (ChIP) may tell us how Ets-2 controls those genes in macrophages.

Combinatorial control is a characteristic property of Ets family members, involving interaction between Ets and other key transcriptional factors such as NFkB.

NFkB is a ubiquitous transcription factor involved in immune, inflammatory and stress responses (Li and Verma 2002). Adjacent or overlapping binding sites for Ets and NFkB are present in many inducible inflammatory genes, such as IL-12 (Gri et al. 1998) and genes important for cell survival, such as Bclx (Lee et al. 1999; Smith et al. 2000). Co- transfection of NFkB and Ets factors contributes to synergistic transcription activation of

IL-12 (Gri et al. 1998). The functional cooperation between NFkB and Ets proteins require the physical interaction between NFkB and Ets proteins (Li et al. 2000). Further genetic and biochemical approaches may address the role of the interactions between

NFkB and Ets-2 in inflammation and other immune responses.

Implications of Ets-2 in human inflammation disease.

Deregulation of macrophage can lead to auto inflammation disease in human.

Many of Ets-2 target genes contribute to those diseases. For example, several studies have shown that excessive levels of many MMPs, most of them are Ets target genes, are present in chronically inflamed tissues throughout the body. These observations have led to the often held concept that excessive proteolysis damages tissues and impairs healing.

Unregulated, excessive proteolysis is indeed probably responsible for the destruction of matrix proteins in emphysema, as well as in arthritis, aneurysms, and other conditions of structural tissues (Parks and Shapiro, 2001). Ets-2 overexpression was observed in 30% 134

of rheumatoid arthritis patient (Dooley et al. 1996). Our study has demonstrated that Ets-

2 phosphorylation was required in an inflammation animal model, and it was required for

Ets-2 to activate transcription of many genes that are important for macrophage action.

The study of Ets-2 phosphorylation state in human autoimmune diseases may disclose the impact of Ets-2 phosphorylation and leads to develop new strategies to treat patients with those diseases.

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CHAPTER 4

ETS-2 AND EMBRYO DEVELOPMENT

4.1 Introduction

The previous chapter illustrated the role of Ets-2 in a pathological condition. In this chapter, the role of Ets-2 in a physiological process, embryo development, was examined. Ets proteins are crucial in many important cellular activities, including cell growth, proliferation, differentiation, migration, oncogenic transformation and survival, and therefore are required in many biological processes, such as hematopoiesis, angiogenesis, embryo development and tumorgenesis. Disruption of ets proteins many lead to developmental abnormalities, even embryonic or neonatal lethality (Chapter 1).

Both Ets-1 and Ets-2 are reported to be involved in many important events. Many of those events, such as cell transformation, survival, angiogenesis, was demonstrated in cellular and biochemical models. However, the minimal phenotype of ets-1 knockout mice, tetraploid aggregation rescued ets2-/- mice, or ets-2T72A/T72A mice did not provide much in vivo evidence of their roles in these processes. Ets-1 and Ets-2 are highly homology to each other. They are phosphorylated at the conserved threonine residue by

Ras/MAPK pathways and regulate similar spectrum of genes (such as uPA, MMP3).

Their expression also overlaps. Therefore, genetic redundancy between the two genes

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may account for the minimal phenotype in ets-1-/- mice and ets-2T72A/T72A mice. To test this hypothesis, we used genetic approaches to generate and test the phenotype of ets-1-/-, ets-2T72A/T72A mice. Those mice were embryonic lethal from E11.5 to E14.5, with severe angiogenesis and cardiovascular defects. The expression of many genes involved in angiogenesis was affected by ets-1/2 mutation. For example, Ang1 and Tie2, whose disruption led to embryonic lethality with similar phenotype as the ets-1-/-, ets-2T72A/T72A mice, were downregulated in ets-1-/-, ets-2T72A/T72A mice compared to that in control

animals. The expression of macrophage marker genes was also reduced in ets-1-/-, ets-

2T72A/T72A mice, indicating that Ets-1/2 may be involved in hematopoiesis. Furthermore, embryonic fibroblasts from ets-1-/-, ets-2T72A/T72A mice expressed much less of several

extracellular matrix proteases, including MMP3 and MMP9, which are important for

inducing a proteolysis cascade that lead to degradation of extracellular matrix, and are

important for tumor metastasis. Fibroblasts, macrophages and vascular cells are important

components of tumors. The results from this chapter suggested that ets-1 and ets-2 are

important in cancer, especially in tumor angiogenesis and metastasis.

4.1.1. Angiogenesis.

Many mutations lead to embryonic lethality because they affect blood vessel

formation. Blood vessels are formed by two processes: vasculogenesis and angiogenesis.

In vasculogenesis, a primitive vascular network is established during embryogenesis from multipotential mesenchymal progenitors. In angiogenesis, preexisting vessels (both in embryo and adult) send out capillary sprouts to produce new vessels. Angiogenesis is not only crucial for embryo development, but also indispensable in adult physiological 137

processes, such as ovulation, implantation and wound healing, and in pathological

conditions, such as tumor progression (Hanahan 1997; Papetti and Herman 2002; Marx

2003).

Angiogenesis can be divided into a phase of activation (sprouting) and a phase of resolution. The activation phase encompasses several stages: increased vascular permeability, vessel wall disassembly, basement membrane degradation, cell migration and ECM invasion, endothelial cell proliferation, and capillary lumen formation. The resolution phase include: inhibition of endothelial cell proliferation and migration, basement membrane reconstitution and vessel wall assembly, including recruitment and differentiation of pericytes and smooth muscle cells (Hanahan 1997; Papetti and Herman

2002; Marx 2003). Endothelial cells are centrally involved in each process of angiogenesis: They migrate and proliferate and then assemble into tubes with tight cell- cell connections to contain the blood. Peri-endothelial support cells are recruited to encase the endothelial tubes, providing maintenance and regulatory functions to the vessels. Those cells include pericytes for small capillaries and smooth muscle cells for larger vessels (Hanahan 1997; Papetti and Herman 2002; Marx 2003).

4.1.2. Genes involved in angiogenesis.

The establishment and remodeling of blood vessels is controlled by paracrine signals, many of which are protein ligands that bind and modulate the activity of transmembrane receptor tyrosine kinases (RTKs) (Table 4.3). One major regulator of vasculogenesis and angiogenesis is vascular endothelial growth factor (VEGF, also called vascular permeability factor, VPF) (Achen and Stacker 1998; Drake et al. 2000; Ferrara 138

2001; Ferrara and Gerber 2001). Deletion of one of the VEGF alleles is sufficient to

disturb normal embryonic development of the mouse and cause midgestational lethality

(E9.5) because of compromised endothelial proliferation and/or differentiation that

prevents the establishment of a normal circulatory system (Carmeliet et al. 1996; Ferrara

et al. 1996). Homozygous VEGF-/- embryos generated by tetraploid rescue show even

more severe vascular defect. They die at midgestation (E9.5) due to cardiovascular

dysfunction (Carmeliet et al. 1996; Ferrara et al. 1996). There is a requirement for

precise VEGF dosage regulation during development. Moderate overexpression of VEGF

from its endogenous locus results in embryonic lethality at E12.5-E14 with severe

abnormalities in heart development, such as attenuated compact layer of myocardium,

defective ventricular septation and abnormalities in remodeling of the outflow track of

the heart (Miquerol et al. 2000).

VEGF signaling is mediated by two RTKs, Flt1 (VEGFR1) and Flk1 (VEGFR2)

(Achen and Stacker 1998; Drake et al. 2000; Ferrara 2001; Ferrara and Gerber 2001). The two receptors are primarily expressed in endothelial cells but send distinctive signals. Flk knockout mice, which die by embryonic day 8.5 (E8.5), lack both endothelial cells and a developing hematopoietic system (Shalaby et al. 1995). In contrast, Flt knockout mice, which also die around E8.5, have normal hematopoietic progenitors and abundant endothelial cells, which migrate and proliferate but do not assemble into tubes and functional vessels (Fong et al. 1995).

Two additional RTKs critical for endothelial cells are Tie1 and Tie2 (Tek) (Ward and Dumont 2002). Both of them are specifically expressed in developing vascular endothelial cells. Embryos deficient in Tie-1 failed to establish structural integrity of 139

vascular endothelial cells, resulting in edema and subsequently localized hemorrhage, and

die between days 13.5 and 14.5 of gestation (Puri et al. 1995; Sato et al. 1995; Puri et al.

1999). In contrast, Tie2 knockout mice die in E9.5 to E12.5. They have decreased sprouting, incomplete heart development, absence of myocardial projections, simplification of vessel branching, decreased EC survival and lack of recruitment of pericytes (Dumont et al. 1994; Puri et al. 1995; Sato et al. 1995; Puri et al. 1999). It was indicated that Tie-1 and Tie-2 were indispensable for vasculogenesis because vasculogenesis proceeds normally in embryos lacking both Tie-1 and Tie-2, although such embryos die early in gestation of multiple cardiovascular defects (Puri et al. 1999).

In contrast, both receptors are required in the microvasculature during late organogenesis

and in essentially all blood vessels of the adult, indicating that they are essential for

maintaining the integrity of the mature vasculature (Puri et al. 1999).

Tie2 has two ligands with opposite function, angiopoietin-1 (Ang1) and Ang2

(Ward and Dumont 2002). Ang1 is the major physiological ligand for Tie2. It is involved

in almost all of Tie2's functions, such as vessel branching, recruiting and sustaining

pericytes. Ang1 knockout mice die with vascular defects similar to the Tie2 knockout mice (Davis 1996; Suri et al. 1996). Ang1 induces autophosphorylation of Tie2 in cultured endothelial cells, while Ang2 presents a negative signal to Tie2, and allows vascular remodeling. Transgenic mice overexpressing the negative ligand Ang2 also die during embryogenesis, again with similar vascular defects (Maisonpierre et al. 1997).

Other growth factors were also shown to have roles in angiogenesis. Basic and acidic fibroblast growth factors (bFGF and aFGF) stimulate endothelial cell proliferation, migration and production of plasminogen activators and collagenases. bFGF also causes 140

endothelial cells to form tube-like structures in three-dimensional collagen matrices

(Schweigerer 1989; Papetti and Herman 2002). Mice deficient in bFGF or both aFGF and bFGF have moderate defects in brain development, blood pressure regulation, and wound healing (Miller et al. 2000). Platelet-derived growth factor (PDGF) stimulates the proliferation of cultured pericytes and smooth muscle cells. Although mice deficient in

PDGF-B or PDGFR-β develop blood vessels that appear normal, they lack pericytes and die perinatally from hemorrhage and edema (Leveen et al. 1994; Soriano 1994). At low dose, transforming growth factor-β (TGFβ) stimulates endothelial cell growth, migration and in vitro endothelial tube formation. However, at high dosage, it inhibits those activities (Papetti and Herman 2002). TGFβ1-/- mice are embryonic lethal because of defects in the yolk sac vasculature and hematopoietic system. Blood vessel walls in those mice are fragile because of disrupted endothelial cell contacts (Dickson et al. 1995).

TFG-βR2 deficient mice show a similar phenotype: blood vessels are formed but are dilated with disrupted cell contacts (Oshima et al. 1996)

Extracellular proteinases, including matrix metalloproteinases, have been implicated in many stages of angiogenesis, including basement membrane degradation, cell migration/ECM invasion, and capillary lumen formation. They also regulate growth factor activity, for example, their activation could lead to activation of latent TGF-β, release of matrix-bound bFGF and VEGF (Papetti and Herman 2002). MMP-9 is frequently expressed at sites of active tissue remodeling and neovascularization. It degrades components of the ECM with high specific activity for denatured collagens

(gelatin). Chondrocytes growth plates from MMP9-/- mice show delayed vascularization

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(Vu et al. 1998). Cadherins are membrane proteins that mediate many functions involved in blood vessel assembly. VE-cadherin is localized to adherent junctions exclusively in endothelial cells. VE-cadherin knockout mice exhibit extreme vascular abnormalities, such as progressively disconnected endothelial cells, severely diminished branching and sprouting, and vessels eventually regress and disintegrate (Carmeliet et al. 1999).

Endothelial nitric oxide synthase (eNOS) catalyzes an oxidative reaction to produce

Endothelium-derived nitric oxide (NO), which plays a crucial role in controlling cell growth, apoptosis, postdevelopmental vascular remodeling and angiogenesis. eNOS deficient mice are viable, but have high blood pressure, lower heart rate (Shesely et al.

1996), defects in vascular remodeling (Rudic et al. 1998) and abnormal aortic valve development (Lee et al. 2000).

In summary, angiogenesis can be divided into several stages. Growth factors and their receptors, extracellular proteinases and transmembrane proteins binding to extracellular matrix components or to receptors on other cells regulate different aspects of angiogenesis. Deficiency or overexpression of those genes often leads to defects in angiogenesis. Therefore, the expression of those genes is tightly regulated. One layer of the regulation is through Ets transcription factors.

