The Pennsylvania State University
The Graduate School
Intercollege Graduate Degree Program in Genetics
REQUIREMENT OF THE DYNLRB FAMILY DYNEIN LIGHT CHAINS
IN TRANSFORMING GROWTH FACTOR BETA SIGNALING
A Thesis in
Genetics
by
Guofeng Gao
2007 Guofeng Gao
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
May 2007 The thesis of Guofeng Gao was reviewed and approved* by the following:
Sarah K. Bronson Associate Professor, Department of Cellular and Molecular Physiology Thesis Advisor Chair of Committee Co-Chair, Intercollege Graduate Degree Program in Genetics
Keith C. Cheng Associate Professor, Department of Pathology
Mark Kester Distinguished Professor, Department of Pharmacology
Jiyue Zhu Associate Professor, Department of Cellular and Molecular Physiology
*Signatures are on file in the Graduate School iii ABSTRACT
Transforming growth factor β (TGFβ) is the prototype for a superfamily of related members. TGFβ family signaling controls various fundamental cellular functions, including cell proliferation and migration. Alterations in the TGFβ signaling pathways have been implicated in a vast array of human cancers and other diseases as well.
Despite advances in our understanding of TGFβ signaling transduction, the mechanism of the multifunctional TGFβ signaling is not completely clear yet. Therefore, further studies are required for deeper understanding of its diverse biological responses. DYNLRB1 was identified in the laboratory through a screen for TGFβ receptor-interacting proteins, and it is also a dynein light chain. Dynein is molecular motor that plays many important functions in the cell. I hypothesized that DYNLRB family dynein light chains may play important functions in TGFβ signaling. In this thesis, we investigated the regulation of the function of DYNLRB dynein light chains by TGFβ and their role in TGFβ signaling in mammalian cells and in primary zebrafish (Danio rerio) ovarian follicle cells.
The work in Chapter 2 aimed to determine the involvement of DYNLRB1 in
TGFβ signaling and characterizing the function of DYNLRB1. The results from this chapter have demonstrated that the phosphorylation of DYNLRB1 on serine residues is stimulated by TGFβ in Cos-1 cells and requires TβRII. It is further demonstrated that
DYNLRB1 expression knockdown significantly impaired TGFβ-induced fibronectin expression in MDCK cells. TGFβ-induced DYNLRB1phosphorylation has been shown to be responsible for its recruitment to the dynein motor complex. Therefore, these results indicate a potential role for DYNLRB1 as a TGFβ signaling intermediate, and its iv requirement in TGFβ induction of fibronectin (a major component of the extracellular matrix), which plays important roles in cell adhesion, migration and differentiation.
The experiments in Chapter 3 was designed to test the hypothesis that DYNLRB2 might be involved in Smad3-dependent TGFβ signaling. Human DYNLRB2 is 77% identical to human DYNLRB1. Results in Chapter 3 have demonstrated that TGFβ induction of SBE2-Luc, plasminogen activator inhibitor-1 expression in HaCaT cells and of Smad7-Luc in Hep3B cells, is significantly impaired, after blocking endogenous
DYNLRB2 expression by siRNA. It is known that the induction of these genes is
Smad3-dependent signaling event. However, similar blocking DYNLRB2 expression does not inhibit the induction of ARE-Lux by TGFβ, which has been demonstrated to be
Smad2-dependent signaling event. Therefore, these results suggest that DYNLRB2 is specifically required in Smad3-dependent signaling. Further, it is demonstrated that
TGFβ-stimulated preferential interaction between DYNLRB2 and Smad3 may be the underlying mechanism for the requirement of DYNLRB2 in Smad3-dependent TGFβ signaling. In addition, results have shown that TGFβ stimulated a rapid recruitment of the DYNLRB2 to the dynein complex, and the TGFβ-induced phosphorylation of
DYNLRB2 is responsible for this TGFβ stimulated recruitment. Collectively, results in this Chapter have demonstrated for the first time that DYNLRB2 is required for Smad3- dependent TGFβ signaling.
The experiments in Chapter 4 tested our hypothesis that in zebrafish ovarian follicle cells the function of zDYNLRB might be regulated by TGFβ, and zDYNLRB might play an important role in TGFβ signaling in such cells. It is shown that zDYNLRB v is rapidly phosphorylated after TGFβ stimulation, for which the TβRII is required. In addition, it is shown that the phosphorylation of zDYNLRB facilitates its rapid recruitment to the dynein complex, which is stimulated by TGFβ. Knockdown experiments in zOFCs by morpholino have demonstrated that zDYNLRB was required for TGFβ induction of TRE-Luc, 3TP-Lux and ARE-Lux. Thus, the results suggest a potential role for zDYNLRB in TGFβ signaling in zebrafish ovarian follicle cells.
Collectively, the experiments in this thesis demonstrated for the first time a requirement of DYNLRB2 dynein light chain in Smad3-dependent TGFβ signaling in mammalian cells, and a requirement of DYNLRB1 as a potential TGFβ signaling component in TGFβ induction of fibronectin, respectively, as well as potential role for zDYNLRB in TGFβ signaling in zebrafish ovarian follicle cells. However, the physiological significance of their functions needs to be addressed in vivo in the future. vi TABLE OF CONTENTS
List of figures...... x
List of tables ...... xiv
List of abbreviation...... xiv
Acknowledgements...... xix
1. Chapter 1. Literature review…..……………………………..……………… .1
1.1 Introduction…………………………………………………….…………….1 1.2 TGFβ signaling pathways……………………………………….…………...3 1.2.1 The Smad pathway……………………………………….. ..…………..6 1.2.1.1 R-Smads…………………………………………………….………... 7 1.2.1.2 Co-Smads………………………………………………….………...... 7 1.2.1.3 I-Smads…………………………………………………….…………..8 1.2.2 The MAPK pathways………………………………………….………...9 1.2.3 Other pathways……………………………………………….……...…11 1.3 Role of TGFβ signaling in cancer and development……………….………..12 1.3.1 Role in cancer………………………………………………….…..…...12 1.3.2 Role in development…………………………………………….……...18 1.3.3 Role in ovarian follicle development…………………………….……..21 1.4 Regulation of TGFβ signaling………………………………………….…….24 1.4.1 Regulation at Ligand production level…………………………….…….25 1.4.2 Regulation of Ligand Processing and Activation…………………….…26 1.4.2.1 Regulation of Ligand availability for Processing………………….…..27 1.4.2.2 Regulation of Ligand Processing……………………………………....29 1.4.2.3 Regulation of TGFβ Ligand activation…………………………….…..29 1.4.2.4 Regulation of the availability of active mature TGFβ ligand……….....31 1.4.3 Regulation of TGFβ receptor level and activity……………………..….32 vii 1.4.3.1 Enhancing effects of TGFβ co-receptors……………………………...32 1.4.3.2 Negatively regulating the activity of TGFβ receptors………………....33 1.4.3.2.1 Physical Blockade of TGFβ receptors……………………...33 1.4.3.2.2 Enzymatically regulating the activity of TGFβ receptors……………………………………...…35 1.4.3.3 Regulating the level of TGFβ receptors………….………..….36 1.4.4 Intracellular regulation…………………………………………………...37 1.4.4.1 Regulating the Smad2/3 activity by phosphorylation…………………..37 1.4.4.2 Terminating the Smad2/3 signaling by dephosphorylation…………….39 1.4.4.3 Terminating the Smad2/3 signaling by irreversible degradation……………………….…………..…….40 1.4.4.4 Regulating the level and activity of the Co-Smad, Smad4……………...41 1.4.4.5 Regulating the subcellular compartmentalization of Smad2/3………….43 1.4.5 Transcriptional regulation of target gene expression in the nucleus…………………………………………..…….48 2. Chapter 2. Requirement of DYNLRB1 for TGFβ-mediated induction of fibronectin………………………………….53 2.1 Introduction…………………………………………………………………….54 2.2 Materials and methods…………………………………………………………56 2.3 Results………………………………………………………………………….61 2.3.1 TGFβ stimulates DYNLRB1 phosphorylation, which occurs on serine residues……………………………61 2.3.2 DYNLRB1 detection by rabbit polyclonal DYNLRB1 anti-serum……….65 2.3.3 DYNLRB1 protein has a short half-life and DYNLRB1 specific siRNAs knock down its expression in MDCK cells………...68 2.3.4 DYNLRB1 knockdown reduced fibronectin induction by TGFβ in MDCK cells…………………………………...70 2.3.5 TGFβ stimulates the interaction between DYNLRB1 and dynein motor, and TβRII is required for this interaction…………...72 2.4 Discussion……………………………………………………………………...74 viii 3. Chapter 3. Requirement of DYNLRB2 in Smad3-dependent TGFβ signaling………78 3.1 Introduction……………………………………………………………….……79 3.2 Materials and methods………………………………………………………….81 3.3 Results…………………………………………………………………………..87 3.3.1 DYNLRB2 siRNAs specifically block DYNLRN2 expression…………...87 3.3.2 DYNLRB2 knockdown significantly inhibits TGFβ induction of Smad3-dependent transcriptional activation of SBE2-Luc and Smad7-Luc……………………...90 3.3.3 DYNLRB2 knockdown significantly interferes with TGFβ induced PAI-1 gene expression……………………..…94 3.3.4 DYNLRB2 is in early endosomes with Smad3 after TGFβ stimulation…..98 3.3.5 TGFβ induces a preferential interaction between DYNLRB2 and Smad3…100 3.3.6 TGFβ stimulates the recruitment of DYNLRB2 to the dynein motor……104 3.3.7 DYNLRB2 is phosphorylated after TGFβ stimulation and TβRII is required for this phosphorylation…………………..108 3.3.8 DYNLRB2 phosphorylation mediates its rapid recruitment to the dynein motor, which is stimulated by TGFβ………………..114 3.4 Discussion…………………………………………………………………..…114 4. Chapter 4. R e q u i r e m e n t o f z e b r a fi s h d y n e i n l i g h t c h a i n z D Y N L R B i n T G Fβ s i g n a l i n g i n z e b r a f i s h o v a r i a n f o l l i c l e c e l l s………….122 4.1 Introduction………………………………………………………………...…123 4.2 Materials and methods……………………………………………………...…127 4.3 Results…………………………………………………………………………131 4.3.1 Cloning and expression detection of zDYNLRB…………………………131
4.3.2 Primary zebrafish ovarian follicle cells (OFCs) are TGFβ responsive…...135
4.3.3 zDYNLRB is phosphorylated after TGFβ receptor activation……….…..137
4.3.4 TGFβ stimulates the interaction between zDYNLRB and DIC,
which requires the TβRII kinase……………………………….140 ix 4.3.5 zDYNLRB specific morpholinos knock down zDYNLRB expression…..148
4.3.6 zDYNLRB knockdown interferes with some TGFβ-induced transcription activation………………………………………..149 4.4 Discussion……………………………………………………………..………157 5. Chapter 5. Overall discussion and future directions………………………………....165 References…………...………………………………………………………………….179 Appendices……………...………………………………………………………………209 Appendix A. Blocking dDYNLRB2 partially impairs TGFβ-mediated DNA synthesis inhibition in MDCK cells with high passage numbers (25-30), but not in MDCK cells with low passage numbers (<15)………………………….………...…210 Appendix B. dDYNLRB2 siRNA specifically knockdown exogenous dDYNLRB2 protein expression…………………...…211 Appendix C. dDYNLRB2 siRNA specifically knockdown endogenous dDYNLRB2 mRNA expression…………..……...…212 Appendix D. dDYNLRB1 siRNA specifically knockdown endogenous dDYNLRB1 protein expression……………..….…..213 Appendix E. Nonspecific development arrest of 24 h and 48 h embryos injected with the ATG MO……….…..…214 x LIST OF FIGURES
Figure Page
1 Model for TGFβ regulation of transcription through Smad signaling proteins...4
2 TβR internalization by clathrin- and lipid-raft-mediated endocytosis………....45
3 Comparison of Smad2 and Smad3……………………………………………..50
4 DYNLRB1 is phosphorylated upon activation of TGFβ receptors…………….64
5 Activation of the TGFβ receptors results in phosphorylation of DYNLRB1
primarily on serine residues….………………………………………...66
6 Specificity assessment of a rabbit polyclonal
antiserum for detection of DYNLRB1 protein expression…………….67
7 Determination of DYNLRB1 protein half-life…………………………………69
8 The siRNA blockade of DYNLRB1 expression reduces TGFβ
induction of fibronectin expression.……………………………71
9 Phosphorylation of DYNLRB1 is required for
recruitment of DYNLRB1 to the DIC…………………………73
10A dDYNLRB2 siRNA specifically knockdown exogenous
dDYNLRB2 protein expression………………………….88
10B hDYNLRB2 siRNA specifically knockdown endogenous
hDYNLRB2 mRNA expression………………………….89
10C siRNA blockade of endogenous hDYNLRB2 expression
results in significant inhibition of Smad3-dependent
SBE2-Luc activation in HaCaT cells……………………...92
10D siRNA blockade of endogenous hDYNLRB2 expression has xi no effect on inhibition of Smad2-dependent
ARE-Lux activation in HaCaT cells………………...…93
10E siRNA blockade of endogenous hDYNLRB2 expression
inhibits TGFβ-mediated transcriptional activation of
human the Smad7 promoter in Hep3B cells……………….....95
11 siRNA blockade of endogenous hDYNLRB2 inhibits TGFβ-mediated induction
of endogenous PAI-1 gene expression……………………...97
12 hDYNLRB2 is present in EEA1-enriched early endosomes
together with TβRII and Smad3 after TGFβ treatment……….…..99
13A hDYNLRB2 interacts preferentially with Smad3
in IP/blot analyses in 293T cells……………………………….....101
13B hDYNLRB2 interacts preferentially with Smad3
in LUMIER analyses in IEC4-1 cells …………………….103
14A TGFβ stimulates the recruitment of hDYNLRB2 to DIC
in 293T cells……………………………………………………...105
14B TGFβ stimulates the recruitment of hDYNLRB2 to DIC
in HaCaT cells………………………………………………………..107
15 DYNLRB2 interacts with the TβRII in 293T cells…………………………..109
16A hDYNLRB2 is phosphorylated upon activation of TGFβ receptors.………...111
16B TGFβ stimulated hDYNLRB2 phosphorylation in both
Mv1Lu epithelial cells and in R1B cells, but not in DR26 cells …….113
17 The interaction between DIC and hDYNLRB2 requires
TβRII kinase activity…………………………………………………..115
18 Amino acid sequence alignment of hDYNLRB1, hDYNLRB2 xii and zDYNLRB………………………………………………..133
19 Detection of zebrafish DYNLRB……………..……………………………..134
20 zOFCs are responsive to TGFβ treatment……………………….………….136
21A zDYNLRB is phosphorylated upon activation of TβRs in 293T
cells and this phosphorylation is blocked by TGFβ KNRII…………139
21B zDYNLRB is phosphorylated in zOFCs upon TGFβ treatment
and KNRII significantly decreased this phosphorylation…………141
22A TGFβ stimulates the recruitment of zDYNLRB to the DIC
in Mv1Lu cells……………………………………………………143
22B The recruitment of endogenous zDYNLRB to the DIC
in zOFCs is stimulated by TGFβ…………………………………..144
22C The interaction between zDYNLRB and the DIC in zOFCs is
confirmed by opposite direction IP/blot analyese …………...146
22D A functional TβRII is required for the TGFβ induction
of the DIC-zDYNLRB interaction……………………………147
23A The zDYNLRB MOs specifically knock down
exogenous zDYNLRB expression in 293T cells………………150
23B The zDYNLRB MOs specifically knock down
endogenous zDYNLRB expression in zOFCs………………....151
24A MOs knocking down zDYNLRB expression markedly
inhibited TRE-Luc induction by TGFβ……………………..…153
24B Blocking zDYNLRB expression also inhibited
3TP-Lux induction by TGFβ…………………………………...154
24C zDYNLRB expression knocking down did not interfere with xiii phTG5-Lux transcriptional regulation……………………….…156
24D Blocking zDYNLRB expression significantly repressed
ARE-Lux induction by TGFβ……………………………….…..158
25 Hypothetical model………………………………………………………166-167 xiv LIST OF TABLES
Table Page
1 Knockout mouse models of TGFβ family signaling proteins……………………...16-17
2 Comparision of DYNLRB1 to some other DYNLRB/robl/LC7 family members…...62
3 TGFβ family signaling pathway component mutants
with ovary development defects………………161 xv LIST OF ABBREVIATIONS
α alpha
AP-1 activator protein 1
ARE activin response element b bases
β beta
BMP bone morphogenic protein
CamKII Calcium-calmodulin-dependent protein kinase II cdk cyclin-dependent kinase
Co-Smad common Smad cPML cytoplasmic form of the ProMyelocytic Leukemia tumor
suppressor protein
Dab2 Disabled 2
°C degrees Celsius
DIC dynein intermediate chain
CREB cyclic AMP response element binding protein
DLC dynein light chain
DNA deoxyribonucleic acid
ECM extracellular matrix
EGF epidermal growth factor
ELF Embryonic Liver Fodrin
EMT epithelial-to-mesenchymal transdifferentiation
ERK extracellular signal-related kinase xvi γ gamma
GADD34 growth arrest and DNA-damage-inducible 34
GDF growth and differentiation factor h hour
HCCC human colon carcinoma cells
HC heavy chain
Hrs Hepatocyte growth factor-Regulated tyrosine kinase Substrate
IC intermediate chain
IP Immunoprecipitation
I-Smad inhibitory Smad
JNK c-jun-N-terminal kinase
KNRII kinase deficient TβRII
LC light chain
LIC light-intermediate chain
LTBP latent TGF-beta binding proteins
LUMIER luminescence-based mammalian interaction mapping
MAPK mitogen-activated protein kinase
MEK mitogen-activated protein kinase min minute mRNA messenger ribonucleic acid
MT1-MMP membrane type 1-matrix metalloprotease
µg microgram
µl microliter xvii ml millililiter
µM micromoles per liter
MO Morpholino phosphorodiamidate oligonucleotide
MT microtubule ng nanogram pmol picomole
PAI-1 plasminogen activator inhibitor –1
Pak-1 p21-actvivated kinase
PCR polymerase chain reaction
PI3K phosphatidylinositol-3k-kinase
PI3P phosphatidylinositol 3-phosphate
PIASy protein inhibitor of activated STATy
PKA protein kinase A
PKC protein kinase C
PP1 protein phosphatase 1
PP2A protein phosphatase 2A p70s6k p70 S6 kinase
RL renilla luciferase
RNA ribonucleic acid
R-Smads receptor-activated Smad proteins
ROS reactive oxygen species
SAPK stress-activated protein kinase
SARA Smad anchor for receptor activation xviii SBD Smad-binding domain
SBE Smad binding element sEng soluble TGFβ co-receptor endoglin siRNA small interfering RNA
Smurf Smad ubiquitination-related factor
Sp1 stimulatory protein 1
STRAP Serine-Threonine kinase Receptor-Associated Protein
TβRI TGFβ receptor type I
TβRII TGFβ receptor type II
TGFβ transforming growth factor beta
TIF1γ transcriptional intermediary factor 1 gamma
TK thymidine kinase
TLP TRAP-1 Like Protein
TPA 12-O-tetradecanoyl phorbol-13-acetate
TRAP1 TβRI-associated protein-1
TRE TPA response element
TRIP-1 TGFβ-receptor interacting protein-1
TSP-1 thrombospondin-1
UEC untransformed epithelial cells
UTR untranslated region
YAP65 Yes-Associated Protein zOFC zebrafish ovarian follicle cells xix ACKNOWLEDGEMENTS
To my family and my previous mentors, especially my elder brother, my parents and my wife, your encouragement, support, understanding, and love have given the strength to follow my dream and to come through the tough years in my life.
I wish to express my great appreciation for my current and former thesis committee members, current and former interim committee Chairs, and former committee
Chairs, including Drs. David J. Spector, Anita K. Hopper, Sarah K. Bronson, Mark
Kester, Keith C. Cheng, Hui-Ling Chiang, Jiyue Zhu, Patrick G. Quinn, Michael F.
Verderame, Kathleen M. Mulder and Maricarmen D. Planas-Silva. I am grateful to current thesis committee for their guidence, encouragement and dedication to me to support me finish my graduate study here. I am also grateful to my former thesis committee for your expertise, constructive criticism and support, which not only guided me though my study here and will steer my future as well. Special thanks I want to give to Drs. David J. Spector, Kent E. Vrana, Anita K. Hopper, Mark Kester, Sarah K.
Bronson, Keith C. Cheng, Michael F. Verderame and Mala Chinoy. Without your support and critical guidance, I would probably not make it here now, therefore I really don’t know how to put in words to thank you, and I will keep doing research the way you guided me through my study here.
To the Genetics faculty, the faculty at the Pharmacology Department, staff and graduate student of the Genetics program and of the Pharmacology Department, I thank you all for your support and encouragement that have made my graduate study here xx memorable. Special thanks I want to give to Drs. Keith C. Cheng and Robert Levenson’s lab (especially Jessica A. Croushore) to teach and help me with the zebrafish experiments.
I’d like to thank Dan Krissinger and Rob Brucklacher at our Functional Genomics
Core Facility. Their work was a big help to my research, especially their help with
Quantitative Real-Time PCR analyses.