4.1.3 Ets factors and angiogenesis.

Many of these genes discussed above contain Ets binding sites in their promoter, and have been shown to be targets of Ets proteins (Sementchenko 2000; Lelievre et al.

2001; Oettgen 2001). Several Ets proteins are expressed in endothelial cells or supporting cells. Disruption or dysregulation of their expression often leads to embryonic lethality or 142

developmental abnormalities, including angiogenesis defects (Table 1.4). Fli-1 null

embryos die at E11.5 with loss of vascular integrity leading to bleeding in the

midbrain/forebrain boundary and in the hindbrain, and downregulation of Tie2 gene

expression (Hart 2000a). Tel null mice are embryonic lethal and die between E10.5-11.5 with defective yolk sac angiogenesis and intro-embryonic apoptosis of mesenchymal and neural cells (Wang et al. 1997).

Ets-1 is expressed at high levels in vascular endothelial cells during the formation of new blood vessels of the developing embryo, and in blood vessels of the adult during angiogenesis (Bhat et al. 1987; Wernert et al. 1992; Lelievre et al. 2001; Tokuhara et al.

2003). Ets1 increases endothelial cell adhesion and stimulates endothelial organization into capillary-like structures in vitro (Mattot et al. 2000). Expression of matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) and integrin β3, and invasiveness, are correlated with level of ets-1 expression in endothelial cell lines (Sato et al. 2000). Ets-2 is ubiquitously expressed throughout embryonic and neonatal development, including endothelial cells (Fig1.3). As in other cells types, Ets-2 is activated by phosphorylation at threonine 72 residue by Ras/MAPK pathway in endothelial cells, and activate the expression of target genes, such as CD13 (Petrovic et al. 2003). Cells containing Ets-2 inhibitory siRNAs were completely incapable of forming the organized networks characteristic of endothelial morphogenesis (Petrovic et al. 2003). A dominant negative constructs, constituted only the Ets DNA binding domain of Ets-1, inhibits angiogenesis

(Nakano et al. 2000). However, despite ets-1 and ets-2 expression in embryonic blood vessels, and indications from the above experiments, there was no defect in vascular development reported from mice with ets-1 and ets-2 gene inactivation or mutation. Ets- 143

1-/- mice are viable and fertile (Barton et al. 1998). Tetraploid rescued ets-2-/- mice

develop to basically normal adults, with a few minor defects in skin (waved hair) and

eyes (Yamamoto et al. 1998). Mice homozygous for the ets-2T72A allele are viable and fertile, and develop normally (Man et al. 2003).

Ets-1 and Ets-2 are closely related Ets family transcription factors. Overall, they share 53% identity and 67% similarity. However, they have much higher conservation in two conserved domains, the Ets DNA binding domain and the pointed domain. They share 96% identity and 99% similarity in the Ets DNA binding domain. In the pointed domain, they share 70% identity and 80% similarity, and 100% identity in the MAPK site

(Fig. 1.1, 1.2). Ets-1 and Ets-2 are also functionally related. They both are activated by phosphorylation of a conserved threonine residue in the pointed domain by MAPK, and activate the persistent expression of similar spectrum of genes, such as MMP-1, MMP-9 and uPA (Yang et al. 1996; Fowles et al. 1998a). Their expression patterns also overlap in many tissues (Fig. 1.3). Therefore, the absence of phenotype in Ets1-/- and Ets-2

T72A/T72A mice could be caused by genetic redundancy between the two genes. Ets-1 and

Ets-2 may replace each other in angiogenesis and other embryo development processes in

ets-1-/- mice or ets2T72A/T72A mice. To test this hypothesis, we used genetic approaches to generate and test the phenotype of ets-1-/-, ets-2T72A/T72A mice. Those mice died between

embryonic day 11.5 to 14.5, with severe angiogenesis and cardiovascular defects.

Consistent with the phenotype, the levels of transcripts of many genes, which were reported to be involved in angiogenesis, were affected by ets-1/2 mutation. Preliminary experiments suggested that the endothelial cell defects might be the primary lethal cause in those mice. The levels of several macrophage markers mRNA were reduced in ets-1-/-, 144

ets-2T72A/T72A mice. Embryonic fibroblasts from ets-1-/-, ets-2T72A/T72A mice expressed much less of several extracellular matrix proteases. Fibroblasts, macrophages and vascular cells are crucial components of tumors and greatly affect tumor development.

Thus ets-1 and ets-2 may be required in tumorgenesis, especially in tumor angiogenesis and metastasis.

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4.2. Results

4.2.1. Ets1-/-, Ets-2 T72A/T72A mice were embryonic lethal and died between E11.5-

E14.5.

To reveal possible genetic redundancy between ets-2 and ets-2, a genetic

approach was undertaken to characterize the in vivo role of ets-2 phosphorylation in mice.

Ets-2 T72A/T72A mice were mated with Ets-1 knockout mice. Brother-sister mating of their

F1 offspring generated F2 mice homozygous for one gene and heterozygous for the other,

but not mice homozygous for both mutations (data not shown). Ets1+/-, Ets-2T72A/T72A

mice and Ets1-/-, Ets-2 T72A/+ mice were phenotypically normal and fertile. Ets1+/-, Ets-2

T72A/T72A male and female mice were bred to try to generate Ets1-/-, Ets-2 T72A/T72A mice.

More than 350 pups from this mating have been analyzed, however, no live Ets1-/-, Ets-

2T72A/T72A pups were born (Table 4.1). Therefore, Ets1-/-, Ets-2 T72A/T72A mice were

embryonic lethal. Further analysis with embryos at different embryonic development

stages indicated that those mice died between embryonic days 11.5 to 14.5 (Table 4.2).

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Total Ets1-/-,Ets2T72A/T72A Ets1+/-,Ets2T72A/T72A Ets1+/+,Ets2T72A/T72A # of pups 355 0 219 136 Actual Percentage 0% 62% 38% Mendelian ratio 25% 50% 25%

Table 4.1. Ets1-/-, Ets-2 T72A/T72A mice were embryonic lethal. This table shows the results of one of the mating strategies to obtain ets-1-/-, ets-2T72A/T72A mice. Both male and female breeders were homozygous for ets-2T72A allele, and heterozygous for ets-1 knockout allele. Thus 100% of their pubs should be ets-2T72A/T72A, while the distribution of ets-1 knockout allele should follow the Mendelian ratio, i.e. ets- 1-/- : ets-2+/- : ets-1+/+ should be 1:2:1. However, no ets-1-/- pups were born.

Embryonic Total Total Ets1-/-, Angiogenesis Resorbed Normal Day embryo Ets2T72A/T72A defect 9.5 14 4 0% 0% 100% 10.5 43 14 29% 0% 71% 11.5 43 9 33% 11% 56% 12.5 43 10 40% 0% 60% 13.5 54 12 50% 17% 33% 14.5 47 14 79% 14% 7% >14.5 75 10 20% 80% 0%

Table 4.2 Ets1-/-, Ets-2 T72A/T72A mice died from E11.5 to E14.5. The embryos were classified by their appearance during dissection. “Angiogenesis defect” refers to mutant phenotype similar to that observed in Figure 4.1, such as abundant dilated blood vessel, edema and/or hemorrhage. Out of those mice, some were alive during dissection, some were dead, but no significant resorption was evident. The criteria for living and dead embryo is based on heart beating and/or blood flowing in blood vessel presented in the yolk sack. “Normal” means no obvious difference between the double mutant mice and control mice. “Resorbed” indicates embryos were being resorbed, but was still able to be genotyped. Embryos that were resorbed too much that genotyping of them was failed, were not counted in.

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4.2.2. Ets1-/-, Ets-2 T72A/T72A mice had numerous dilated blood vessels.

The mutant embryos were first analyzed by their appearance. All the mice earlier than E9.5 appeared normal. From E10.5, some embryos started to have massive dilated blood vessels and/or edema. The percentage of mice that had this appearance increased with age (up to 80% in E14.5), until they were all dead and resorbed (Table 4.2). From

E12.5, besides the dilated blood vessel phenotype, some embryos had evident edema

(Figure 4.2 B, D). Hemorrhage was also found in some of the embryos (Figure 4.2, D).

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, Ets-2 +/+ ive s s1 s t a A: An E

e same age. M are pointed by arrows). s at th o mice embry 72A E11.5 day embryos are shown. 72A/T T mice (some of these regions , Ets-2 -/- t throughout the body in those mice had abundant dilated blood vessels. T72A/T72A , Ets-2 -/- 1 s t E

1 . mouse embryo, shown normal appearance. B and C: Ets1 4

e 72A r u g i 72A/T F T enlarged/dilated blood vessels were eviden 149

Figure 4.2 Ets1-/-, Ets-2 T72A/T72A mice had dilated blood vessels, edema and hemorrhage. Representative embryos at E13.5 and E14.5 days are shown. The embryos were at E13.5 (A, B) and E14.5 (C, D). A and C: Ets1+/+, Ets-2 T72A/T72A mouse embryos, shown normal appearance. B and D; Ets1-/-, Ets-2 T72A/T72A mice embryos. They had abundant dilated vessel (indicated by long arrows), edema (indicated by short arrows) and/or hemorrhage (indicated by arrow heads). 150

4.2.3. Ets1-/-, Ets-2 T72A/T72A mice had cardiovascular and angiogenesis defects.

The Ets1-/-, Ets-2 T72A/T72A mice embryos were sectioned along with their

littermate control to be examined for abnormalities. One obvious defect of the double

mutant embryos was defects in the heart structure. The most striking phenotype of the

Ets1-/-, Ets-2 T72A/T72A mice heart was the thin endocardium layer and reduced

trabeculation. There were just very few layers of cells constituting the ventricle wall,

contrary to several layers of cells in Ets1+/+, Ets-2 T72A/T72A controls. There was also much

less trabeculation in the double mutant heart than that in control ones (Fig 4.3).

The abundant diluted vessel phenotype of Ets1-/-, Ets-2 T72A/T72A embryos were

similar to several mutant mice with angiogenesis defects, including Tie-2 and Ang-1

knockout mice (Dumont et al, 1994, Sato et al, 1995, Suri et al, 1996), over-dose of

VEGF mice (Drake and Little 1995) and PDGF-B or PDGFR-β knockout mice (Leveen

et al. 1994; Soriano 1994).To test whether angiogenesis was impaired in the Ets1-/-, Ets-2

T72A/T72A embryos, they were whole-mount immuno-stained with an endothelial cell marker, PECAM (CD31), to visualize the vascular system. Ets1+/+, Ets-2 T72A/T72A litter

mate embryos were used as control. There was a striking difference in blood vessel

density between embryos of the two genotypes. These mice with double mutations had

large, diluted blood vessels and lacked the characteristic branching of the vascular system

that occurs in control embryos (Fig4.4). In some regions, the vessels were uniformly

dilated and there was no clear distinction between large vessels and smaller vessels as

seem in control embryos (Fig4.4, C, D, E and F). This defect was a general defect

throughout the body, similar to that in Tie-2 or Ang-1 knockout mice. Although

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harvested and stained exactly at the same time and conditions, there was less intensity

and sharpness, but more background of staining in the double mutant mice than in the

control ones. The fuzzy staining in double mutant mice was unlikely due to total PECAM

level, because PECAM was expressed at similar level in the double mutant mice and their

control littermates (Figure 4.5). In some regions, specked PECAM staining was evident

(Fig 4.4 F), indicating that some endothelial cells might be able to migrate out (and

proliferate) but not able to form tube structures. It is worth pointing out that those Ets1-/-,

Ets-2 T72A/T72A embryos shown in Fig 4.4 had not shown the massive enlarged blood vessel phenotype and thus indistinguishable from the control mice before staining.

Therefore, the angiogenesis defects preceded the massive enlarged blood vessels appearance, and was likely to be the direct cause of the red appearance of the double mutant mice.