I would also like to acknowledge former graduate student Dr. Yangrong Zhang in
Dr. Hancock’s lab at University Park for her help with gel filtration experiments to provide valuable clues to the research in this thesis. I also want to acknowledge summer undergraduate student (Weeda Nejrabi) who helped me and contributed to the research in this thesis. 1
Chapter 1
Literature Review
1.1 Introduction
Transforming growth factor β (TGFβ) is the prototype for a superfamily of highly conserved ubiquitous peptides. More than 60 distinct TGFβ ligands have been identified, including TGFβs, Activins, bone morphogenic proteins (BMPs) and growth and differentiation factor (GDF), and phylogenetic studies have identified homologous TGFβ family members in humans and all animal model organisms (Feng and Derynck, 2005;
Newfeld et al, 1999). TGFβ signaling pathways have been shown to be essential for embryonic patterning, organogenesis, and adult tissue homeostasis, since its discovery over 20 years ago (Massague et al, 2000; Yue and Mulder, 2001; Shi and Massague,
2003). Genetic alterations in the TGFβ signaling pathways, which inactivate tumor suppressor genes or activate oncogenes, have been implicated in a vast array of human cancers and other diseases as well, highlighting the importance of TGFβ family signaling throughout the organism. For these reasons, there is a growing interest in understanding and therapeutically targeting TGFβ-mediated processes (Yingling et al, 2004; Kaklamani and Pasche, 2004; Gupta et al, 2004; Ishisaki and Matsuno, 2006). Despite advances in our understanding of the mechanisms of TGFβ signaling transduction, the mechanisms of the multifunctional TGFβ signaling is not completely clear yet. Therefore, further studies are required for deeper understanding of the underlying mechanisms of its diverse 2 biological responses. With increasing numbers of laboratories studying TGFβ signaling, additional TGFβ signaling components and pathways are likely to be discovered to mediate the diverse biological responses of this polypeptide growth factor. DYNLRB1 was identified as TGFβ receptor-interacting proteins (Ding and Mulder, 2004; Tang et al,
2002). In this thesis, the regulation of the function of DYNLRB1 and DYNLRB2 by
TGFβ, and the role of DYNLRB1 and DYNLRB2 in TGFβ-mediated signaling in mammalian cells have been investigated, as well as the function of a zebrafish homologue zDYNLRB and its role in TGFβ signaling in zebrafish ovarian follicle cells.
TGFβ family members have been shown to exert a growth inhibitory effect in most cell types (epithelial, endothelial, neuronal and haematopoietic cells) from mature tissues and hematopoietic precursor cells, which involves antiproliferation and apoptosis responses in these cells, thus to maintain tissue homeostasis under normal physiological conditions (Massague et al, 2000; Yue and Mulder, 2001; Shi and Massague, 2003;
Derynck and Zhang, 2003). However, growth inhibition is not the only biological action of TGFβ family members, since they also control various other fundamental processes, such as cell growth inhibition, migration, differentiation, apoptosis, the extracellular matrix (ECM), angiogenesis, and immune response (Massague et al, 2000; Yue and
Mulder, 2001; Shi and Massague, 2003; Derynck and Zhang, 2003). Genetic evidence from gene-ablation studies of TGFβ signaling components does not indicate a growth inhibitory effect from TGFβ during early embryogenesis (Massague et al, 2000;
Massague, 2000). For example, Dickson et al, demonstrated that the primary effect of loss of TGFβ1 in vivo in TGFβ1 null mice is not increased haematopoietic or endothelial 3 cell proliferation, but defective haematopoiesis and endothelial cell differentiation, suggesting the primary role of TGFβ1 in endothelial and haematopoietic precursors is to regulate their differentiation rather than inhibit their proliferation (Dickson et al, 1995). A variety of studies also showed that many cell types can lose the ability to respond to the growth inhibitory effect of TGFβ, and the nature of the cells and their cellular context determine together the final role of TGFβ in the regulation of growth of the cells: growth inhibition, or growth proliferation (Massague, 2000). Loss of responsiveness to growth inhibition by TGFβ in tumor cells may allow such tumor cells gain an advantage by selective inactivation TGFβ’s tumor suppressor activities, while maintaining its tumor promoting activities (Massague et al, 2000; Wakefield and Roberts, 2002). Thus, tight regulation of TGFβ cytokine signal transduction is essential for its role in maintaining adult tissue homeostasis in normal conditions and regulating normal embryonic development.
1.2 TGFβ signaling pathways
There are two types of TGFβ receptors, which are single-pass transmembrane serine/threonine kinase receptors, known as type I TGFβ receptor (TβRI) and type II
TGFβ receptor (TβRII). The TβRII kinase is constitutively active. As sown in Fig. 1,
TGFβ family ligands initiate signaling by binding and bringing together a pairs of each receptor TβRI and TβRII, to form a heterotetrameric receptor complex (Massague, 2000;
Yue and Mulder, 2001; Shi and Massague, 2003; Derynck and Zhang, 2003). This then triggers phosphorylation of intracellular signaling components, initiated by the TGFβ 4
TβRII TβRI cytoplasm P
P P Smad2/3
P Smad4
Nucleus P
P Co-activator TF
Fig. 1 Model for TGFβ regulation of transcription through Smad signaling proteins. P, phosphorylation; TF, transcription factor. 5 constitutive kinase activity of TβRII, which transphosphorylates the adjacent TβRI in the heterotetrameric receptor complex, and thereafter the activated TβRI kinases are capable of phosphorylating and activating other downstream signaling components, like the receptor-activated Smad proteins (R-Smads) (Massague, 2000; Yue and Mulder, 2001;
Shi and Massague, 2003; Derynck and Zhang, 2003). These signaling components either directly exert cytoplasmic effects or translocate into the nucleus to regulate target gene transcription. The final TGFβ signaling outcome is diverse, dependent upon the tissue and cell type, the microenvironment, and presence of other growth factors (Massague,
2000; Yue and Mulder, 2001; Roberts, 2002; Shi and Massague, 2003; Derynck and
Zhang, 2003). TGFβ signaling plays a very important role in maintaining tissue homeostasis in adults as well as in embryonic development processes. Because of such important functions, TGFβ signaling is a frequent target of dysregulation during carcinogenesis, and alteration of many signaling components have been identified in various human cancers (Levy and Hill, 2006).
TGFβ signal transduction is unique in that it activates two signaling pathways from the plasma membrane to the nucleus, the one-step amplification Smad pathway and the multiple-step amplification mitogen-activated protein kinase (MARK) pathway, to regulate its cellular and tissue activities (Massague, 2000; Yue and Mulder, 2001; Shi and
Massague, 2003). There is general agreement that most TGFβ target genes and end points are regulated by the Smad pathway, and that a few of them are regulated by the
Ras-MAPK pathway (Yue and Mulder, 2001; Piek and Roberts, 2001; Roberts, 2002;
Derynck and Zhang, 2003). In certain specific cellular contexts, TGFβ may also involve 6 other signaling pathways, like protein kinase A (PKA), protein kinase C (PKC), protein phosphatase 2A (PP2A), and phosphatidylinositol-3k-kinase (PI3K) and Rho GTPases pathways. Crosstalk exists between these pathways (Yue and Mulder, 2001; Piek and
Roberts, 2001; Roberts, 2002; Derynck and Zhang, 2003).
1.2.1 The Smad pathway
In the Smad pathway, there is only one step of signal amplification between the plasma membrane, where the TGFβ signal is received, and the nucleus, where target genes’ transcription is activated or repressed. The Smad proteins belong to a family of genes now accepted as the major known downstream signaling components of TGFβ family ligands in both invertebrates and vertebrates (Shi and Massague, 2003; Derynck and Zhang, 2003), and their nomenclature as “Smad” in the vertebrates is a merger of Sma in Caenorhabditis elegans and Mad in Drosophila melanogaster (Derynck et al, 1996). They were originally identified as downstream signaling components from genetic screens for second-site mutations to enhance the phenotype of known TGFβ signaling component mutants in Drosophila melanogaster and Caenorhabditis elegans
(Savage et al, 1996; Padgett et al, 1997). To date, three types of Smads have been identified in the family of eight mammalian Smad proteins: receptor-activated
Smads (R-Smads), which includes Smad1-3, Smad5 and Smad8; common Smad
(Co-Smad), which includes only Smad4; and inhibitory Smads (I-Smads), which includes Smad6 and 7. 7 1.2.1.1 R-Smads
Broadly speaking, R-Smads are pathway specific: Smad2 and 3 transduce signals downstream of the TGFβ/Nodal/Activin ligands, while Smad1, 5 and 8 function downstream of BMP and GDF. However, exceptions have been reported, like
TGFβ signaling through Smad1 in human breast cancer cells (Liu et al, 1998), and in intestinal epithelial cells (Yue et al, 1999a and b), and through Smad1 and
5 endothelial cells (Oh et al, 2000; Goumans et al, 2002).
The Smad2 and 3 R-Smads are phosphorylated by the activated TβRI kinases in the heterotetrameric receptor complex, and then translocate into the nucleus and regulate target gene transcription through the interaction with more than 60 nuclear proteins (Feng and Derynck, 2005). Although Smad2 and 3 are over 95% homologous and each activated by TGFβ (or Activin, etc.), recent studies showed that they have distinct functions (Felici et al, 2003; Ju et al, 2006;
Kim et al, 2005; Kretschmer et al, 2003; Kurisaki et al, 2001; Levy and Hill,
2005; Liu et al, 2003; ten Dijke and Hill, 2004; Uemura et al, 2005). However, the final signaling outcome is dependent upon other signaling pathways initiated by TGFβ as well as signaling pathways initiated by other growth factors in the specific cellular context (Yue and Mulder, 2001; Roberts, 2002; Derynck and
Zhang, 2003; Feng and Derynck, 2005).
1.2.1.2 Co-Smads
Until very recently, there has been only one mammalian Co-Smad, Smad4, 8 identified. Smad4 has had a central role, since TGFβ family signaling is very much dependent on it in that it forms complexes with all R-Smads to render them transcriptionally active (Feng and Derynck, 2005). A recent new finding by Massague and colleagues demonstrated that transcriptional intermediary factor 1γ (TIF1γ) is possibly a second Co-Smad that can compete with Smad4 and mediate different transcriptional effects (He et al, 2006). They showed that TIF1γ competes with Smad4 to bind Smad2/3 in hematopoietic, epithelial and mesenchymal cells in response to TGFβ, and that TIF1γ mediates the differentiation response of hematopoietic cells by the TGFβ-
Smad pathway, while Smad4 mediates the antiproliferative TGFβ effect on hematopoietic cells (He et al, 2006). However, since both TIF1γ and Smad4 are ubiquitously expressed, it remains to be determined whether TIF1γ regulates the differentiation of cells other than hematopoietic cells, and what role TIF1γ plays in epithelial and mesenchymal cells. TIF1γ selectively binds Smad2/3 in competition with
Smad4, but it is unknown whether similar molecules exist to bind to Smad1, 5 and 8 in competition with Smad4 for the BMP and GDF pathway.