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l (B). The less h mate contro litter ) and muc the left ventricle region of A w 72 o r 72A/T T e wall (ar l , Ets-2 c i -/- r t A and B were sections of d by simple ven controls (A). e z i 72A r e t 72A/T T mice embryo (A) and Ets1 72A , Ets-2 72A/T +/+ mice had heart defects. T t (B) was charac , Ets-2 T72A/T72A +/+ 1 s t E mice hear

5 , Ets-2 . 72A 3 -/- 1 E

72A/T Ets1 T m

o r f

t r a , Ets-2 e -/- h

e h Ets1 Figure 4.3. t trabeculation (arrowhead) than Ets1

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Figure 4.4. Ets1-/-, Ets-2 T72A/T72A embryos had defects in vessel branching and reduced vascular complexity. The mouse embryos were whole-mount immunohistochemistry stained with anti-PECAM antibody to show the vascular system. Ets1-/-, Ets-2 T72A/T72A embryos (B, D, F and H) lacked the characteristic branching of the vascular system that occurs in control embryos (arrows and arrow heads) and have much less vessel network complexity than Ets1+/+, Ets-2 T72A/T72A control littermate (A, C, E and G). In some regions, the vessels in the double mutant mice were uniformly dilated and there was no clear distinction between

154

large vessels and smaller vessels (D, F, long arrows) as seem in control embryos (C,E, long arrows and narrow arrows). A and B were side view of the head. C and D were the top view of the mid-brain region. E and F were the lateral region between front and back limbs. G and H were the vertebrate region. E and F were 11.5 day embryos, and others were 10.5 day embryos.

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4.2.4. Gene expression in embryos was affected by Ets-1 and Ets-2 mutations.

Real time PCR analysis was used to analyze expression of potential ets-target

genes in the mutant embryos. Because the double mutant mice had severe angiogenesis

defects, we focused on the expression of genes related to angiogenesis. In Ets-1-/- mice,

the expression of none of the genes analyzed was significantly effected (Fig 4.5).

Although Ets1+/+, Ets-2 T72A/T72A mice are phenotypically normal, the expression of some

genes were down regulated in those embryos, such as Bcl-2, Bclx and eNOS. For

example, there was no significant difference of the mRNA levels of Bcl-2 or Bclx

between Ets1+/+, Ets-2 T72A/T72A mice and Ets1-/-, Ets-2 T72A/T72A mice, but Bcl-2 or Bclx

mRNA levels were less than half of those in Ets-1-/- mice or wild-type mice. There are

some genes whose expression was not affected by Ets-2 mutation alone, but was only

reduced in Ets1-/-, Ets-2 T72A/T72A double mutant embryos. Those genes included receptor

ligand (Ang1), RTK (Tie2), cell cycle regulatory genes (CDC2 and Cyclin D),

macrophages specific genes (fms, lysozyme, MSR1 and TNFα), transcription factor (Fli-

1), extracellular proteases (MMP1, MMP14), and other genes (HIF1α). For example,

Ets1-/-, Ets-2 T72A/T72A embryos expressed less than half of Tie2 transcripts compared to mice with other three genotypes. The ligand of Tie2, Ang1, was also expressed less than half in the double mutant embryos. Fli-1 was expressed at least 3-fold less in Ets1-/-, Ets-2

T72A/T72A embryos compared to control mice. The levels of BRCA1 transcripts in Ets1-/-,

Ets-2 T72A/T72A embryos were only about one-third of those in control animals. TNFα,

which is normally primarily produced by macrophages, and three macrophages marker

genes, fms, lysozyme and MSR1, were all expressed at much reduced levels.

Surprisingly, contrary to the down-regulation or unchanged expression of other genes, 156

VEGF was expressed at a significantly higher level in double mutant mice, with 6-8 fold higher in Ets1-/-, Ets-2 T72A/T72A embryos than in littermate controls.

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Figure 4.5. Ets-1 and Ets-2 mutations affected target gene expression in mutant embryos. The graphs shows the expression of the following genes grouped by their functions: A and F, Growth factors; B and G, Receptors; C and H, Genes control cell cycle and survival; D and I, Macrophages genes; E and J, Other genes. Total RNA was harvest from one embryo for each genotype at E10.5 days or E11.5 days, to compare the gene expression level by real time PCR. A-E were real time PCR results from E10.5 days embryos. F-J were real time PCR results from E11.5 days embryos. Error bars indicate the standard deviation of two measurements.

Continued on next page 158

Fig.4.5. Continued.

Continued on next page 159

Fig.4.5. Continued.

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4.2.5. Ets-1 and Ets-2 mutations reduced the expression of matrix proteinases in

embryo fibroblasts.

Stromal fibroblasts secret matrix proteinases that help tumor cells metastasis.

There are reports that Ets-1/2 correlates with MMP1, MMP9 expression. Several ets

factors are able to activate matrix proteinases expression in transient transfections, where

those Ets factors are overexpressed. However, there is no direct evidence to identify

which Ets proteins are responsible for the activation of those genes in vivo. Since the

double mutant mice are embryonic lethal, it is impossible to get tumor stromal fibroblast

from them. Therefore, we used embryonic fibroblasts to monitor gene expression. Mouse

embryonic fibroblasts (MEFs) were isolated from Ets1-/-, Ets-2 T72A/T72A mice embryo and

Ets1+/+, Ets-2 T72A/T72A littermate mice embryo at E12.5. Total RNA was extract from

fibroblasts at passage 2. Quantitative Real time PCR was used to measure gene

expression in those cells. MEFs from Ets1-/-, Ets-2 T72A/T72A mice expressed significant

lower levels of MMP1, MMP3, MMP9 and uPA than MEFs from Ets1+/+, Ets-2 T72A/T72A

mice. Especially, in MEFs from Ets1-/-, Ets-2 T72A/T72A, MMP3 and MMP9 were

expressed less than one sixth of that in cells from Ets1+/+, Ets-2 T72A/T72A mice (Fig.4.6).

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Figure 4.6. Matrix proteinases expression was reduced in Ets1-/-, Ets-2 T72A/T72A mice embryonic fibroblasts. Total RNA was harvest from MEFs at passage 2 to compare the gene expression level by real time PCR. The average result of two experiments is represented. Error bars indicate the standard deviation of two measurements.

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4.3. Discussion

Ets-1 knockout mice are viable and fertile. Tetraploid rescued ets-2-/- mice are

also basically normal, only with minor defects. Mice homozygous for the ets-2T72A

mutation are also phenotypically normal and indistinguishable from wild type mice.

However, Ets1-/-, Ets-2 T72A/T72A mice were embryonic lethal, with distinct cardiovascular

and vessel complexity defects, indicating that ets-1 and ets-2 were crucial for embryo development, and there was genetic redundancy between the two genes.

Genetic interactions between Ets proteins and Tie2 and Ang1.

The angiogenesis and heart defects in the double mutant mice are similar to what observed in Tie2 knockout mice or Ang-1 null mice. They all had decreased sprouting, simplification of vessel branching and similar heart defects (Dumont et al. 1994; Sato et al. 1995; Suri et al. 1996). Although Tie2 deficient mice died earlier than the ets-1/2 double mutant embryos, Tie-2 was shown to be required in the microvasculature during late organogenesis and in essentially all blood vessels of the adult (Puri et al. 1999).

Tie2 promoter has Ets binding sites which are necessary for Tie2 expression

(Dube et al. 1999; Iljin et al. 1999; Hart 2000a; Gaspar et al. 2002; Minami et al. 2003).

Several reports attribute ets proteins as regulators of Tie2 expression. Ets-1, Ets-2, Elf-1,

Fli-1 and NERF are all expressed in endothelial cells and able to activate Tie-2 gene expression in transient transfections (Dube et al. 1999; Iljin et al. 1999; Hart 2000a;

Gaspar et al. 2002). However, it is not clear which Ets proteins play a major role in Tie2 regulation. As mentioned before, the Ets-1/Ets-2 double mutant mice displayed similar phenotypes as in Tie2 knockout mice. Furthermore, Tie-2 or Ang1 mRNA level in the 163

double mutant mice was only half of that in the control mice (Fig 4.5). Therefore there

may be close genetic relationships among ets-1/2 and Tie2 and Ang1, and Tie2 and Ang1

may be direct targets of Ets-1/2 action. The combination reduction of Tie2 and Ang1

level may lead to the compromised vascular complexity. Alternatively, ets-1/2 may

regulate Tie2 expression through Fli-1. Fli-1 is shown to be regulated by Ets-1 (Lelievre

et al. 2002), and was expressed at a much reduced level in ets-1/2 double mutant embryos

(Fig. 4.5). Disruption of Fli-1 also leads to reduced Tie2 expression (Hart et al. 2000a).

Chromatin immunoprecipitation experiments with endothelial cells may define which Ets

factor(s) is (are) binds to Tie2 promoter and directly regulates its expression.

Ets-1, Ets-2 and RTKs interactions in angiogenesis.

Ets-1/2 activity is regulated by signal transduction pathways. For example, Ets-1

and Ets-2 are activated by phosphorylation at a conserved residue by MAPK in response

to Ras signaling, which is activated by RTK upon ligand binding. However, some RTK

or their ligands contain Ets binding sites at their promoters and possibly regulated by Ets

proteins. The expression of some RTKs (e.g., Tie1 and Tie2) and their ligands (eg., Ang-

1) were restricted in ets-1/2 double mutant mice (Fig 4.5, Table 4.3) and could be direct

target genes of Ets-1/2 action. However, there was no significant changes of several other

RTKs (such as Flt) or ligands (e.g. bFGF, PDGFB and TGFβ) in ets-1/2 mutant embryos

(Fig 4.5, Table 4.3), indicating that they might be upstream of ets-1/2 and they were not ets-1/2 target genes, or there was further redundancy between ets-1/2 and other ets factors.

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VEGF was overexpressed in the ets-1/2 double mutant embryos (Fig. 4.5, Table

4.3). VEGF has reported to be able to induce Ets-1 expression in endothelial cell

(Iwasaka C et al. 1996; Sato et al. 2001). VEGF also induces the expression of Ets target genes, such as MMP-3, MMP-9 (Iwasaka et al. 1996; Sato et al. 2001). Ets-1 antisense oligonucleotides inhibits VEGF-induced endothelial cell migration (Chen et al. 1997).

Ets-1 or Ets-2 does not change VEGF expression in a transient transfection system (Man et al. 2003). Therefore, VEGF might be upstream of Ets-1 and Ets-2. The absence of Ets-

1 and the phosphorylation form of Ets-2 may block VEGF signaling, and lead to downregulation of some Ets target genes in the double mutant mice. Insufficient expression of some of these genes may trigger the overexpression of VEGF. However, expression of some Ets target genes were not rescued by overexpression of VEGF, indicating that Ets-1 and Ets-2 may be key effectors of VEGF signaling (Fig 4.7). Furthermore, the overexpression of VEGF may cause further problems, such as abnormal heart structure or vessel fusion. Hypoxia condition was reported to lead to VEGF overexpression

(Ylikorkala et al. 2001). Since the double mutant mice had sparse vascular network, the cells located too far away from blood vessel may not get much access to oxygen and nutrients and suffer hypoxia, which induces VEGF expression. Thus the overexpression of VEGF may be a secondary effect in the double mutant mice.

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s Et EBS: e mutant mice embryos. pression in ets-1/2 doubl tors ex their recep factors and inding site. Table 4.3. Some growth b

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Figure 4.7. Model for the role of Ets-1 and Ets-2 in angiogenesis. See text for detailed description.

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Endothelial cells or pericytes.

Development of a vascular system involves the assembly of two principal cell

types, vascular endothelia cells and peri-vascular supporting cells, including pericytes

and smooth muscle cells, into many different types of blood vessels. Ets1-/-, Ets-2

T72A/T72A mice embryos had numerous dilated blood vessels. This phenotype is also

present in PDGF B, PDGFRβ deficient mice (Leveen et al. 1994; Soriano 1994),

although PDGF B, PDGFRβ deficient mice die at a much later time. PDGF and PDGFR

are critically involved in the recruitment of pericytes to embryonic blood vessel (Lindahl

et al. 1997; Hellstrom et al. 1999). Tie2 knockout mice also have defects in recruiting

pericytes. Tie2 was implicated to control the capability of endothelial cells to recruit

stromal cells to encase the endothelial tubes so as to stabilize the structure and modulate

the function of blood vessels (Dumont et al, 1994, Sato et al, 1995, Hanahan, 1997, Ward

and Dumont, 2002). Thus Ets-1/2 might also regulate pericytes recruitment to newly

formed vessel tubes. Immunohistochemistry with pericyte or smooth muscle markers

(Desmin or smooth muscle actin, respectively) will tell us whether Ets1-/-, Ets-2 T72A/T72A

mice have normal pericytes surrounding the endothelial tube. Since there is a complicated

interaction loop between endothelial cell and pericytes, it is hard to tell whether the phenotype we observed was due to autonomous endothelial cell defects. The ets-2floxed

allele (Chapter 6) is being introduced to mice harbor Tie-2 cre transgene (Kisanuki et al.

2001). The Tie-2 promoter can drive endothelial specific expression of cre in the transgenic mice. Preliminary experiments demonstrated that mice with endothelial cell specific deletion of ets-2 in ets-2-/- background were embryonic or perinatal lethal,

indicating the endothelial function of ets-1/2 is required for angiogenesis and embryo 168

development. Further characterization of the ets-1-/-, ets-2floxed/T72A, Tie2-cre mice will

dissect the endothelial specific roles of Ets-2 in angiogenesis and embryo development.