1.2.1.3 I-Smads
I-Smads, the third type of Smads, structurally distinct from the other two types of
Smads, antagonize the Smad signaling pathway as well as other TGFβ signaling pathways. Whereas Smad6 is implicated in preferential inhibition of BMP-like signaling pathway, Smad7 is important to control TGFβ-like signaling (Nakao et al, 1997; Hata et al, 1998; Fujii et al, 1999; Ishisaki et al, 1999; Hanyu et al, 2001; Miyazono, 2000). 9 Several mechanisms have been identified so far for Smad7 to negatively regulate TGFß signaling: physical blockage of R-Smad’s phosphorylation, recruiting phosphatase such as protein phosphatase 1 (PP1) to dephosphorylate TßRI, scaffold for assembling a complex of TAK1- MKK3 and p38 MAPK to activate p38 MAPK, and recruiting ubiquitin E3 ligase such as Smurf-1 and Smurf-2 for proteasome-mediated degradation of the activated receptors (Ebisawa et al, 2001; Edlund et al, 2003; Kavsak et al, 2000; Shi et al, 2004). The expression of Smad7 is induced by TGFβ, thus forming an autoinhibitory feedback loop to terminate TGFβ signaling (Brodin et al, 2000; Nakao et al, 1997). Other cytokine signaling pathways have also been shown to induce Smad7 expression, such as the Jak/Stat signaling pathway initiated by epidermal growth factor
(EGF) and interferon-γ (Ulloa et al, 1999), and the NF-κB signaling pathway initiated by tumor growth factor-α and interleukin-1β (Bitzer et al, 2000; Nagarajan et al, 2000).
Therefore, Smad7 integrates signals from multiple signaling pathways and utilizes multiple mechanisms to negatively regulate TGFβ signaling, suggesting the central role of Smad7 as a potent natural inhibitor of TGFβ responses.
1.2.2 The MAPK pathways
The MAPK pathways also transduce signals initiated by TGFβ. MAPK pathways lead to rapid phosphorylation and activation of nuclear transcription factors in response to numerous extracellular signals. Activation of these signaling pathways results in transcriptional regulation of proteins involved in diverse cellular processes including cell proliferation, differentiation, apoptosis, cytokine production, and cytoskeletal reorganization. Three distinct groups of MAPKs have been identified in mammalian 10 cells, including the extracellular signal-regulated kinases (ERK1 and ERK2, also known as p44/p42 MAPKs), the stress-activated protein (SAP) kinases known as c-Jun N- terminal kinases (JNK1, JNK2 and JNK3), and the p38 MAPK (Yue and Mulder, 2001;
Piek and Roberts, 2001; Derynck and Zhang, 2003). These pathways incorporate sequential steps of signal amplification between the plasma membrane and the nucleus, and each step allows catalytic protein phosphorylation and activation, and amplification of the received signal, so that a much larger response can be produced from a small amount of stimulus.
TGFβ has been demonstrated in various cell types to activate ERKs (Hartsough and Mulder, 1995; Frey and Mulder, 1997; Hu et al, 1999; Ravanti et al, 1999; Funaba et al, 2002), JNKs (Frey and Mulder, 1997; Hartsough and Mulder, 1997; Atfi et al, 1997;
Engel et al, 1999; Hocevar et al, 1999; Mazars et al, 2000; Brown et al, 2002; Tian et al,
2004), and p38 MAPK (Hanafusa et al, 1999; Ravanti et al, 1999; Adachi-Yamada et al,
1999; Yu et al, 2002; Tian et al, 2004). ERK activation by TGFβ may involve Ras as upstream regulator (Yue and Mulder, 2001; Wakefield and Roberts, 2002). The rapid activation of MAPK within 5-30 minutes of TGFβ treatment was demonstrated as evidence for direct activation of MAPK by the TGFβ signaling pathway (Yue and
Mulder, 2001). More evidence were obtained from experiments employing over- expression of a dominant-negative mutant of Ras, and specific MAP/ERK kinase
(MEK1) inhibitor showing that TGFβ’s ability to activate ERK1 is blocked (Hartsough et al, 1996; Yue et al, 1999a and b), and experiments in cells over-pressing a Smad-binding defective mutant of TβRI or dominant-negative Smads and in Smad4-deficient cells 11 displaying maintained ability to activate p38 and JNK MAPK by TGFβ (Engel et al,
1999; Yu et al, 2002). Recent data suggest that the balance between the Smad pathway and the MAPK pathway as well other pathways plays an important role in the final TGFβ response, and changing the balance toward the MAPK pathway may allow cells to escape growth inhibition and apoptosis and acquire invasive and metastatic features of the tumor cells (Lehmann et al, 2000; Park et al, 2000; Yan et al, 2001; Zavadil et al, 2001; Liu et al, 2004; Tian et al, 2004). However, the in vitro feature of many such studies as well the specificity of the kinase inhibitors frequently used in such studies raised questions on the specific role of activating the MAPK pathway by TGFβ (Moustakas and Heldin, 2005).
The very recent identification of a possible alternative Co-Smad other than Smad4 for
TGFβ signaling (He et al, 2006) also raised questions on many studies previously performed in Smad4-deficient cells. Therefore, the exact mechanisms for the activation of these MAPK pathways by TGFβ are poorly understood, and remain to be explored and clarified.
1.2.3 Other signaling pathways
TGFβ has been shown to activate PKC and PKA (Choi et al, 1999; Hirota et al,
2000; Sylvia et al, 2000; Yakymovych et al, 2001), Calcium-Calmodulin-dependent protein kinase II (CamKII) (Wicks et al, 2000), PP2A (Griswold-Prenner et al, 1998;
Petritsch et al, 2000), the Rho GTPase (Edlund et al, 2002; Bhowmick et al, 2001), PI3K
(Bakin et al, 2000; Runyan et al, 2004), and NF-κB (Arsura et al, 1996; Kon et al, 1999;
Bitzer et al, 2000), etc, but their effects appear to be highly cell type specific. For example, the protein phosphatase 2A (PP2A) was recently identified due to the specific 12 interaction between its regulatory subunit Bα and the activated TβRI. It has been shown that Bα is directly phosphorylated by the activated TβRI to regulate PP2A catalytic activity to dephosphorylate and inactivate cell cycle regulatory proteins such as Akt,
ERK and p70 S6 kinase (p70s6k), and has been linked to control cell cycle progression
(Griswold-Prenner et al, 1998; Petritsch et al, 2000). TGFβ has also been shown to directly activate PI3K, as indicated by phosphorylation of its downstream signaling component Akt, and such activation has been implicated in TGFβ induced epithelial-to- mesenchymal transdifferentiation (EMT) (Bakin et al, 2000; Runyan et al, 2004;
Moustakas and Heldin, 2005). However, a lot remains to be clarified on the roles of these pathways in TGFβ-mediated responses and a deeper understanding of the mechanisms of their activation by TGFβ.
1.3 Role of TGFβ signaling in cancer and development
1.3.1 Role in cancer
It is now commonly accepted that TGFβ suppress tumorigenesis in early stages of tumor development, but promote tumor growth and metastasis in later stages (Tian et al,
2004; Wakefield and Roberts, 2002). The tumor suppressor activity of the TGFβ signaling pathway derives largely from its growth inhibitory effects in most normal cell types of adult tissues and tumor cells during the early stages of carcinogenesis. TGFβ arrest cells in the G1 phase of the cell cycle. Central to such growth inhibitory effects is its ability to downregulate the expression of c-Myc, a key cell cycle regulator, and to upregulate the expression of cyclin-dependent kinase (cdk) inhibitors p15, p21 and p27, 13 also important regulators of cell cycle (Chen et al, 2001; Claassen and Hann, 2000; Datto et al, 1995; Moustakas and Kardassis, 1998; Pardali et al, 2000; Wakefield and Roberts,
2002; Warner et al, 1999). In addition to cell cycle control, TGFβ signaling is also required for apoptosis induction and maintenance of genomic stability, etc. However, the exact mechanisms of such effects are not clear.
Because of TGFβ receptors’ importance to initiate intracellular TGFβ signaling, they naturally become frequent targets during carcinogenesis. Direct evidence that suggest involvement of disruption of TGFβ signaling in cancers actually comes from studies on the TβRII gene, TGFBR2 (Yue and Mulder, 2001; Piek and Roberts, 2001;
Levy and Hill, 2006; Sjoblom et al, 2006). Due to a 10-base pair poly-adenine repeat in
TGFBR2 gene exon3, mutations have been found in this region in 90% of colon cancers with microsatellite instability (either hereditary non-polyposis colorectal cancer or sporadic cases), and also in about 15% of colon cancers without microsatellite instability
(Yue and Mulder, 2001; Piek and Roberts, 2001; Levy and Hill, 2006). These mutations usually result in a premature stop codon and truncated protein unable to transduce TGFβ signals. There are also other missense mutations found in TβRII in various cancers, such as E526Q, D404G and T315M (Yue and Mulder, 2001; Piek and Roberts, 2001; Levy and Hill, 2006). D404G mutation has been reported as a dominant-negative mutation by inhibiting the function of wild-type TβRII through preventing its appearance on the cell plasma membrane (Tanaka et al, 2000). But mutations are not the only way to inactivate the function of the tumor suppressor TβRII. TβRII expression is frequently decreased in various advanced cancers through epigenetic silencing of the TGFBR2 promoter (Yue 14 and Mulder, 2001; Piek and Roberts, 2001; Levy and Hill, 2006). There is strong evidence that when signaling from TβRII is decreased, growth inhibitory effect of TGFβ is selectively lost, while oncogenic effects (such as invasive growth) are promoted (Chen et al, 1993; Oft et al, 1998). This is what is exactly has been demonstrated for T315M mutation of TβRII: selectively blocking TGFβ-mediated growth arrest, while enhancing the metastatic potential of tumor cells (Yue and Mulder, 2001; Piek and Roberts, 2001).
Similarly, mutation of TβRI and epigenetic silencing of its promoter have been reported in prostate, colon, gastric and ovarian cancers (Levy and Hill, 2006; Piek and Roberts,
2001).
The major TGFβ signaling pathway, the Smad pathway, is also frequently targeted during carcinogenesis. Smad4, another tumor suppressor, was originally cloned and named as DPC4 (deleted in pancreatic cancer, locus 4). It has been frequently mutated in pancreatic and colorectal cancers, making cells lose the growth inhibitory effects of TGFβ (Hahn et al, 1996; Sjoblom et al, 2006). Subsequent studies found alteration of TGFβ signaling in almost all pancreatic cancers (Goggins et al, 1998). The tumor suppressor role of Smad4 is corroborated by observation that Smad4 heterozygous mice have increased risk for gastrointestinal cancers (Friedl et al, 1999; Taketo and
Takaku, 2000). Mutations of Smad2 have been found in sporadic colorectal carcinomas and lung cancers, and the proteins of such mutations are non-functional in transducing the growth inhibitory effects of TGFβ, thus strongly implicating the role of disruption of the
TGFβ signaling in such cancers (Eppert et al, 1996; Sjoblom et al, 2006). However, only very recently, Smad3 mutation has been found in human colorectal cancers (Sjoblom et 15 al, 2006). Smad3 knockout mice harboring a deletion in exon 2 were generated and exhibited a predisposition to colorectal adenocarcinomas, but the incidence of cancer was background dependent (Zhu et al, 1998). In contrast, other groups generated different
Smad3 knockout mice with different genetic backgrounds targeting exon 8 (Yang et al,
1999) or exon 1 (Datto et al, 1999), but have not found colon cancer in their mice. Table
1 lists the phenotypes of the TGFβ signaling component knockout mouse mutants. The reason for this discrepancy is unknown. It could be due to a gain-of-function of the
Smad3 allele that causes the colon cancer, or influences on penetrance from some genetic modifiers in the different genetic backgrounds, or something else currently unknown yet.