An alternative way to study endothelial cell autonomous gene expression

alteration is to obtain endothelial cells from double mutants mice and culture them ex

vivo. Tie2- GFP mice (Motoike et al, 2000) express GFP specifically in endothelial cells.

They were mated with mice harboring ets-1 knockout allele and ets-2T72A allele. Ets1+/-,

Ets-2 T72A/T72A, Tie2-GFP mice were generated. Brother-sister mating of these mice can yield Ets1-/-, Ets-2 T72A/T72A, Tie2-GFP mice. Endothelial cells from these mice can be

sorted out by flow-cytometry and cultured in the absence of pericytes. Gene expression,

and the ability to assemble into tube structure, can be examined in those cells with or

without exogenous growth factors.

Embryo or placenta.

Ets-2 plays an important function in placenta. Ets2-/- mice or Ets-2T72A/- mice

have defected placenta (Yamamoto et al. 1998; Man et al. 2003). However, there was no

major visible structure defect in the placenta of the double mutant mice (data not shown).

It is possible that the placenta from these mice have defects in secreting some growth

factors important for embryo development. To further define the function of ets-2 in

placenta and embryo development, and test whether the ets-1/2 double mutant defect are

due to embryo autonomous defects, or a secondary defect from the placenta defect,

conditional ets-2 knockout allele (Chapter 6) can be deleted specifically in embryo by

Mox-2-cre in ets-1-/- background, or deleted in placenta specifically by trophoblast

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specific cre expression. Characterization of these mice will further define the biological roles of ets-1/2.

Ets-1, Ets-2 and macrophage differentiation.

The mRNA level of all three macrophage markers (fms, lysozyme and MSR1) were much lower in Ets1-/-, Ets-2 T72A/T72A embryos than in control animals. TNFα was primarily secreted by macrophages (Chapter 3). There was much reduced TNFα mRNA in Ets1-/-, Ets-2 T72A/T72A embryos. Therefore, Ets-1 and Ets-2 might affect hematopoiesis, especially macrophage differentiation. It has been reported that ets-2 was not required for macrophage differentiation from ES cells (Henkel et al. 1996). However, ets-1 was intact in that case, and ets-1 and ets-2 may play redundant roles in hematopoiesis. Alternatively, ets-1/2 is required for macrophage proliferation and survival. Colony formation assays using the hematopoietic precursor cells from the ets-1/2 double mutant mice may define the roles of ets-1/2 in hematopoiesis. Macrophages are involved in drilling of metalloelastase-positive tunnels to facilitate the endothelial cell migration step in angiogenesis (Moldovan et al. 2000), or can serve as pericytes (Thomas 1999). Therefore, deficiency of macrophages may also contribute to the angiogenesis defects in the double mutant embryos.

Implications of Ets-1 and Ets-2 functions in cancer.

This chapter demonstrated that Ets-1 and Ets-2 were required for angiogenesis and embryo development. Tumor angiogenesis and embryo angiogenesis share similar mechanisms and players (Papetti and Herman 2002). Therefore, one immediate 170

implication of the functions of Ets-1 and Ets-2 in tumor is in tumor angiogenesis.

Furthermore, ets-1-/-, ets-2T72A/T72A mice also had less number of macrophages and

reduced extracellular matrix proteases expression in fibroblasts. Fibroblasts, macrophages

and vascular cells are important components of tumors, and play crucial roles in tumor

development. The results from this chapter suggested that ets-1 and ets-2 are important in cancer, especially in tumor angiogenesis and metastasis. Further experiments with the ets-2 conditional allele would define the role of Ets-2 in each cell type in tumorgenesis

(see Chapter 7 for a detailed discussion).

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CHAPTER 5

ETS-2 AS A TRANSCRIPTIONAL REPRESSOR

5.1 Introduction

Previous chapters described the in vivo function of Ets-2 phosphorylation by genetics methods. This chapter is about biochemical approaches to get out the role of Ets-

2 phosphorylation. Ets proteins, modulated by discrete signal transduction pathways, are able to form complexes with other transcription factors, coactivators or corepressors.

Such interactions may strengthen the transactivating activity and/or define target gene specificity (Chapter 1). For example, Ets-2, at different phosphorylation state, associates with different set of nuclear proteins (Baker et al. 2003).One major group of proteins associated with Ets proteins encompasses proteins involved in chromatin structure.

Chromatin and gene expression.

Eukaryotic gene expression can be viewed within a conceptual framework in which regulatory mechanisms are integrated at two levels. The first is the sequence level, i.e. the linear organization of transcription units and regulatory sequences. The second is the chromatin level, that allows switching between different functional states.

Biochemical and genetic findings have established that the condensation of eukaryotic

DNA in chromatin functions not only to constrain the genome within the boundaries of the cell nucleus but also to suppress gene activity in a general manner. This genetic 172

repression extends from the level of the nucleosome, the primary unit of chromatin organization, where coiling of DNA on the surface of the nucleosome core particle impedes access to the transcriptional apparatus, to the higher order folding of nucleosome arrays and the organization of silent regions of chromatin (Horn and Peterson 2002; Tyler

2002).

Chromatin structure is inextricably linked to transcriptional regulation, and recent studies show how chromatin is perturbed so as to facilitate transcription. Switching between a state that suppresses transcription and one that is permissive for gene activity involves changes in chromatin structure that are controlled by the interplay between histone modification, DNA methylation, and a variety of repressive and activating mechanisms. This regulatory level is combined with control mechanisms that switch individual genes on and off, depending on the properties of the promoter (Luo and Dean

1999; Fyodorov and Kadonaga 2001; Tyler 2002).

Acetylation occurs at specific lysines in the flexible N-terminal histone tails that protrude from the nucleosome surface. Hyperacetylation of histones is associated with transcriptional activity or the potential for transcriptional activity, whereas histone hypoacetylation is correlated with transcriptionally silent chromatin and heterochromatin.

Histone acetylation is catalyzed by histone acetyltransferase (HAT), while histone deacetylase (HDAC) removes the acetyl modification of the histones (Luo and Dean

1999; Goodsell 2003).

Chromatin can also be switched between an active state and a repressed state by

ATP-dependent chromatin remodeling complexes (Suharsanam and Winston 2000;

Fyodorov and Kadonaga 2001). Genetic and biochemical evidence demonstrate that the 173

products of the SWI/SNF genes, first defined in Saccharomyces cerevisiae as co- activators of gene expression, form a complex with the ability to remodel chromatin. The complex has ATP-dependent chromatin remodeling activity and can alter the conformation of the nucleosome core in a reversible fashion. The SWI/SNF complex is conserved in mammals. Brg1, the ATPase hydrolyzing subunit, is an essential component in this complex (Harbour and Dean 2000). Brg1 can interact with specific protein partner and regulate gene expression. For example, Brg-1 interacts with the retinoblastoma tumor suppressor protein (Rb) and is required for E2F/Rb-mediated repression of gene expression (Harbour and Dean 2000; Strobeck et al. 2000; Wang et al. 2002). Yeast

Swi/Snf complex can controls the chromatin structure of the SER3 promoter and directly repress transcription of the SER3 gene. This repression depends primarily on one Swi/Snf component, Snf2 (Martens and Winston 2002). Therefore, SWI/SNF appears to act as both co-activator and co-repressor, depending on the interaction partner and chromatin context.

Regulation of Ets-2 activity by signal transduction pathway and protein partners.

Two major mechanism regulating Ets proteins specificity and activity are modification of discrete family members by signal transduction pathways, and association with distinct protein partners (Chapter I). As described earlier, Ets-2 is phosphorylated at threonion 72 residue at the N-terminal of the Pointed domain by

Ras/MAPK pathways. The pointed domain appears similar to domains in other transcription factors, for example in the cAMP responsive enhancer-binding protein, which are regulated by phosphorylation-dependent protein-protein interactions with 174

transcription co-activators (Kwok et al. 1994). Some Ets factors containing Pointed

domain, such as TEL, can act as repressors of gene expression, and the Pointed domain

has been implicated in this activity (Wang and Hiebert 2001). Thus, whereas Ets-1 and

Ets-2 have been considered to be activators of gene expression, it is possible that they

also repress target gene expression.

In breast cancer cells lines, the mRNA expression levels for some Ets-2 target

genes, such as uPA, MMP3/ and MMP14, are positively correlated with phospho-Ets-2

expression (Baker et al. 2003). The BRCA1 promoter also contains a consensus ets- binding site conserved in both mouse and human promoters. However, BRCA1 mRNA level is negatively correlated with phosphorylated Ets-2 in breast cancer cell lines.

Exogenous ets-2 represses the activity of a BRCA1 promoter-luciferase reporter, and this repression is dependent on a conserved ets-2-binding site in this promoter in MCF-7 cells. Conditional overproduction of ets-2 in MCF-7 cells results in repression of endogenous BRCA1 mRNA expression (Baker et al., 2003). Components of the mammalian SWI/SNF chromatin remodeling complex interact with ets-2, both in vitro and in vivo. The pointed domain of ets-2 directly interacts in vitro with the C-terminal region of Brg-1, the ATP-hydrolyzing component of the SWI/SNF complex, in a phosphorylation-dependent manner (Baker et al., 2003).

Therefore, signaling pathways may regulate Ets-2 activity by altering its association with different classes of nuclear proteins. In response to various signal transduction pathways, Ets-2 may interact with either co-repressors or co-activators in a phosphorylation-dependent manner, and affect either repression or activation of target genes. 175

5.2 Results

Brg1 and Ets-2 cooperatively repressed BRCA1 expression.

To test the functional significance of the Brg-1/ets-2 interaction, the effects of

these nuclear factors on the activity of the BRCA1-luciferase reporter were studied. For

these experiments, the tumor cell line SW13, which lacks detectable Brg-1 and Brm-1

proteins (Strobeck et al. 2000), was used. First, the BRCA1-luciferase reporter was

introduced into cells with the combination of expression vectors for ets-2 and Brg-1 in

transient transfection assays. The results of the experiments, expressed as fold-repression,

indicated that neither expression vectors for ets-2 nor Brg-1 alone repressed the BRCA1 reporter. However, the combination of the two resulted in an approximate 3-fold repression of reporter activity (Fig. 5.1 A). If an expression vector for a Brg-1 gene encoding a protein with a mutation in the ATP-binding domain, Brg-1 (K798R), was used in the assay, repression of the BRCA1 reporter was not observed in either the presence or absence of ets-2 (Fig. 5.1 A). Because transient DNA templates may not always be organized as chromatin (Fryer and Archer 1998; Hager 2001), we constructed

SW13 cells with stably integrated BRCA1-luciferase reporter genes. The cells were used in assays to detect Ets-2 and Brg1 action on BRCA1 promoter (Fig. 5.1B). In these experiments, ets-2 expression by itself had little effect on integrated reporter gene expression. Brg-1 expression alone could repress the BRCA1 reporter ~7-fold, whereas the combination of ets-2 and Brg-1 resulted in a further dose-dependent repression of

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BRCA1 promoter activity (Fig. 5.1B). A 14-fold reduction was observed with the higher concentration of ets-2 expression vector. As in the transient assays, Brg-1 (K798R) did not repress BRCA1 reporter activity alone or in combination with ets-2 (Fig. 5.1B).

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Figure 5.1. Brg-1 and ets-2 repressed BRCA1 promoter reporter in SW13 cells. A, transient transfections performed in SW13 cells. 5 µg of human BRCA1-luciferase reporter was co-transfected with 100 ng of expression vector for ets-2 and 250 ng of expression vector for Brg-1, or Brg-1 K798R, or with a combination of 100 ng of ets-2 vector and 250 ng of Brg-1, or Brg-1 K798R vectors, as indicated (left bar graph). B, transfection with stably integrated BRCA1-luciferase reporter. For these experiments, 1 µg of Brg-1 or Brg-1 K798R alone or in combination with ets-2 (0.2 or 1 µg, as

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indicated) were transfected into SW13 cells that contained stably integrated copies of the BRCA1-luciferase reporter. For both panels A and B, fold-repression is the ratio of relative luciferase activity for the BRCA1-luciferase reporter alone (with empty expression vectors) to the activity in the presence of ets-2, Brg-1 (Brg-1 K798R), or a combination of both ets-2 and Brg-1 (Brg-1 K798R). Results of three independent experiments performed in duplicate are presented. Error bars indicate standard deviation of the measurements.

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Ets-2 interacts with the co-repressor BS69 through the pointed domain.

In order to find other proteins that interact through the "Pointed" domain to modulate Ets-2 transcription activity, a yeast two-hybrid screen was carried out using human ets-2 pointed (amino acid residues 67-170) as bait. A mouse embryonic library

(E8.5 and E9.5) with insert sizes selected at 500-800 bp fused to the gal4 activation domain was screened (Hollenberg et al. 1995). About 200,000 colonies (approximately

1/3 of the library) were screened initially, and 16 interacting clones were isolated and shown to be specific ets-2 interactors. Of these clones, four encoded BS69, a known adenovirus E1A interacting protein that is a repressor of gene repression (Hateboer et al.

1995; Masselink and Bernards 2000). The clones isolated encoded for a region in the C- terminal domain of BS69 with the minimal region represented corresponding to amino acid residues 435-522, which includes the MYND domain known to be essential for repression (Hateboer et al. 1995; Masselink and Bernards 2000).

To determine if the ets-2 pointed/BS69 interaction were direct, we next studied the interaction of the proteins in GST-pulldown assays (Figure 5. 2 A). In these experiments, the terminal 150 amino acids of BS69 were produced as a GST-fusion protein. This BS69 fusion protein specifically interacted with his-tagged ets-2 pointed region, while GST alone did not (Figure 5.2A). In contrast, if threonine 72 residue within the his-tagged ets-2 pointed domain were first phosphorylated using recombinant Erk-1 kinase in vitro, less than 1% of the radioactively tagged, phosphorylated ets-2 pointed peptide was found in the GST-BS69 pellet (Figure 5.2.B). Western analysis confirmed that unphosphorylated ets-2 present in the labeled mixture still formed a complex with

GST-BS69 (Figure 5.2.B). 180

BS69 has been shown to directly interact with the co-repressor N-CoR, and this interaction is necessary for BS69 repressor activity (Masselink and Bernards 2000). Thus we wished to determine if ets-2 and N-CoR could be found in a common complex. COS cells were co-transfected with vectors for N-CoR and Flag-tagged ets-2 (Fig 5.2C). The material expressed was precipitated with an N-CoR antibody, and the presence of ets-2 in the N-CoR complex was determined by western blotting using an anti-Flag antibody (Fig

5.2 C). This experiment indicated that N-CoR and ets- 2 were in a common complex.

BS69 is constitutively expressed in most cell types, including COS cells (Hateboer et al.

1995; Masselink and Bernards 2000), and thus it is difficult to demonstrate with certainty a requirement for BS69 in the ets-2/N-CoR interaction in this type of assay.

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Figure 5.2. Phosphorylation-dependent direct interaction between BS69 and Ets-2 in vitro. A. Illustration of BS69 protein. B. Recombinant GST-BS69 (1 µg) was immobilized on GST beads and then incubated with recombinant, unphosphorylated 6X-hhis tagged pointed domain of ets-2 (1 ug). Protein bound to beads was analyzed by SDS PAGE and western blotting, using GST or 6X-His specific antibodies as indicated. Lane 1: GST only +ets-2 pointed; Lane 2 GST-BS69 + unphosphorylated ets-2; Lane 3: 50% of input of ets- 2. C. Pulldown assay with GST-BS69 and 6X-his ets-2 pointed phosphorylated by Erk using 32P γ- ATP. Upper panel is an autoradiograph to detect 32P labeled ets-2 pointed. Radioactivity was quantified using a Molecular Dynamics Phosphoimager. Two lower panels are western blots probed with GST and 6X-his antibodies, respectively. Lane 1: GST only + 32P-labeled ets-2 pointed; Lane 2 GST-BS69 + 32P-labeled ets-2; Lane 3: 10% of input of 32P -labeled ets-2. D. Expression vectors for N-CoR and Flag-tagged ets- 2 were co-transfected in COS cells. NCoR was immunoprecipitated from whole cell 182

extracts and ets-2 in the immune complex detected by western using anti-Flag antibody. Lane 1: Western of total extract with anti-Flag antibody; lane 2: N-CoR immunoprecipitate probed with anti-Flag antibody; Lane 3: Nonimmune serum control probed with anti-Flag antibody. Upper arrow indicates position of Flag tagged ets-2; Bottom arrow is position of rabbit IgG.

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Ets-2 and BS69 repressed expression of BRCA1 and uPA-luciferase reporter.

To determine if the ets- 2/BS69 interaction was functional, we studied the action of these factors in combination on the expression of two ets-target genes. One was a well- defined ets-2 target, the urokinase plasminogen activator (uPA) promoter/enhancer, the second was the human BRCA1 proximal promoter region. Because BS69 was reported to be associated with NCoR/HDAC complex, its action may be involved in modulating chromatin structure. Since transiently transfected DNA does not form proper chromatin structure, we cloned uPA promoter and BRCA1 promoter into a pREP4-luciferase episomal vector (Liu et al. 2001a; Liu et al. 2002). pREP4 is an episomal mammalian expression vector that contains the Epstein-Barr virus replication origin and a nuclear antigen to permit nucleosome structure and extra-chromosomal replication in mammalian cells (Yates et al. 1985). For these experiments, the luciferase reporters were co- transfected with ets-2 or BS69 alone, or with the combination of the two, into the tumor cell line SW13 (Figure 3). This analysis showed that ets-2 could activate the uPA reporter in these tumor cell line about 2.5-fold. However, the addition of BS69 resulted in significant repression: 8-fold repression of the uPA reporter compared to the activity in the presence of ets-2, and about 3-fold lower than the basal level activity of the reporter

(Figure 3A). Similar results were obtained with the BRCA1 reporter, with 8-fold repression over ets-2 alone, and 3-fold repression of the basal activity of the promoter

(Figure 3B). BS69 alone had little significant effect alone on either on the uPA-luciferase or BRCA1-luciferase reporter (Figure 3).

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Figure 5.3. BS69 and ets-2 repressed uPA and BRCA1 reporter genes in SW13 cells. Transient transfections performed in SW13 cells. 1 µg of mouse uPA-luciferase reporter (Panel A) or human BRCA1-luciferase reporter (Panel B) were co-transfected with 1µg of expression vector for ets-2 alone, 1µg of BS69 alone, or the combination of both (1µg each), as indicated. Relative luciferase activity (see Materials and Methods) is presented. Results of two independent experiments performed in duplicate are presented. Error bars indicate standard deviation of the measurements.

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5.3 Discussion

Ets-2 activity is regulated by signal transduction pathways and protein partners.

The results presented here provide one potential molecular explanation for Ets-2

activity and specificity. The pointed domain of ets-2 interacted with distinct sets of

proteins depending on phosphorylation of residue threonine 72. Brg-1, the ATPase

subunit of a chromatin-remodeling complex, directly interacts only with

unphosphorylated ets-2, at least in vitro (Baker et al., 2003). BS69 also interacted with

unphosphorylated Ets-2 in vitro (Fig 5.3). The fact that pools of both phosphorylated and non-phosphorylated ets-2 are present even in cells that have high activation of ras signaling pathways makes it difficult to determine whether the interaction with Brg-1 or

BS69 is strictly phospho-specific in vivo. Despite this caveat, our results still suggest that phosphorylation-specific interactions with distinct sets of nuclear factors partially

accounts for the specificity of ets-2.

BS69 is a potent repressor of gene expression and contains the MYND domain

located at the C-terminal of the protein (Hateboer et al. 1995; Masselink and Bernards

2000). The MYND domain is predominantly found in proteins associated with chromatin

and is indicated to mediate transcriptional repression (Ansieau and Leutz 2002). Recently, it has been shown that the MYND domain of BS69 can recognize a conserved five amino-acid sequence in several proteins (Ansieau and Leutz 2002). This sequence is also present in Ets-2, and more interestingly, it coincides with the phosphorylation site of Ets-

2 (Fig 5.4). Therefore, phosphorylation of Ets-2 may interfere with Ets-2-BS69 interaction, and relieve the repression activity.

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E1A (Ad5, 112) MPNLVP E1A (Ad12, 112) MPQLHP EBNA2 (EBV A, 382) MPELSP EBNA2 (EBV A, 436) APILFP EBNA2 (EBV B, 349) MPELSP EBNA2 (EBV B, 403) PPFLFP EBNA2 (HVP, 420) MPQLSP EBNA2 (HVP, 481) PPILFP hMGA(2927) MPKLTP hMGA (2952) MPALAP Ets-2 (68) LPLLTP Ets-1 (34) VPLLTP Pointed P2 (147) LPPLTP

Figure 5.4 Ets-2 contains a consensus PXLXP motif. The alignment of the PCLXP motif of several proteins shows that Ets-2 and its close relative Ets-1 and Drosophila Ets factor pointed P2 have the consensus BS69 binding motif. The number in the figure indicates the start location of this motif within the protein

Chromatin dependent regulation of transcription.

Ets-2 has previously been characterized as an activator of gene expression, and

the results presented in this chapter indicate that this factor can also act as a repressor.

Ets-1 and Ets-2 recruit transcription adapter proteins p300 and CBP (cAMP-responsive element-binding protein) during the transcriptional activation of the human MMP-3 promoter (Jayaraman et al. 1999). The Ets-2-Brg1 or Ets-2-BS69 complexes behaved as transcriptional repressors. Ets-2 was shown to be in the same complex as Brg1 and other

Brg1 chromatin remodeling complex proteins, such as BAF57/p50 and Ini1 (Baker et al.,

2003). The Brg1 complex was shown to activate or repress gene expression (Liu et al.

2001a). We were also were able to demonstrate that ets-2 and N-CoR, known to associate with multiple histone deacetylase complexes (Huang et al. 2000), could be detected in the 187

same complex when overexpressed in COS cells. CBP, p300, Brg1 and BS69 all have

bromodomain and are found (directly or indirectly) to be associated with chromatin.

Bromodomain is an approximately 110-amino-acid module found in many chromatin-

associated proteins and it can interact specifically with acetylated lysine in the histone tail

(Dhalluin 1999). Therefore, both transcriptional coactivator (e.g. p300, CBP) and corepressor (e.g. Brg1, BS69) can interact with Ets-2 in a chromatin environment and modulate its activity.

Defining the exact composition of the Ets-2/BS69 complex, and demonstrating that this complex contains histone deacetylase activity, are important questions that need to be addressed by future work. However, our results suggest the following model

(Figure 5.5). Ets-2 may recruit Brg1/chromatin remodeling complex and/or BS69/HDAC complex to target genes resulting in their repression. Phosphorylation of ets-2 by the ras/Erk pathway will lead to lower concentrations of the Ets-2-Brg1 or Ets-2/BS69 complex, and subsequent activation of gene expression. In addition, phosphorylation- dependent recruitment of co-activators by Ets-2 can lead to increased expression of target genes. Whether all genes that are repressed by Ets-2 can also be activated following signaling events may depend on the context of the Ets binding site as well as the cell type being studied. Thus, some targets like BRCA1may be only repressed by ets-2, while others like uPA may be both repressed and activated following signaling. Future work will need to address the biological role of ets-2 as both repressor and activator in normal and transformed cells.

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Figure 5.5. Model for ets-2 as a signaling-regulated repressor and activator. A. Phosphorylated form of Ets-2 acts as a transcriptional activator. The target gene promoter region does not have compact chromatin structure. B. Non-phosphorylated form of Ets-2 binds to Brg1 chromatin remodeling complex and/or BS69/HDAC repressor

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complex. C. The chromatin remodeling complex and/or HDAC repressor complex turn the promoter region into compact chromatic structure, make it not accessible for transcriptional machinery, and repress gene expression. D. In response to Ras signaling, Ets-2 is phosphorylated. Phosphorylated Ets-2 does not interact with Brg-1 or BS69. Instead, Ets-2 interact with p300/CBP, which is a coactivator and has histone acetyltransferase (HAT) activity. E. Acetylation of histone renders the chromatin to an active (loose) state.

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Ets-2 and breast cancer.

Breast cancer is the second leading form of cancer in women in the United States.

New mechanisms by which the tumor suppressor BRCA1 might be down-regulated in mammary tumor cells are biologically significant. Germline mutations in BRCA1 account for approximately one-half of inherited breast cancers, but mutations of BRCA1 are infrequent in sporadic breast cancer (Ford et al. 1998). Several studies indicate that

BRCA1 expression is down-regulated in primary breast tumors versus normal breast tissue (Thompson and Jensen 1995; Magdinier et al. 1998; Rice et al. 1998). Aberrant methylation of CpG islands in the BRCA1 promoter may be one mechanism that leads to decreased gene expression in sporadic breast cancer(Rice et al. 1998). However, hypermethylation of the BRCA1 promoter region is only found in ~13% of sporadic breast cancer cases (Catteau et al. 1999; Bianco et al. 2000), suggesting that additional mechanisms may be involved in BRCA1 silencing. Ets-2 presents in two forms within cells: phosphorylated and unphosphorylated form. Phosphorylated Ets-2 used to be regarded as the only oncogenic form because it activates expression of some enzymes that promote tumor metastasis. However, the finding that unphosphorylated Ets-2 repressed the level of BRCA1 in breast cancer cells indicates unphosphorylated form of

Ets-2 as a potential oncogenic form. Thus Ets-2, as both a repressor of BRCA1 and an activator of uPA, MMP3, and MMP14 in a subset of breast cancer cases, provides an attractive model.