The embryonic lethality of the homozygous knockout mice targeting Smad2 or Smad4 prevents studies on tumorigenesis in these mice. Smad7 acts to limit the duration of
TGFβ signaling. Smad7 amplification and/or over-expression have been reported in patients with colorectal, pancreatic and endometrial cancer (Boulay et al, 2001 and 2003;
Dowdy et al, 2005; Kleeff et al, 1999), and Smad7 over-expression has also been shown to result in complete loss of TGFβ’s growth inhibitory response and enhanced tumorigenicity in pancreatic cancer, but no effect on induction of PAI-I by TGFβ (Kleeff et al, 1999). But, Smad7 knockout mouse has not been reported yet.
Overproduction of TGFβ ligand is also commonly found in many cancers, like colorectal cancer, gastric carcinoma, and prostate cancer, and has even been correlated with late stage tumor progression (Levy and Hill, 2006; Yue and Mulder, 2001). This is consistent with TGFβ’s ability to induce epithelial-mesenchymal transition, migration and invasion of tumor cells, and its paracrine effects (fibrosis, angiogenesis, 16
Table 1. Knockout mouse models of TGFβ family signaling proteins
Knock out Phenotype References mouse model Dickson et al, 1995; 50% die from yolk sac defects; 50% postnatal TGFβ1 Kulkarni et al, 1993; death from inflammatory disorders Shull et al, 1992 Craniofacial defects; skeletal defects; heart defects; TGFβ2 Sanford et al, 1997 renal defects
TGF 3 Kaartinen et al, 1995; β Cleft palate; delayed lung development Proetzel et al, 1995
Embryonic lethal at midgestation, severe defects Dong et al, 1996; TβR1 in the vascular development of the yolk sac and Elvin et al, 2000; placenta, and lacked circulating red blood cells; Larsson et al, 2001; endothelial cells enhanced cell proliferation, Larsson et al, 2003 improper migratory behavior, and impaired fibronectin production in vitro.
Disrupted hematopoesis and vasculogenesis of the Oshima et al, 1996; T R1I β yolk sac conditional knockout a lethal Leveen, et al, 2002 inflammatory
Smad2 homozygous No mesoderm development; no anteroposterior axis; Perigastrulation lethality. Nomura and Li, 1998; Defective in extra embryonic ectoderm and Waldrip et al, 1998; mesoderm induction/formation. Abnormalities in Weinstein et al, 1998. anterior-posterior axis
Severe mucosal infection and immune Datto et al, 1999; dysregulation. Colon cancer. Accelerated wound Smad3 Tomic et al, 2002; healing. Osteoporosis, osteoarthritis and Yang et al, 1999; skeletal defects. Reduced litter sizes; defective Zhu et al, 1998; immune response; accelerated wound healing; Ashcroft et al, 1999; excisional ear wound enlargement; colorectal Arany et al, 2006. tumors; defective folliculogenesis
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Knock out Phenotype References mouse model Homozygotes: Perigastrulation lethality. Defective in extraembryonic ectoderm and mesoderm induction/formation. Lack of ectodermal cell Sirard et al, 1998; Smad4 proliferation Yang et al, 1998; Xu et al, 2000 Heterozygotes: nodevelopmental abnormality, but increased proliferation of gastric polyps and tumors.
liver- Pronounced accumulation of iron in Wang et al, 2005 specific liver, kidney, and pancreas. Smad4 knockout skin specific Skin hair follicle defects and squamous cell Qiao et al, 2006 Smad4 carcinoma knockout
Smad1 mutant mice die at approximately 9.5- Smad1 10.5 days postcoitum. Extraembryonic defects: Lechleider et al, 2001; no or few primordial germ cells, lack of placenta Tremblay et al, 2001 and failure to establish a definitive embryonic circulation.
Embryonic lethal; Angiogenesis defects (enlarged Chang et al, 1999; Smad5 blood vessels surrounded by decreased numbers of vascular smooth muscle cells); mesenchymal Yang et al, 1999 apoptosis, gut, heart, and craniofacial defects.
Perinatally normal, but exhibit an age-dependent Smurf1 Yamashita et al, 2005 increase of bone mass.
Homozygous was embryonic lethal before E6.5 Dab2 due to defective cell positioning and structure Yang et al, 2002 formation of the visceral
FKBP12 Severe dilated cardiomyopathy and ventricular Shou et al, 1998 septal defects, but normal skeletal muscle. 18 inflammation and immunosuppression) on stromal tissues (Roberts, 1998).
1.3.2 Role in development
During development, the multi-functional TGFβ family signaling acts to promote the differentiation of pluripotent cells into specific cell types, and plays an important role in early embryo development processes such as axis formation (the dorsal-ventral axis, the anterior-posterior axis, and the left-right axis), tissue patterning and development of the three germ layers (ectoderm, mesoderm, and endoderm), or late development stage processes like organ specification, or the complex processes of morphogenetic movements of cells during the entire development period. A combination of genetic, embryological and molecular analyses in many model organisms (like frog, zebrafish, mouse and chicken) has provided much insight into the mechanisms of genetic control of these processes (Schier and Talbot, 2005; De Robertis et al, 2000; Kimelman and Griffin,
2000). In frog and zebrafish, many TGFβ family ligands (including Activin, Nodal,
BMPs) play important roles in dorsal-ventral patterning of the embryo. BMPs are known to promote ventral mesoderm development through a dorsal-ventral morphogen gradient across the embryo, while dorsalization of the embryo is achieved through dorsalizing factors (such as TGFβ family members Activin and Nodal), as well as the action of antagonists (like Noggin, Follistatin, and Chordin) to counter ventralizing factors (such as
BMP2) (Jones et al, 1995; Fainsod et al, 1997; Rodaway et al, 1999; Toyama et al, 1995;
Schier and Talbot, 2005). TGFβ family signaling has also been shown to play important role in dorsal-ventral patterning in Drosophila (Arora et al, 1994; Ashe and Levine, 1999; 19 Decotto and Ferguson, 2001). However, the role of TGFβ family members in dorsal- ventral axis formation has not yet been established in mouse or chicken.
The major TGFβ downstream intracellular signaling components, the Smad2, 3 and 4, are expressed ubiquitously throughout development in all adult tissues and are also essential for normal embryonic development. Their targeted knockout mouse models
(Table 1) have revealed different roles for them at different stages of embryogenesis.
Smad2 and Smad4 knockout mouse studies have shown that both are essential for gastrulation, since both knockout mice show perigastrulation lethality, and defective extraembryonic ectoderm and mesoderm induction, as well as abnormal anterior- posterior formation, although Smad4 null embryos exhibit more severe phenotype (Sirard et al, 1998; Waldrip et al, 1998; Weinstein et al, 1998; Yang et al, 1998). Since Smad2 and Smad3 are nearly identical, targeted inactivation of Smad3 gene generates surprisingly distinct phenotypes (Ashcroft et al, 1999; Datto et al, 1999; Yang et al,
1999). Two Smad3 knockout mice targeting exon 8 (Yang et al, 1999) or exon 1 (Datto et al, 1999) are viable and have been reported to display severe mucosal infection and immune dysfunction, osteoporosis and other skeletal defects (Datto et al, 1999; Yang et al, 1999), suggesting an important role for Smad3 in mucosal immune response and proper development of the skeleton. But such phenotypes have not been found in the viable Smad3 null mice harboring a deletion in exon 2 (Zhu et al, 1998), which might be due to some genetic modifiers influencing penetrance in the different genetic backgrounds or something else currently unknown yet, as indicated above. So, these studies suggest that Smad2 and Smad3 may regulate distinct TGFβ responses through 20 different target genes, despite their high homology, which is consistent with the results of functional differences between these Smads reported in cell culture studies (Kim et al,
2005; Kretschmer et al, 2003; Levy and Hill, 2005; Roberts et al, 2006).
TGFβ family ligands also activate the Ras/MAPK pathway, including p38
MAPK, JNKs and ERKs. Recent studies in zebrafish and Drosophila show that the
MAPK p38 is required specifically on the dorsal side to control the cell fate in dorsal blastomeres, and activation of p38 occurs in the future dorsal region of the embryo, but not in ventralized embryos after the dorsalizing signals are inhibited, indicating p38’s important role in dorsal-ventral axis formation (Suzanne et al, 1999; Fujii et al, 2000).
MKP3, a MAP kinase phosphatase, whose earliest expression is restricted to the future dorsal region of the embryo, was also shown to be required for dorsoventral patterning in zebrafish at the onset of gastrulation (Tsang et al, 2004). But, it remains to be determined whether p38 MAPK acts in parallel to, or crosstalk with the Smad signaling pathway to specify the initiation of dorsalizing signals, and the presence of such an intrinsic signaling pathway in other organisms.
TGFβ family signaling is also essential for organogenesis of many systems, including the nervous system and skin (Hawley et al, 1995; Bellusci et al, 1996; Zhao et al, 2000; Hogan and Kolodziej, 2002). TGFβ family members promote organogenesis by regulating a variety of cellular processes such as branching morphogenesis, EMT, cell proliferation and apoptosis (Hogan and Yingling, 1998; Hogan, 1999; Hogan and
Kolodziej, 2002). Branching morphogenesis is very important for organ development such as the lung and kidney. BMP4 is highly expressed in lung buds. BMP4 over- 21 expression in the lung buds reduces overall lung size, while blocking its signaling reduces it (Bellusci et al, 1996; Hogan and Yingling, 1998; Hogan and Kolodziej, 2002), suggesting BMP4’s important role in promoting the differentiation of lung buds into mature lung cells. TGFβ3 null mice exhibit perinatal death from unique developmental defects in the lung and palate, suggesting an essential function for TGFβ3 in the normal morphogenesis of palate and lung (Kaartinen et al, 1995). TGFβ2-null mice also exhibit perinatal mortality, but with a wide range of developmental defects, including cardiac, lung, craniofacial and urogenital defects, which involve various processes such as epithelial-mesenchymal interactions, ECM production and tissue remodeling (Sanford et al, 1997). But, surprisingly, there is no overlap on phenotypes between the knockout mice targeting different TGFβ isoforms (Table 1), indicating numerous non-compensated biological roles for these different isoforms. The processing and activation of the different TGFß isoforms may play important roles in determining such different phenotypes (Annes et al, 2003).