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CHAPTER 6

ETS-2 CONDITIONAL KNOCKOUT MICE

6.1 Introduction

Mice homozygous for the ets-2db1 allele (deletion of the Ets domain of ets-2)

(Yamamoto et al. 1998), and ets-1-/-, ets-2T72A/T72A mice are embryonic lethal. The

embryonic lethality hampered the study of Ets-2 in later development stages. Moreover, all the cells have the same deletion or mutation and it is hard to dissect the role of Ets-2 in different cell types in some complicated biological processes, such as cancer.

Therefore, conditional gene targeting strategy was used in this chapter.

Gene targeting is defined as the introduction of site-specific modifications into the genome by homologous recombination (Muller, 1999). Targeted gene disruption/modification in mice is a powerful tool for generating murine models for

human development and disease (Muller, 1999). Gene targeting is accomplished by

homologous recombination in embryonic stem (ES) cells. To achieve homologous

recombination, a replacement vector is linearized in such a way that the vector sequences

remain collinear with the target sequences. Chromosomal sequences are replaced by

vector sequences by a double crossover event involving the flanking homologous regions.

ES cell lines are derived from pluriopotent, uncommitted cells of the inner cell mass of

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pre-implantation blastocysts. When gene-targeted ES cells are injected into blastocysts, they can contribute efficiently to both somatic and germ-line tissues. Colonization of the germ line allows generation of intact animals with the desired genetic alteration (Muller,

1999).

Conventional gene targeting leads to inactivation or modification of a gene in all tissues of the body from the onset of development throughout the whole lifespan.

However, in many cases, complete gene inactivation leads to a lethal or otherwise adverse phenotype that prevents a more detailed analysis. If a given gene has a widespread pattern of expression, it is hard to distinguish the primarily and secondary affected tissue. Moreover, if a selection cassette is left in the mouse genome, the strong promoter driving the introduced selection maker may interfere with the expression of neighboring genes, and even cause lethality which is not present in the true knockout mice (Fiering et al. 1995; Scacheri et al. 2001). To avoid those problems, conditional knockout strategies, using cre-LoxP or Flp-FRT systems, have been developed.

The majority of the conditional knockout mice are generated with the Cre-LoxP system. Cre is the 38-kDa product of the cre (cyclization recombination) gene of bacteriophage P1. Cre recognizes a 34-bp sequence called loxP (locus of X-over of P1).

The loxP site consists of two 13-bp inverted repeats flanking an 8-bp nonpalindromic core region that gives the loxP site an overall directionality. Cre efficiently catalyzes reciprocal conservative DNA recombination between pairs of LoxP sites.

As its human orthologue, the mouse ets-2 gene has 10 exons (Figure 6.1). The first exon is non-coding. The second exon encodes 24 amino acids and begins with the start codon ATG. The pointed domain of Ets-2 is encoded by exon 4 and 5, and the Ets 193

DNA binding domain is encoded by exon 9 and 10. The first ets-2 targeting allele, ets-

2db1, referred as ets-2 knockout allele (ets-2- allele) in previous chapters, was generated by targeting 3’ end of exon 8, exons 9 and 10. This allele abrogates a nuclear localization signal and the DNA binding domain. However, the exons upstream of the Ets domain are still transcribed and fused to the neo transcript (Yamamoto et al., 1998). Therefore, ets-

2db1 has a deletion of a critical portion of the gene but does not result in a null allele. ets-

2db1/db1 mice are embryonic lethal before E8.5 from extraembryonic defects (Yamamoto et al. 1998). Ets-2T72A allele is a targeted knock-in allele that the conserved threonine 72 residue is mutated to alanine. As described earlier, this allele is possibly a hypomorphic allele. To generate a conditional null allele, the cre/LoxP system was used.

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6.2 Results

Mouse Ets-2 deletion strategy.

As mentioned earlier, the mouse ets-2 gene has 10 exons (Figure 6.1). The first exon is non-coding. The second exon begins with the start codon ATG and encodes 24 amino acids, which is not conserved in Ets-1. Deletion of ets-2 exon 3-5 will delete the conserved MAPK phosphorylation site and the pointed domain, which is important for protein-protein interactions. Moreover, this deletion creates a frameshift that generates multiple stop codons directly after exon 2 (Fig. 6.1). Therefore, this strategy shall yield a null allele. For convenience, this null allele was named ets-2∆3, because 3 exons from exon 3 were targeted.

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Figure 6.1. Illustration of ets-2 mRNA transcribed from different targeting alleles. Squares with numbers represent exons. Exons encodes the Pointed domain are colored with green, and exons encoding Ets DNA binding domain is depicted in red color. Arrow indicates translation start.

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Cloning of Ets-2 conditional knockout construct

A DNA fragment corresponding to mouse ets-2 exon 4 and 5 was used as a probed to screen RPCI-22 129 mouse BAC library (Osoegawa et al. 2000). One positive clone, 210JS, was identified. The BAC DNA was isolated and confirmed to have at least exon 2 to exon 10 of ets-2 through PCR and Southern blots (data not shown). Several portions of the BAC DNA were subcloned into pBluescript II, and then assembled into the Triple-lox targeting vectors (Fig. 6.2). The final construct (pLox-Ets-2) is illustrated in Figure 6.3. The integrity of this vector was confirmed by PCR, restriction mapping and

Southern blotting. All LoxP site and flanking region were confirmed by sequencing and in vitro recombination with purified Cre recombinase (Novagen) followed by PCR analysis (data not shown and Fig. 6.6). Flp-FRT is an alternative conditional knockout system for cre-LoxP. Ets-2 fragments were also cloned in the triple-FRT vectors in the same way as in the cre-LoxP system (data not shown).

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Figure 6.2. Outline of ets-2 conditional targeting vector cloning. A. sequence corresponding to 1kb upstream of EcoR I site located in intron 2 was amplified by PCR, and used as the short arm for homologous recombination. The central 4.3 kb region between EcoR I site in intron 2 and BamH I site in intron 5 was the targeted region, and was flanked by two loxP sites in the targeting construct. The 8.5 kb region between BamH I site in intron 5 and EcoR I site at 3’ end of the gene was used as the long recombination arm.

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Figure 6.3. Ets-2 gene targeting illustration. A. Targeting vector construct. B. Wild type ets-2 gene. C. After homologous recombination, ets-2 exons 3-5 are flanked by 5’ and middle loxP sites. Neo/tk cassette is flanked by middle and 3’ loxP sites. D. After transient Cre expression, neo/tk cassette is deleted and only the intended deletion region (exon 3 – 5) is marked in ES cells by the 5’ and 3’ loxP sites. E. Tissue specific expression of Cre will delete exon 3-5 of ets-2, result in a frameshift and generate a null allele. The position of PCR primers and Southern blot probe are also shown.

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Generation and selection of ES clones with homologous recombination.

The ets-2 gene targeting vector, pLox-ets-2, was linearized by Not I and used to

transfect ES cells. After 8 days of Neo selection, at least 400 ES clones appeared. 144

clones were picked and screened for the expected homozygous recombinants. To screen

for 5’ homologous recombination, a PCR reaction (94oC, 30 sec, 55 oC, 30 sec, 72 oC, 2 min, 35 cycles) was carried out with two primers (Fig. 6.4 A). Primer I2P5 is located in intron 2 and about 100bp upstream of the 5’ target construct homologous region (Fig 6.3

C). PGK Rev2 is located at 5’ of neo cassette at the reverse complementary direction (Fig

6.3 A,C). Positive clones were further screened for the integrity of the 3’ loxP site by a second PCR reaction (94oC, 30 sec, 55 oC, 30 sec, 72 oC, 1 min, 35 cycles) with two

primers (LoxNB and E6) (Fig. 6.4 B). Primer loxNB is located at the 3’ loxP site. Primer

E6 is located at exon 6 at the reverse complementary direction (Fig. 6.3 A, B and C). The

positive clones were further confirmed with a third PCR reaction (94oC, 30 sec, 60 oC, 30

sec, 72 oC, 1 min, 35 cycles) with two other primers (Ets2I5P2 and Ets2I5P3) that are

located in intron 5 and flank the 3’ loxP site (Fig. 6.3 A, B and C). This reaction

amplifies both the endogenous and targeted locus (Fig 6.4 C). The positive clones were

further studies and confirmed to be correct by Southern blotting (Fig 6.4 D). ES genomic

DNA was digested with Bam HI. A 0.5 kb PCR product, corresponding to exon 2 and

about 460 bps downstream of exon 2 in intron 2, located upstream of the target construct

(Fig. 6.3. C), was used as the probe. 3 clones, clone 18, 82 and 153 were tested positive

in all the PCR screening and confirmed by Southern blots (Fig. 6.6).

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Figure 6.4. ES clones having desired homologous recombination were generated. A. Screening for 5’ homologous recombination. Primers I2P5 and PGK Rev2 were used in a PCR reaction (94oC, 30 sec, 50 oC, 30 sec, 72 oC, 2 min, 35 cycles). B. Screening for 3’ homologous recombination. Positive clones from first screening were further selected

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by a PCR reaction (94oC, 30 sec, 55 oC, 30 sec, 72 oC, 1 min, 35 cycles) with primers loxNB and E6. C. Confirmation PCR reaction results using primer I5P2 and I5P3. The two primers flank the 3’ LoxP site. PCR condition: (94oC, 30 sec, 60 oC, 30 sec, 72 oC, 1 min, 35 cycles). The lower band is wt allele, while the top band is the targeting allele, which contains the 3’ LoxP site. D. Southern blotting of ES clones. ES genomic DNA was digested with Bam HI. A 0.5 kb PCR product, corresponding to exon 2 and 460 bps downstream of exon2 in intron 2, which is upstream of the target construct, was used to make probes to identify clones that had correct homologous recombination. “1” refers to clone 18, “2” refers to clone 82, “+” and “-“ represent positive and negative controls, respectively.

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Generation and selection of ES clones with neo/tk cassette deleted.

ES clone 18 was expanded under continuous selection for neo resistance to minimize revertants. Then the cells were electroporated with pPGK-cre plasmid (Abuin and Bradley 1996) and plated in 2 100mm dish with feeder cells. 2 days later, a serial dilution of cells at density of 106, 105, 104, 103 cells were plated, rested for one day and selected with 2 µM gancyclovir. About one week later, positive clones were picked, expanded and screened by PCR (94oC, 30 sec, 60 oC, 30 sec, 72 oC, 1 min, 35 cycles) with primer I2P10 and I2P9 for deletion of neo/tk cassette. Primer I2P10 is located upstream of the 5’ loxP site and primer I2P9 is located downstream of the middle loxP site (Fig. 6.3 B, C and D). If the neo/tk cassette is still present, a 5 kb fragment is amplified by the two primers. However, if the neo cassette is deleted, a 360bp fragment will be amplified. Furthermore, the two primers can also amplify the wt allele (about 320 bp), which can serve an internal control in the PCR reaction (Fig. 6.5.A., left 6 lanes).

Because transient expression of cre could lead to deletion of both the neo cassette and exon 3-5, another primer set (primer I2P10 and I5P3, as illustrated in Fig 6.3 D and E) was used to screen for the complete deletion (Fig 6.5 A, middle 6 lanes). Four conditional knockout clones (clone 14, 60, 137 and 140), one complete knockout clone (clone 44) and one mixed clone (clone 23) were identified. The integrity of 3’ end loxP site was confirmed by PCR (Fig6.5, B).

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Figure 6.5. ES clones with neo cassette deletion were generated. The drug resistant clones were expanded and screened by PCR (94oC, 30 sec, 60 oC, 30 sec, 72 oC, 1 min, 35 cycles) with primer I2P10 and I2P9 for deletion of neo/tk cassette. Primer I2P10 is located upstream of the 5’ loxP site. Primer I2P9 is located downstream of the middle loxP site. If the neo/tk cassette is still present, there is about 5 kb distance between the two primers. However, if the neo cassette is deleted, there will be only about 400bp between the two primers. Furthermore, the two primers also amplify the wt allele (about 320 bps), which can serve a nice control in a PCR reaction. Primer set (I2P10 and I5P3) was used to amplify the complete knockout allele. A mixture of three primers (I2P10, I2P9 and I5P3) amplifies the conditional knockout allele, the complete knockout allele and the wild type allele. B. Primer I5P2 and I5P3 were used to confirm for the integrity of 3’ end loxP site. 1 and 3 represent clones with conditional knockout allele; 2 represents a clone with complete knockout allele; 4 is a negative control of ES cells with homologous recombination but no cre treatment; 5 is a negative control of wild type ES cells; and 6 is a positive control that the targeting construct plasmid was treated with recombinant Cre.

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Generation of mice harboring Ets-2 floxed and knockout allele.

ES cells from clone 60 (neo cassette deleted) were injected into blastocysts. Three

highly chimeric mice were obtained. They were mated with C57Bl/6 female mice. Agouti

pups were tested for germline transmission by PCR with primers I2P10 and I2P9, and

confirmed by PCR with primers I5P2 and I5P3. Brother-sister mating of mice

heterozygous for the floxed allele (ets-2floxed/+) yielded mice homozygous mice with

floxed allele ets-2floxed/floxed (Fig. 6.6 A). These mice are phenotypically normal and fertile

(data not shown).

EIIA-cre mice (Holzenberger et al. 2000; Xu 2001) express cre in the mouse germ-line in a chimeric pattern. They were mated with ets-2floxed/+ mice. The loxP sites flanked region was successfully deleted in some mice in the germ-line and yielded ets-

2∆3/+ mice (Fig. 6.6 B). Ets-2∆3/+ mice are phenotypically normal and fertile. However,

preliminary data indicated that there were no live ets-2∆3/∆3 mice embryos found from 3

litters (total 27 embryos) at E9.5 from ets-2∆3/+ mice brother-sister matings, suggesting

that they were embryonic lethal before E9.5, probably due to similar reason as in ets-

2db1/db1 mice.

Mox-2-cre mice express cre in embryos but not in extra-embryonic tissue

(Tallquist and Soriano 2000). A mating between Mox-2-cre/+, ets-2floxed/+ mice and ets-

2floxed/floxed mice gave rise to Mox-2-cre/+, ets-2floxed/floxed mice. Cre expressed in Mox-2

cre mice excised ets-2 very efficiently in most tissues (data not shown), so the genotype

of these mice was almost ets-2∆3/∆3. Phenotypically, these mice were similar to the

tetraploid rescued ets-2 knockout mice (Yamamoto et al. 1998). They could be

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recognized from about 10 days after birth by waved hair phenotype. The adult Mox-2-

cre/+, ets-2floxed/floxed mice were fertile and had no obvious defects except the waved hair

phenotype.

Figure 6.6. Gene targeted mice harboring Ets-2 floxed and knockout alleles were generated. A. Genotyping of conditional knockout mice. 1 and 6 represent mice with homozygous floxed ets-2 allele; 2,3,4 and 7 represent mice with only wild type allele; and 5 represents a mouse with heterozygous floxed ets-2 allele. The arrow points to the conditional knockout band and the arrow head points to wt band. B. Genotyping of the complete knockout allele. Lane 1 and 5 represent mice with wild type allele. Lane 2, 3, 4, and 6 represent mice with the knockout allele. The arrow points to the knockout band and the arrow head points to the wt band.

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6.3 Discussion

An ets-2 conditional knockout allele (ets-2floxed) was generated and transmitted

through germ-line. Ets-2floxed/floxed mice were normal, indicating the LoxP tags did not

interfere with ets-2 function. The targeted ets-2 regions were successfully deleted in mice

express EIIA-cre (Holzenberger et al. 2000; Xu 2001), Mox-2-cre (Tallquist and Soriano

2000) and lysozyme-cre (Clausen 1999). Germ-line expression of cre created an ets-2

knockout allele (ets-2∆3), which was genetically similar to ets-2db1 allele, according to our

preliminary experiments with embryonic lethality of homozygous ets-2 knockout mice

and waved fur phenotype of mice specific lacking ets-2 in embryonic tissue but not extraembryonic tissue. Further experiments are needed to further characterize the ets-2db1

allele and ets-2 functions, such as Western blotting for Ets-2 protein, characterization of

ets-2∆3/∆3 during earlier embryo development stages, and study of ets-2∆3/T72A embryos.

Ets-2 is reposted to be involved in many important biological processes. The generation of the ets-2 conditional knockout allele is helpful to define the function of ets-

2 in these processes. The previous three chapters illustrated some examples of ets-2 functions, which can be better tested by the conditional deletion of ets-2 in specific tissues and/or at specific times. For example, deletion of ets-2 specifically in macrophages in mev mice could define macrophage cell autonomous functions of ets-2 in inflammation. Endothelial cells or pericytes specific deletion of ets-2 in the ets-1-/- background will further define the role of ets-2 in angiogenesis and mice embryonic development. Deletion of ets-2 specifically in trophoblasts or other cells types in the

placenta will demonstrate a definite role of ets-2 in placenta function or differentiation.

Deletion of ets-2 by Mox-2 cre in ets-1-/- mice may further define ets-1 and ets-2 207

function in embryogenesis. When Mox-2-cre and the ets-2 conditional knockout allele are

introduced into disease mouse models, such as PyMT breast cancer model, the

contribution of ets-2 to those diseases could be defined. The role of ets-2 in those

diseases can be further defined by specifically deletion in different tissues, such as

epithelial cells, fibroblasts and macrophages. Bringing ets-2 dosage to normal level by

conditional knock out of ets-2 in Down syndrome mice may define the role of ets-2 in

this disease. Comparing gene expression in cells with wild-type ets-2, the ets-2T72A/T72A allele, or deficient for ets-2 by microarray may define genes activated by ets-2 as well as genes repressed by ets-2.

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CHAPTER 7

DISCUSSION AND FUTURE DIRECTIONS

Ets family transcription factors share an evolutionarily-conserved Ets domain of about 85 amino acid residues that mediate binding to purine-rich DNA sequences with a central GGAA/T core consensus and additional flanking nucleotides (Graves and

Petersen, 1998). They are regulated by signal transduction pathways, activate or repress transcription of genes in cooperation with other transcription factors and co-factors, and play crucial roles in regulation of a variety of cellular function including growth, apoptosis, development, differentiation and oncogenic transformation (Graves and

Petersen 1998a; Wasylyk 1998; Oikawa 2003). Ets-2 is phosphorylated by Ras-MAPK pathway at a conserved threonine 72 residue. The phosphorylation of Ets-2 leads to the activation of Ets-2 and persistent target gene expression. In this work, we found that unphosphorylated Ets-2 interacted with co-repressors, such as Brg1 chromatin remodeling complex or BS69 co-repressor complex, and repressed target gene expression. The biological importance of Ets-2 phosphorylation was demonstrated in vivo in animal models. The first model was a pathological model (mev mice) where Ets-2 phosphorylation was deregulated. Ets-2 regulated many genes important for macrophage action, and the phosphorylation was required for the inflammation phenotype in mev

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mice. The phosphorylation of Ets-2 was also crucial for angiogenesis and embryo

development, because ets-1-/-, ets2T72A/T72A mice were embryonic lethal, with severe

cardiovascular defects. Embryonic fibroblasts from ets-1-/-, ets2T72A/T72A mice had defects

in expression of several matrix proteases. Because macrophages and fibroblasts are

important components of tumors microenvironment and crucial for tumor metastasis, ets-

2 may play important roles in tumor development by regulating target expression in

tumor cells as well as in cells constituting tumor microenvironment, and regulate tumor

angiogenesis and metastasis.

7.1 Phosphorylation of Ets-2 regulates protein partnership and activity.

One major way of regulating specificity within the Ets family is through

posttranslational modifications. Many Ets family proteins are downstream nuclear targets

of signal transduction cascades (Wasylyk 1998; Yordy 2000). Ets-1 and ets-2 are

phosphorylated at a conserved residue (threonine 38 and threonine 72, respectively) at the

N-terminal of the Pointed domain, a domain conserved in evolution from Drosophila to

human, by the well characterized ras/MAPK pathway. Phosphorylation of Ets-1 and Ets-

2 by ras-dependent pathways leads to persistent expression of target genes including uPA, Bclx and MMP9. The ras induced persistent expression of target genes is mediated by Ets-1 and Ets-2, but not other ets factors (Yang et al. 1996; McCarthy et al. 1997;

Fowles et al. 1998a; Patton et al. 1998). Phosphorylation of Ets-2 also alters its protein partnership. Phosphorylated and unphosphorylated Ets-2 binds to discrete sets of nuclear proteins (Baker et al. 2003). For instance, the unphosphorylated form of Ets-2 interacted with Brg-1 chromatin remodeling complex (Baker et al. 2003) or BS69/N-CoR co- 210

repressor complex (Fig. 5.2). The expression of target genes was repressed by the

collaborative action of Ets-2 and Brg1 (Fig. 5.1) or Ets-2 and BS69 (Fig. 5.2) in

transfections assays. These data suggested that the phosphorylated form of Ets-2 is an

activator, while the unphosphorylated form of Ets-2 may act as an active repressor by

recruiting co-repressors. BRCA1 was down regulated in Ets-1-/-, ets-1T72A/T72A mice but not in Ets-1+/+, ets-1T72A/T72A mice. Both Ets-1 and Ets-2 might be able to bind to BRCA1

promoter, and BRCA1 level was not changed in Ets-1+/+, ets-1T72A/T72A mice because of the presence of phosphorylated Ets-1. In the absence of ets-1, unphosphorylated Ets-2 binds to BRCA1 promoter, and recruits co-repressors to down-regulate BRCA1 expression in the double mutant embryos. Alternatively, BRCA1 may be a positive target for Ets-1 or Ets-2 in vivo during development. Further experiments may be used to test the role of Ets-2 phosphorylation in regulating gene expression. Antibodies specific for phosphorylated or unphosphorylated form or Ets-2, Brg1 or BS69, can be used in chromatin immunoprecipitation assays to test whether Brg1 or BS69 is recruited by Ets-2 to the promoter of BRCA1 or other Ets target genes. The target gene expression levels can be further investigated between cells deficient for ets-2 (may be generated by conditional deletion from the ets-2 conditional knockout mice), cells homozygous for the ets-2T72A mutation and wild type cells. If the level of BRCA1 and other Ets target genes mRNA is lower in ets-2T72A/T72A cells than that in ets-2-/- cells, it will implicate that the

unphosphorylated form of Ets-2 acts as an active repressor.

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7.2. Ets-2 is important for inflammation and other immune disease.

Our data suggested that Ets-2 is important for macrophage survival, and regulate

genes involved in macrophage migration, adhesion, cytokines and chemokines

production. The inflammation phenotype of mev mice was corrected, at least partially, by

lowering the dosage of ets-2 by half, or homozygous T72A mutation (Chapter 3). Ets-1-/-,

ets-2T72A/T72A double mutation caused significant reduction of macrophages (Fig. 4.5), whereas deletion (Henkel et al. 1996) does not affect macrophage differentiation ; mutation of ets-2 (Chapter 3) did not affect macrophage proliferation. Both ets-1 and ets-

2 are expressed in macrophages (Fig. 1.3). Thus Ets-1 and Ets-2 may play redundant roles in macrophages. Deletion of ets-2 by fms-cre or Lysozyme-cre in ets-1-/- background may further reveal the roles of Ets-2 in macrophages. Deregulation of macrophage can lead to auto inflammation disease in human. Many of Ets-2 target genes, especially several matrix metalloproteinases, contribute to those diseases. Besides in macrophages, ets-2 is also expressed in other cell types involved in inflammation, such as

T-cells, B-cells and granulocytes, and is reported to regulate their activities. Transgenic mice that express ets-2T72A in T-cells have small thymus and T-cells from those mice

have increased apoptosis (Fisher et al., personal communication). Consistent with data

from mice models, Ets-2 overexpression has been observed in 30% of rheumatoid

arthritis patient (Dooley et al. 1996). Further studies of Ets-2 expression and

phosphorylation state in human immune diseases may disclose the impact and

mechanisms of Ets-2 action in these diseases and leads to develop new treatment.

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7.3. Ets-2 and cancer.

Ets-2 was shown to be important for tumor cell transformation and regulated

target gene (e.g. uPA) expression in tumor cells. In addition, Ets-2 was required in

angiogenesis (Chapter 4) and regulates the expression of many genes, including matrix

proteases MMP1, MMP9 and uPA, in macrophages (Chapter 3) and fibroblasts (Chapter

4). Macrophages, fibroblasts and blood vessels are important components of tumors microenvironment and are crucial for tumor metastasis. Therefore, ets-2 plays important roles in cancer by regulating target expression in tumor cells as well as cells constituting tumor microenvironment.

7.2.1. Ets-2 and tumor cells.

Ets-2 is overexpressed in many cancer cells (Fig 1.3 C) and regulates transformation of cancer cells and the expression of many target genes in cancer cells.