1.3.3 Role in ovarian follicle development
The TGFβ family signaling has profound effects on wide-ranging processes in many tissues and organ systems, including the ovary (Massague, 1998; Knight and
Glister, 2003; Pangas and Matzuk, 2004). Once established, the ovary has two major functions: first, to produce mature oocytes for fertilization, and, second, to produce hormones essential for the sexual maturation and reproductive ability of the female, as well as the development of secondary sexual characteristics. An ovarian follicle undergoes a series of complex developmental processes to become a preovulatory 22 follicle. During the process of folliculogenesis, the oocytes enlarge and mature. It has been established that the somatic cells (granulosa and theca cells) of the follicle provide a proper yet dynamic microenvironment that supports and nurtures the appropriate development of the oocyte (Eppig et al, 2002; Knight and Glister, 2003; Ge, 2005).
However, it is not completely known yet whether one cell type, either the somatic cell or the germ cell, or both determine the overall rate of follicle development (Erickson and
Shimasaki, 2000; Peng et al, 2000; Eppig et al, 2002; Ge, 2005). In recent years, exciting progress has been made towards unravelling the complex intraovarian control mechanisms. Although hormonal (such as Gonadotrophin and steroid hormones) regulation of follicle growth, apoptosis, and differentiation is critical for normal ovarian follicle development, it is the locally produced autocrine and paracrine signals in the ovarian follicles (particularly the TGFβ family signals) that allow individual follicles to grow and develop (Gougeon, 1996; Robker and Richards, 1998).
The role of the TGFβ family ligands in ovarian folliculogenesis has been studied extensively in animals. It has been demonstrated that various TGFβ family members
(TGFβ, Activin, Inhibin, GDF9, etc.) and their signaling components are expressed by ovarian somatic cells and oocytes in a developmental, stage-related manner and function as important intraovarian local regulators of folliculogenesis (McNatty et al, 1999;
Montgomery et al, 2001; Tomic et al, 2002 and 2004; Xu et al, 2002; Kohli et al, 2003 and 2005; Knight and Glister, 2003; Pangas and Matzuk, 2004). For example, all three mammalian TGFβ isoforms and their receptors are expressed in the sheep ovarian follicles, and their major intracellular signaling components Smad2 and Smad3 are 23 expressed in a stage specific manner in the rat ovarian follicle development, thus allowing different effects of the TGFβ family ligands to use the same signaling pathways during the ovarian follicle development.
Unfortunately, due to the embryonic lethality or perinatal mortality of the knockout mice targeting TGFβ1, TGFβ2, TGFβ3, TβRII, Smad2 and Smad4 (Dickson et al, 1995; Oshima et al, 1996; Kaartinen et al, 1995; Sanford et al, 1997; Weinstein et al,
1998; Yang et al, 1998), little can be gained about TGFβ signaling in ovarian follicle development. However, viable knockout mice do provide some insights about the role of
TGFβ signaling in ovarian follicle development. Smad3 knockout mice targeting exon 1, exon 2 or exon 8, are viable. Smad3 null mice targeting exon 8 exhibit slowed follicle growth, atretic follicles and degenerated oocytes, and reduced fertility, indicative of impaired ovarian follicle development and abnormal ovarian function (Tomic et al, 2002 and 2004). However, Smad3 null mice targeting exon 2 are fertile (Zhu et al, 1998).
Very recently, TGFβ1 null mice are reported to be viable and show severely impaired reproductive capacity and infertility (Ingman et al, 2006), suggesting that TGFβ1 plays a critical role in normal ovarian follicle development. Originally a different line of TGFβ1 null mice on a different genetic background was prenatally lethal (Dickson et al, 1995) or died by 3-4 weeks of age (Kulkarni et al, 1993; Shull et al, 1992). However, the reason for such different phenotypes is unknown, but may reflect differences in the genetic background of the generated knockout mice, such as influences on penetrance from genetic modifiers and genes conferring compensatory effect. Thus, for the majority of the components of the pathway, it may be necessary to generate tissue-specific 24 conditional and/or inducible knockout mouse models or to generate mouse models using the RNAi techniques in order to determine the role of the TGFβ isoforms and their signaling components during ovarian follicle development.
In vitro studies show that TGFβ regulates ovarian follicle development by modulating cell proliferation and differentiation. Although TGFβ is generally considered to be an inhibitor of cell proliferation, both inhibitory and stimulatory actions have been reported on follicle cells in vitro, depending on the species, the stage of differentiation and the presence of other growth factors (Attia et al, 2000; Coskun and Lin, 1994; Juneja et al, 1996; Lerner et al, 1995; Knight and Glister, 2003; Kohli et al, 2005; Liu et al,
1999; Pangas and Matzuk, 2004; Skinner et al, 1987). For example, TGFβ has been reported to inhibit proliferation of bovine granulosa cells harvested from large antral follicles (Skinner et al, 1987) and to prevent zebrafish premature oocyte maturation
(Kohli et al, 2003 and 2005), but it has also been reported to stimulate growth of preantral follicles dissected from adult mice ovaries (Liu et al, 1999).
Therefore, more studies are warranted to fully define the precise role of TGFβ signaling in the ovarian follicle development. However, to remember, other TGFβ family members, such as Activin, BMP, and other growth factors, such as epidermal growth factor, are operational with TGFβ signals in ovarian follicle development.
1.4 Regulation of TGFβ signaling
TGFβ family signaling is delicately regulated at multiple levels, both positively and negatively. 25 1.4.1 Regulation at level of ligand production
Ligand is the start point of the cytokine pathway, and the membrane receptor is only the midpoint of the cytokine pathway. So, the function and regulation of the cytokine pathway start with the production and secretion of each cytokine. TGFβ is not an exception. Three highly homologous isoforms of TGFβ (TGFβ1, TGFβ2, and TGFβ3, collectively referred to as TGFβ) have been cloned in mammals and localized to different human chromosomes (19q13, 1q41, and 14q24, respectively) (Kim et al, 1990; Piek and
Roberts, 2001; Roberts, 1998). Expression of the three isoforms is under the control of distinct promoters. TGFβ1 promoter lacks classic TATA box and contains multiple regulatory sites that can be activated by AP1 proteins including c-Jun, c-Fos, and JunD, and various oncoproteins including Ras, Src, and HTLV1 tax, as well as Smad binding elements (SBEs) (Roberts, 1998; Mulder, 2000). TGFβ1 is the most abundant isoform in most cells and tissues, and it is most often found abnormal in disease pathogenesis, including carcinogenesis, consistent with the complex regulation of its promoters
(Roberts, 1998). TGFβ1 stimulates its own production by an autocrine manner (Roberts,
1998; Mulder, 2000). It has been demonstrated in Dr. Mulder’s laboratory that rapid activation of Ras, Erks, and JNKs in proliferating TGFβ1-sensitive untransformed epithelial cells (UECs) and human colon carcinoma cells (HCCCs) is required for AP-1 complex formation at the TGFβ1 promoter and its autocrine production (Mulder, 2000).
It also shows a TGFβ-induced AP-1 complex containing JunD, Fra-2, possibly c-Jun, and
Fos B, as well as a dependence on Smad3 for its autocrine production in UECs, and an
AP-1 complex at the proximal AP-1 site with c-Fos as the major detectable component in 26 HCCCs (Mulder, 2000; Yue and Mulder, 2000; Liu et al, 2006a). However, increased
TGFβ1 levels in most malignant cells result in further increased production of TGFβ1 by malignant cells, which are no longer inhibited by TGFβ (autocrine effects), and stimulation of the still TGFβ1-responsive stromal tissues for enhanced angiogenesis and matrix deposition (paracrine effects) (Piek and Roberts, 2001; Mulder, 2000).
Distinct from TGFβ1 promoter, TGFβ2 and TGFβ3 promoters both contain TATA boxes and a common proximal CREB binding site, which suggest hormonal and developmental control (Piek and Roberts, 2001). Very recently, it is reported in Dr.
Mulder’s laboratory that JNK and p38 MAPK activation is required for TGFβ3 autocrine production in UECs, and the major components of the TGFβ3-induced AP1 complex are
CREB1 and Smad3 (Liu et al, 2006b).
1.4.2 Regulation of ligand processing and activation
It is quite unique that, unlike many peptide growth factors that act on a restricted set of target cells, TGFβ is produced by and can act on nearly every cell type. However, only a very limited fraction of the total produced TGFβ is made mature and available for signaling, and the complexity and ubiquity of TGFβ responses is both cell type- and context-dependent, and isoform-specific (Annes et al, 2003). The release of activated mature TGFβ anywhere other than the appropriate location may produce an unwanted or even deleterious response. These all indicate a critical role of the regulation of TGFβ sequestration, processing, activation and presentation in the precise temporal and spatial control of its functions. 27 TGFβ is first synthesized as large homodimeric biologically inactive precursors.
The inactive precursor is then proteolytically processed by the mammalian convertase, furin, to yield a 25-kD homodimeric the mature protein TGFβ ligand as well as a dimeric propeptide (termed the latency-associated propeptide, LAP) (Annes et al, 2003; Beck et al, 2002; Dubois et al, 1995 and 2001). While earlier reports suggested an intracellular cleavage by furin, recent genetic evidence showed essential extracellular cleavage of the
TGFβ precursor by furin convertases (Beck et al, 2002; Zacchigna et al, 2006). Unlike most propeptides that have little affinity for the mature protein, LAP forms a complex with the mature TGFβ (called latent TGFβ complex) in which it strongly binds to the mature ligand, and thus sequesters the mature TGFβ from binding to its cell surface receptors. The LAP usually also associates with latent TGF-beta binding proteins
(LTBP) via disulfide-bound to form a large latent complex which is targeted to specific locations in the extracellular matrix by the appropriate LTBP. This latent TGFβ complex must be further processed to release the mature ligand to interact with its receptor.
1.4.2.1 Regulation of ligand availability for processing
The TGFβ precursor is proteolytically cleaved by furin to release the mature protein TGFβ ligand and the LAP. It was originally reported that an intracellular cleavage of the TGFβ precursor by furin occurs within the trans-Golgi network in the constitutive secretory pathway of almost every cell type (Dubois et al, 1995 and 2001;
Piek and Roberts, 2001). However, recent studies showed that, in addition to the Golgi apparatus, furin is also located in endosomes and on the plasma membranes in a variety of cell types including intestinal and renal epithelial cells, and endothelial cells (Mayer et 28 al, 2004; Koo et al, 2006; Teuchert et al, 1999), and genetic and biochemical evidence demonstrated essential extracellular cleavage of endogenously produced TGFβ precursors by furin to produce mature ligands (Beck et al, 2002; Koo et al, 2006).
Emilin1 (elastin microfibril interface-located protein 1) is a secreted glycoprotein associated with the ECM of blood vessels (Bressan et al, 1993). Recently, a combination of genetic and biochemical evidence showed that Emilin1 binds specifically to the immature TGFβ precursor but not the free mature TGFβ or the latent TGFβ associated with LAP, thus blocking the TGFβ precursor proteolytic processing to mature TGFβ by furin in the extracellular space (Zacchigna et al, 2006). Zacchigna et al demonstrated that
Emilin1 null mice have increased levels of active TGFβ in the vasculature ultimately leading to hypertension, and this phenotype is rescued by reduction of TGFβ gene dosage
(Zacchigna et al, 2006). It was also demonstrated that Emilin1 over-expression results in accumulation of unprocessed immature TGFβ precursors, whereas the TGFβ precursor is quickly processed in Emilin1 null cells and its stability can be restored with specific furin inhibitors, thus Emilin1 phenocoping furin inhibition (Zacchigna et al, 2006). This places Emilin1 upstream of furin in the TGFβ precursor processing in the extracellular space but downstream of its production in the cells. This also provides more evidence for essential extracellular processing of the TGFβ precursor for its maturation, and is also the most upstream step discovered so far in the regulation of the TGFβ precursor processing.
But, Emilin1 null mice develop normally (Zacchigna et al, 2006), suggesting the presence of redundant genes or modifier genes, etc. It is not clear whether Emilin1 has any role on the TGFβ precursor processing in organ systems other than the vasculature and the lung, 29 and what signal regulates the inhibition of TGFβ precursor processing by Emilin1.
Therefore, future studies are needed.
1.4.2.2 Regulation of ligand processing
Strong genetic and biochemical evidence showed that the TGFβ precursor is proteolytically processed by furin convertase either intracellularly or extracellularly
(Dubois et al, 1995 and 2001; Beck et al, 2002; Koo et al, 2006). But how this processing is regulated is unknown. However, studies showed that in various tissues
TGFβ stimulated the transcription of furin, its own converting enzyme, through crosstalk between the ERK pathway and the Smad pathway (Blanchette et al, 1997 and 2001;
Dubois et al, 2001), thus leading to augmented processing of the TGFβ precursor. But, it is not clear how the transcription of furin is negatively regulated, or how such autoinduction of its own converting enzyme by TGFβ may influence TGFβ availability in normal physiological processes and pathological conditions. Thus, clearly, more studies are required to explore these issues.
1.4.2.3 Regulation of TGFβ ligand activation
To achieve extracellular activation, the mature TGFβ ligand must be dissociated and released from the latent TGFβ complex in the ECM that renders TGFβ inactive and thus finely controls the level of mature TGFβ ligands. The temporal and spatial activation of the mature TGFβ ligand is an important step in the regulation of TGFβ signaling.
Since TGFβ is concentrated in latent TGFβ complexes to the ECM, its activation involves the disassembly of the ECM to release the mature TGFβ ligand. It has been 30 reported that latent TGFβ can be activated by various molecules, including integrins, thrombospondin-1 (TSP-1), reactive oxygen species (ROS), mild acid (pH 4.5), and proteases, which are all known to perturb the ECM environment (Annes et al, 2003).
For the activation of TGFβ in normal physiological conditions, TSP-1-mediated or integrin-mediated activation of TGFβ may be especially important, whereas activation by ROS, mild acid (pH 4.5), and proteases may be important in certain disease conditions. TSP-1 is an extracellular matrix protein and is expressed in various tissues throughout development, which is also consistent with an in vivo role as a latent TGFβ activator (Murphy-Ullrich and Poczatek, 2000). Young TSP-1 null mice demonstrate strikingly similar histological abnormalities to young TGFβ1 null mice in multiple organ systems, especially with regard to lung and pancreas pathologies, which can be reverted to wild type by treatment with TSP-1 derived peptide to activate TGFβ (Crawford et al,
1998; Murphy-Ullrich and Poczatek, 2000). Consistent with the essential role of TGFβ in normal wound healing, TSP1 null mice also exhibit altered dermal wound healing, and a decrease in active TGFβ extracted from wounds of TSP-1 null animals, while addition of active TGFβ to the TSP1 null wounds restores the wild-type phenotype (Crawford et al, 1998; Murphy-Ullrich and Poczatek, 2000). These studies indicate that a major function of TSP1 in vivo is to activate latent TGFβ. Integrin αvβ6 was first identified to control TGFβ activation specifically in epithelial cells where its low expression is restricted, while wounding or inflammation may induce the expression of vβ6 (Breuss et al, 1995; Munger et al, 1999; Miller et al, 2001). Knockout mice targeting this integrin develop exaggerated inflammation and protection from pulmonary fibrosis, which is 31 consistent with TGFβ’s role as fibrosis inducer (Border and Ruoslahti, 1992) and its induction of integrin αvβ6 for subsequent activation of itself. Therefore, this positive feedback loop is interrupted in knockout mice targeting αvβ6, thus resulting in only a minor fibrotic response in these mice (Munger et al, 1999). Recently, integrin αvß8 was reported to activate latent TGFβ, but this activation requires the activity of the membrane type 1-matrix metalloprotease (MT1-MMP) (Mu et al, 2002).
MMP-2 and MMP-9, as well as plasmin, have also been shown in vitro to activate latent TGFβ (Yu and Stamenkovic, 2000), and TGFβ has also been shown to induce the expression of MMP-2 to augment its own activation (McMahon et al, 2003). But, such a role as latent TGFβ activator in vivo has been put in doubt by studies on knockout mice targeting the genes encoding these proteases, due to the absence of any phenotype, although this might also reflect redundancy of the activating enzymes. TGFβ activation by ROS in vivo after irradiation may simply be a result of damage of the ECM and the release of mature TGFβ from the ECM during such processes. Clearly more studies are warranted to further elucidate the mechanisms of latent TGFβ activation and their role in normal physiology.
1.4.2.4 Regulation of the availability of active mature TGFβ ligand
It is known that there are antagonists to mature ligands of the TGFβ family members (Massague and Chen, 2000). For example, Follistatin is a soluble secreted glycoprotein that binds to Activin and BMPs, and antagonizes their signaling by inhibiting interaction with their corresponding receptors (de Winter et al, 1996; Iemura et 32 al, 1998). Both Chordin and Noggin bind specifically to BMPs, but not to Activin or
TGFβ, and antagonize BMP signaling by blocking BMP interaction with their receptors
(Piccolo et al, 1996). However, no such antagonist has been identified for mature TGFβ ligand until the very recent identification of a novel placenta-derived soluble TGFβ co- receptor endoglin (sEng) (Venkatesha et al, 2006). They showed that sEng blocking
TGFβ downstream signaling by preventing binding of TGFβ1 to its receptors in endothelial cells locally where active mature TGFβ1 is produced, while sEng does not impact circulating concentrations of TGFβ1 (Venkatesha et al, 2006). They also demonstrated that increased serum sEng in individuals with preeclampsia (a pregnancy- specific hypertensive syndrome) correlates with their disease severity and falls after delivery (Venkatesha et al, 2006). But how the tissue-specific production of sEng is regulated currently is unknown. Whether there are other mature TGFβ antagonists is also not clear. Future studies may give an answer. Moreover, it is reported that the α2- macroglobulin exhibits reversible binding to TGFβ with relatively low affinity, but it does not appear to compromise the biological activity of TGFβ (Vaughan and Vale,
1993). Since it is an abundant constituent of blood plasma, α2-macroglobulin may serve as a circulating reservoir and/or reduce their degradation and clearance rate.
1.4.3 Regulation of TGFβ receptor level and activity
1.4.3.1 Enhancing effects of TGFβ co-receptors
TGFβ receptors (TβRII and TβRI) are single-pass transmembrane serine/threonine kinase receptors. There are two other TGFβ co-receptors also involved 33 in TGFβ signaling, TβRIII and Endoglin. TGFβ receptor type III (TβRIII), also known as beta-glycan, is non-signaling membrane-anchored co-receptor particular for TGFβ2 isoform, since TGFβ2 itself cannot bind to TβRII to initiate the receptor complex formation and has to rely upon TβRIII to trap and present itself to TβRII to initiate the signaling. Endoglin (Eng), also known as CD105, is another cell-surface co-receptor homologous to TβRIII, and can only bind TGFβ1 and TGFβ3 (St-Jacques et al, 1994).
But, Eng cannot bind ligands by itself, and requires the presence of at least TβRII
(Barbara et al, 1999). Over-expression studies show that TβRIII enhances TGFβ responsiveness. Recently, it has been demonstrated that Eng interacts with both active and inactive TβRII, whereas it only interacts with TβRI when it is inactive, and Eng inhibits phosphorylation levels of TβRII, but increases that of TβRI, resulting in increased phosphorylation of Smad 2 but not Smad 3 and TGFβ response changes
(Guerrero-Esteo et al, 2002). Eng may be involved in the regulation of TGFβ responsiveness in vascular endothelial cells, since Eng is preferentially expressed on vascular endothelial cells (Rius et al, 1998; Botella et al, 2001). However, the mechanisms of their function remain to be elucidated.
1.4.3.2 Negatively regulating the activity of TGFβ receptors
1.4.3.2.1 Physical Blockade of TGFβ receptors
TGFβ receptors need to bind to downstream signaling components (such as
Smad2/3) to phosphorylate them, and TβRII also must bind to TβRI to transphosphorylate it. Therefore, physical blockade of such binding and/or 34 phosphorylation is naturally an important step at which to control TGFβ signaling. As mentioned above, Smad7 is a central negative regulator of TGFβ signaling in multiple ways (Ebisawa et al, 2001; Edlund et al, 2003; Kavsak et al, 2000; Shi et al, 2004). In one mechanism, Smad7 binds to the activated receptor TβRI within the TβRI/TβRII complex, and blocking the binding of R-Smads to the receptor complex, since unlike R-Smads,
Smad7 lacks the C-terminal target site for phosphorylation by TβRI and cannot be phosphorylated and released, resulting in physical blockade of TGFβ receptors activity and preventing access of R-Smads to the activated TβRI (Souchelnytskyi et al, 1998; Shi et al, 2004). Moreover, other studies identified STRAP (Serine-Threonine kinase
Receptor-Associated Protein) and YAP65 (Yes-Associated Protein) both by yeast two- hybrid systems as Smad7 partners to facilitate the recruitment of Smad7 to the activated
TβRI and stabilization of the association between Smad7 and the activated TβRI (Datta et al, 1998; Datta and Moses. 2000; Ferrigno et al, 2002; Halder et al, 2006), thus further strengthening such physical blockade of the TGFβ receptors’ activity. Such a role of
Smad7 in negatively regulating TGFβ signaling is also supported by studies on Smad7’s role on tumorigenesis and metastasis (Azuma et al, 2005; Halder et al, 2005; Kuang et al,
2006), as well as by studies on the role of its partnering proteins STRAP and YAP65 on tumorigenesis (Guo et al, 2005; Halder et al, 2006).