Several oncogenic pathways, including activated ErbB2/neu, Src, Ras, and Raf, activate

Ets-2 in mammalian cells (Langer et al. 1992; Yang et al. 1996; McCarthy et al. 1997;

Fowles et al. 1998a). Ets-2 appears to be a transformation-mediator in mammary epithelium because dominant-negative mutant constructs of Ets-2 can block transformation by Neu/Ras (Langer et al. 1992; Galang et al. 1996; Foos et al. 1998). Ets-

2 dominant negative mutants abolish anchorage-dependent growth and CSF1-stimulated invasion by breast cancer cells (Sapi et al. 1998). The expression of phosphorylated ets-2 correlates with a more invasive, mesenchymal phenotype in ovarian cancer tumor cell lines (Patton et al. 1998). Similarly, expression of phosphorylated ets-2 correlates with expression of target genes, such as MMP3, MMP14 and uPA, and invasiveness in the 213

breast cancer cell lines (Baker et al. 2003). Ets-2 interacts with Brg1 chromatin remodeling complex or BS69 co-repressor complex and repressed the expression of a tumor suppressor, BRCA1. It is interesting that both Brg1 (Reisman et al. 2003) and

BS69 (Masselink and Bernards 2000) were implicated as possible tumor suppressors.

One of their functions as tumor suppressors might be sequestering Ets-2 or inhibiting Ets target gene expression.

7.3.2. Ets-2 and tumor microenvironment.

Our data demonstrated that Ets-2 regulated genes in macrophages and fibroblasts, which are the major components of tumor microenvironment. Thus Ets-2 may play important roles in tumorgenesis by regulating gene expression in macrophages and fibroblasts, and involved in tumor angiogenesis and metastasis.

The conversion of normal epithelial cell to metastatic tumor cell is a multi-stage process that requires progressive genetic revisions within the epithelial tumor cell and has been the focus of intense investigation. However, recently there is growing interest in studying the role of the tumor microenvironment in tumor progression. Stromal cells within the tumor tissue are not inert bystanders, but are active players that shape the frequency and features of tumors. The characteristics of solid tumors, such as uncontrolled proliferation, derangement of cellular and morphological differentiation, invasion, and colonization to distant organs, can be attributed in part to alterations in communications between neoplastic cells and the stromal cells in their immediate microenvironment (Seljelid et al. 1999; Hanahan and Weinberg 2000; Park et al. 2000).

The tumor microenvironment includes: (1) insoluble extracellular matrix (ECM); (2) 214

stroma consisting of fibroblasts, adipocytes, macrophages and other immune cells, and vasculature; (3) cytokines and growth factors (Park et al., 2000).

The extracellular matrix (ECM) is a network of macromolecules in which cells are embedded. It provides not only structural support for tissue integrity but also an interactive environment for specialized cell functions. In many pathological conditions the balance between ECM synthesis and degradation is disrupted, leading to abnormal

ECM remodeling. Excessive ECM deposition occurs in fibrotic diseases such as scleroderma, liver cirrhosis, and glomerulosclerosis; whereas, excessive breakdown of the ECM is associated with rheumatoid arthritis, osteoarthritis, periodontitis, as well as tumor invasion and metastasis (Seljelid et al. 1999; Hanahan and Weinberg 2000; Park et al. 2000).

Members of the integrin family mediate interactions between cells and extracellular matrix proteins. It has been reported that Ets-1 can regulate integrin β3 in endothelial cells(Oda et al. 1999). The phosphorylation of Ets-2 is required for maximal integrin αM and β2 expression in macrophages (Fig. 3.4, 3.5, 3.6). Further study of the

function of Ets-2 on integrins expression in cancer cells and stromal cells may uncover

additional aspects of Ets-2 action.

Ets-2 controls the expression of many extracellular matrix proteases, such as uPA

and several matrix metalloproteinases (MMP1, MMP3, MMP9 and MMP14) in cancer

cells (Chapter 5), fibroblasts (Chapter 4) and macrophages (Chapter 3). Those proteases

induce a cascade of protease activation, which is required for basement membrane

degradation, and facilitate tumor angiogenesis, invasion and metastasis. Furthermore,

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those protease can activate some cytokines or growth factors, such as activation of latent

TGF-β, release of matrix-bound bFGF and VEGF (Papetti and Herman 2002).

Ets-2 was hyper-phosphorylated in stromal fibroblast cells from mice with breast tumors compared to normal mammary fibroblasts (Fig 7.1). The female C3(1)SV40-T antigen transgenic mice express C3(1)SV40-T antigen in mammary epithelial cells and develop visible mammary tumor at 12-14 weeks (Shibata et al. 1996). To explore the role of ets-2 in pre-tumor stromal cells, 10 week old C3(1)SV40-T antigen transgenic mice were used to obtain mammary fibroblast cells and epithelial cells. The cells were cultured in fibroblast or epithelial cell media, respectively, for 7 days. Cells were then serum starved for 6 hours, and 15% serum was added to the medium. 30 minutes later, the cells were harvested for Western blot analysis. In serum starved wild-type mice mammary stromal fibroblasts, there was very little Ets-2 phosphorylation. However, in stromal fibroblasts from the transgenic mice, Ets-2 was still phosphorylated. In contrast, the phosphorylation of Ets-2 in epithelial cells from the transgenic mice was still subject to serum regulation (Fig 7.1). Therefore, the stromal fibroblasts in pre-tumor mammary gland were not normal and Ets-2 phosphorylation may be a molecular marker of tumor stromal fibroblast.

216

Figure 7.1. Constitutive Ets-2 phosphorylation in stromal cells from pre-invasive mice tumors. A. Illustration of experiment procedure. The female C3(1)SV40-T antigen transgenic mice express C3(1)SV40-T antigen in mammary epithelial cells and start to have visible mammary tumor at 12-14 weeks. To explore the role of ets-2 in pre-tumor stromal cells, 10 week old C3(1)SV40-T antigen transgenic mice were used to obtain mammary fibroblast cells and epithelial cells. The cells were cultured in fibroblast or epithelial cell media, respectively, for 7 days. They were serum starved for 6 hours, and 15% serum was added to the medium. 30 minutes later, the cells were harvest for Western blot analysis. B. In serum starved wt mice mammary stromal fibroblasts, there was very little Ets-2 phosphorylation. However, in stromal fibroblasts from the transgenic mice, Ets-2 was still phosphorylated. In contrast, the phosphorylation of Ets-2 in epithelial cells from the transgenic mice was still subject to serum regulation. St: Stromal fibroblast. Ep: Epithelial cells.

217

Macrophages are also important in tumor biology. Growth factors and cytokines

produced by macrophages and other cells are crucial for stroma formation and

angiogenesis. Lytic enzymes provided by stromal cells may be essential for invasion.

PyMT mice with mutation is CSF-1 gene (op) have less macrophages and tumor

metastasis is much delayed in these mice compared to PyMT mice with normal number

of macrophages (Lin et al. 2001). Ets-2 controls the expression of many of those genes

(Chapter 3).

7.2.3. Ets-2 in tumor angiogenesis and metastasis.

Angiogenesis is required for tumor cells to get nutrients and oxygen, and is

crucial for their survival, proliferation and metastasis. There are many reports linking Ets-

1 and Ets-2 to tumor angiogenesis (Nakano et al. 2000; Sato et al. 2001; Khatun et al.

2003). Our data suggest that ets-1 and ets-2 are crucial for embryo angiogenesis. Tumor

angiogenesis and embryo angiogenesis share similar mechanisms and players. Therefore,

ets-1 and ets-2 could well be indispensable for tumor angiogenesis. In order for tumor to

metastasis, degradation of basement membrane, presence of vasculature by angiogenesis,

cell migration and degradation of ECM is required. Macrophages and stromal fibroblast cells secret extracellular proteases to facilitate tumor invasion and metastasis

(Westermarck and Kahari 1999a; De Wever and Mareel 2003). The mRNA level of several extracellular proteases, such as MMP-3 and MMP-9, was much lower in mouse embryonic fibroblast cells deficient for ets-1 and with ets-2 T72A mutation, than in cells with normal ets-1 and ets-2 (Fig 4.6), thus suggesting Ets-1 and Ets-2 are key regulators of the expression of these enzymes and metastasis. 218

7.2.5. Define the role of Ets-2 phosphorylation in tumorgenesis by tissue specific deletion.

Tissue specific deletion of ets-2 may further define the role of ets-2 in discrete cell types in tumorgenesis. Tumors are complex tissues constituted by mutant cancer cells and stromal support cells. Understanding the interactions between the genetically altered malignant cells and these supporting coconspirators is important for cancer pathogenesis studies and for development of novel, effective therapies (Hanahan and Weinberg 2000).

Lowering the dosage of ets-2 to half (Neznanov et al. 1999), or mutation of ets-2 (T72A)

(Man et al. 2003), causes inhibition of mammary gland tumor progression in female mice expressing the PyMT oncogene in epithelial cells of mammary gland. However, it is not known the phenotype of complete removal of ets-2 in this tumor model. Furthermore, the ets-2 mutation is present in every cell types (tumor cells or stromal cells) in those mice. It is yet unknown the mutation of ets-2 in which cell type leads to inhibition of tumor growth. Our study has shown that Ets-2 was involved in the action of cancer cells

(Chapter 5), endothelial cells, fibroblasts (Chapter 4) and macrophages (Chapter 3).

Especially, Ets-2 regulates the expression of genes involved in tumor angiogenesis, invasion and metastasis. Tissue specific deletion Ets-2 experiments would further define the role of ets-2 in tumor cells and cells in tumor microenvironment.

Deletion of ets-2 in tumor cells.

To further define the contribution of Ets-2 in tumor cells, ets-2 may be deleted in epithelial cells and tumor cells by MMTV-cre or WAP-cre in PyMT mice. Ets-1 and ets- 219

2 may be redundant in tumorgenesis, because BRCA1 was down regulated only in ets-1/2

double mutant mice, and our preliminary data suggested that there was no significant

differences in tumorgenesis in ets-1+/+, PyMT mice and ets-2-/-, PyMT mice. Therefore,

the epithelial cell specific deletion of ets-2 shall be carried out in ets-/- background.

These mice will be characterized and compared with PyMT mice that have normal Ets-2,

for tumor occurrence, growth, malignancy and metastasis. The expression of ets target

genes, such as BRCA1 and uPA, can be measured in breast cells to elicit ets-2 function.

Deletion of ets-2 in stromal fibroblast cells.

Conditional knockout Ets-2 in stromal fibroblast cells in Ets1-/-, PyMT

background may define Ets-2 function in tumorgenesis. An ongoing collaborative project

between Dr. Leone’s lab and our lab is to generate mice expressing cre specifically in

breast stoma cells. Metallothionine (MT1) promoter has been reported to drive stromal

specific gene expression (Joseph et al. 1999) and was used to drive cre expression in

transgenic mice. Mice offspring from all 6 MT1-cre founders expressed cre in stromal

cells. Unfortunately, cre was also expressed in breast epithelial cells, although with different degree (data not shown). Therefore, they are not suitable to study stromal cell specific deletion of ets-2. However, transgenic mice with cre driven by FSP promoter express cre specifically in stromal fibroblast and can used to generate mice conditionally deleting ets-2 in tumor stromal fibroblast.

220

Deletion of ets-2 in macrophages.

Conditional disruption of ets-2 in macrophages by fms-cre or lysozyme-cre in

tumor model mice will clarify the role of ets-2 in tumor macrophages, especially their

function in facilitating tumor metastasis.

Deletion of ets-2 in vascular cells.

Conditional knockout ets-2 in endothelial cells by Tie-2-cre, in smooth muscle cells by smooth muscle actin-cre, in the PyMT animal tumor model in the absence of ets-

1 will demonstrate the role of ets-1/2 in tumor angiogenesis. Since ets-1/2 may be required in those tissues for embryo development, deletion of ets-1/2 in those tissues may lead to embryonic lethality. However, this problem can be circumvented by using inducible conditional knockout strategy, such as estrogen receptor fusion inducible Tie2- cre expression (Forde et al. 2002) or the Tetracycline inducible system (Berens and

Hillen, 2003, Tet mouse database: http://www.zmg.uni-mainz.de/tetmouse/).

In summary, ets-2 is involved in normal development and many human diseases, such

as cancer and inflammation. Signaling pathways, such as ras pathway, may modulate Ets-2

through posttranslational modifications. The phosphorylation of Ets-2 affects its protein partnership, and activity (activation/repression). Ets-2 phosphorylation has many important biological roles. It is involved in immune response, angiogenesis, and embryo development. Disruption of Ets-2 phosphorylation caused reduced inflammation, defects in angiogenesis and embryonic lethality in the absence of gene redundancy. Our data also 221

indicate that ets-2 may play important roles in cancer, especially in tumor angiogenesis and metastasis. Understanding how Ets-2 works in cancer and other disease may have important clinical implications, both for early diagnosis and design of novel, effective therapy methods.

222

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