In addition to physically blocking intracellular signaling components' access to and phosphorylation by the TGFβ receptors, another way of physical blocking TGFβ receptor signaling is to inhibit phosphorylation and activation of TβRI by TβRII, which is also an upstream step of such negative regulation. Immunophilin FKBP12 was identified 35 using yeast two-hybrid systems and demonstrated to bind with high affinity to TβRI and to inhibit its signaling function (Wang et al, 1994). It was further demonstrated that
FKBP12 binds to the GS region of TβRI, whose phosphorylation by TβRII can activate
TβRI, and thus masks the TβRII phosphorylation sites and results in stabilization of TβRI it its inactive conformation, but does not disrupt the interaction between TβRI and TβRII
(Chen et al, 1997; Huse et al, 1999; Wang et al, 1996). Therefore TβRI cannot be activated by phosphorylation to initiate signaling. However, studies done on primary mouse embryo fibroblasts and thymocytes from FKBP12 knockout mice showed no difference in TGFβ-mediated transcriptional responses or growth inhibition from such cells from wild-type mice (Bassing et al, 1998), thus questioning the physiological relevance of the FKBP12- TβRI interaction. Therefore more studies are required to determine whether such this interaction has a physiological role in vivo.
Moreover, TRIP-1 (TGFβ-receptor interacting protein-1) has been identified to specifically interact with TβRII in a kinase-dependent way (Chen et al, 1995). TRIP-1 has also been shown to be phosphorylated on serine and threonine by the receptor kinase and to specifically repress TGFβ-mediated induction of plasminogen activator inhibitor-1
(PAI-1), without any effect on TGFβ-mediated growth inhibition, by both receptor- dependent and receptor-independent mechanisms (Choy and Derynck, 1998).
1.4.3.2.2 Enzymatically regulating the activity of TGFβ receptors
Physical blockage is only one way of Smad7 to exercise its inhibitory effects, and it is also a relatively inefficient inhibition, since by this way each Smad7 molecule can 36 only inhibit one TβRI receptor molecule. TβRI’s functions can be inhibited much more efficiently through an enzymatic approach, such as dephosphorylation of TβRI to inactivate it. The protein phosphatase 1 (PP1) is involved in TGFβ signaling, since it has been shown to be recruited to the activated TβRI and to dephosphorylate it, thus negatively regulating TGFβ signaling. Such recruitment is mediated by PP1’s regulatory/targeting subunit GADD34 (growth arrest and DNA-damage-inducible 34).
GADD34 interacts with both TßRI and Smad7 to form a TβRI–Smad7–GADD34 triple complex, which is induced by TGFβ (Hayashi et al, 1997; Nakao et al, 1997), thus terminating TGFβ signaling and limiting the duration of TGFβ signaling.
1.4.3.3 Regulating the level of TGFβ receptors
In addition to modulating the TGFβ receptor activity, the expression level of
TGFβ receptors at the cell membrane can also be regulated, which is also an efficient way of inhibiting the TGFβ signaling. However, this is irreversible way of controlling
TGFβ signaling, since it leads to terminal destruction of the receptors. Several mechanisms have been identified. The ubiquitin-mediated proteasomal degradation pathway is evolutionary conserved, and also plays an important role in control TGFβ signaling. Smurf1 and 2 (Smad ubiquitination-related factor 1 and 2) are E3 ubiquitin ligases and have been identified as Smad interacting proteins to cause the ubiquitin- mediated degradation of Smad proteins (Kavsak et al, 2000; Zhang et al, 2001; Zhu et al,
1999). Smurf1 and 2 are located in the nucleus of cells not stimulated by TGFβ, and exported into the cytoplasm together with Smad7 when cells are treated by TGFβ. 37 Through interaction with Smad7 as an adaptor protein, Smurf1 and 2 are recruited to the
TGFβ receptor complex and ubiquitinate the associated receptors, as well as Smad7, which mark them for degradation by the proteasome machinery (Ebisawa et al, 2001;
Kavsak et al, 2000; Zhang et al, 2001).
Endocytosis-lysosome pathway has long been recognized a means to terminate signaling via degradation of activated receptors after their internalization from the cell surface. TGFβ receptors have also been shown to undergo lysosomal degradation, after
TGFβ ligand binding to the TGFβ receptors and their internalization (Anders et al, 1997 and 1998; Dore et al, 1998). Recently, Di Guglielmo et al demonstrated that the TGFβ receptors are internalized through two distinct endocytic routes: the clathrin-mediated pathway, which is important for promoting signaling, and the caveolin/lipid-raft- mediated pathway, which mediates the degradation of TGFβ receptors (Di Guglielmo et al, 2003). However, receptor down-regulation can also occur via clathrin-mediated endocytosis followed by traffic to the lysosome, without caveolar involvement (Mitchell et al, 2004). Thus, differences in receptor compartmentalization and the outcome of such trafficking are likely to depend on the localization of the proteins that interact with the receptor, and may also be cell type-specific.
1.4.4 Intracellular regulation
There are a number of ways to regulate TGFβ intracellular signaling.
1.4.4.1 Regulating the Smad2/3 activity by phosphorylation
Since the major signaling pathway is the Smad pathway, the activity, level and 38 subcellular location of Smad2/3 are important targets for regulation of the TGFβ intracellular signaling. Smad2/3 are activated through phosphorylation by the activated
TβRI kinase, and translocate into the nucleus to regulate target genes’ transcription
(Massague, 2000; Feng and Derynck, 2005).
However, there are an increasing number of cellular kinases besides TβRI that can also phosphorylate Smad2/3 at distinct amino acid residues to regulate their activity.
It has been reported that cytoplasmic kinases belonging to the MAPK family can also activate Smad2/3 phosphorylation outside the C-terminal site, the TβRI phosphorylation site, (Brown et al, 1999; Engel et al, 1999). For example, activation of Smad2 via phosphorylation by active MEKK-1 (MAPK/Erk kinase kinase 1, an upstream activator of the JNK pathway) results in enhanced Smad2-Smad4 interactions, nuclear localization of Smad2 and Smad4, and selective activation of Smad2-mediated transcription in endothelial cells (Brown et al, 1999). However, the exact amino acid residue(s) phosphorylated by MEKK1 has not been identified yet. It is also reported that TGFβ- induced rapid JNK activation in a SMAD-independent manner phosphorylates Smad3 outside the TβRI phosphorylation site to facilitate both its activation by TβRI and its nuclear accumulation (Engel et al, 1999). But, the exact site of phosphorylated amino acid residues is not clear.
ERK, a member of the MAPK family, has been reported to negatively regulate the
Smad2/3 activity by phosphorylation (Kretzschmar et al, 1999). EGF, hepatocyte growth factor and oncogenic Ras can activate ERK. Activated ERK can cause phosphorylation of
Smad2/3 outside the TβRI phosphorylation site at specific residues in the region linking 39 the DNA-binding domain and the transcriptional activation domain, leading to inhibition of TGFβ-induced nuclear accumulation of Smad2/3 and Smad2/3-dependent transcription in mammary and lung epithelial cells (Kretzschmar et al, 1999). Mutation of these phosphorylation sites in Smad3 yields a Ras-resistant form that can rescue the growth inhibitory response to TGFβ in Ras-transformed cells, providing further strong evidence for a physiological role of such negative regulation by ERK.
Activation of CamKII has also been demonstrated to phosphorylate and inhibit
TGFβ-induced nuclear import and transcriptional activity of Smad2 at several serine residues (Ser110, Ser240 and Ser260), although the exact mechanism of such inhibition is not clear yet (Wicks et al, 2000). Another example is PKC, which is also demonstrated to phosphorylate several serine residues in the MH1 domain of Smad3 and thus to abrogate
DNA binding activity of Smad3 (Yakymovych et al, 2001).
1.4.4.2 Terminating the Smad2/3 signaling by dephosphorylation
Reversible phosphorylation regulates fundamental aspects of cell activity. The phosphorylation state of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. The phosphorylated proteins can be dephosphorylated by certain protein phosphatases. TGFβ-elicited phosphorylation of its major intracellular signaling components Smad2/3 is central to its cellular effects. Thus, dephosphorylation of the Smads by phosphatases is an optimal mechanism for the termination of TGFβ-
Smad signaling, although phosphorylation of Smad2/3 can be prevented or limited by physical blocking their access to the activated receptors by Smad7 and as 40 dephosphorylation of TβRI to inactivate it by phosphatases such as PP1, as mentioned above.
However, the identities of the phosphatases responsible for the R-Smads’ dephosphorylation have remained elusive until very recently (Chen et al, 2006; Lin et al,
2006). Lin et al demonstrated that PPM1A (protein phosphatase 1alpha) is a bona fide
Smad phosphatase, and dephosphorylates TGFβ-induced phosphorylated Smad2/3 (Lin et al, 2006). Both over-expression and depletion studies, as well as early embryogenesis studies in zebrafish, confirmed PPM1A’s critical role in terminating TGFβ signaling.
However, it remains to be determined whether PPM1A’s transcription and/or activity is regulated by TGFβ, or is constitutively active. It also remains to be explored whether there are more phosphatases specific for by TGFβ signaling pathway. Similarly, pyruvate dehydrogenase phosphatase has been identified by biochemical and genetic methods as a phosphatase to directly dephosphorylate a Drosophila Smad (MAD) and to dampen signal transduction of Decapentaplegic, a TGFβ family ligand in Drosophila, thus highlighting the importance of such recently identified phosphatases in TGFβ superfamily signaling (Chen et al, 2006).
1.4.4.3 Terminating the Smad2/3 signaling by irreversible degradation
In addition to the above reversible means of regulation, growing evidence suggests that Smad protein levels are also controlled by irreversible ubiquitin-mediated degradation via the proteome machinery, which may ensure a more thorough elimination of the amplified activated signals. The E3 ligases, Smurf1, Smurf2 and SCF/Roc1 have 41 been implicated in Smad degradation in unstimulated cells and stimulated cells as well
(Fukuchi et al, 2001; Lin et al, 2000; Lo and Massague, 1999).
Smurf2 has been demonstrated to be the E3 ligase responsible for activated
Smad2 degradation in the cytoplasm, and this involves the E2-conjugating enzymes
UbcH5b/c, and, to a lesser extent, Ubc3 (Lin et al, 2000; Lo and Massague, 1999).
Smurf2 exhibits higher binding affinity to activated Smad2 upon TGFβ stimulation and potently reduces the transcriptional activity of Smad2 (Lin et al, 2000; Lo and Massague,
1999). But, it has also been demonstrated that proteasomal degradation of activated
Smad2 also occur in the nucleus (Lo and Massague, 1999), however, the identity of the specific E3 ligase specifically recognizing activated Smad2 in the nucleus has not been determined yet.
Similarly, activated Smad3 has also been shown as target for ubiquitin-mediated proteasomal degradation (Fukuchi et al, 2001). The E3 ubiquitin ligase complex ROC1-
SCF has been demonstrated to induce its ubiquitination and degradation in the cytoplasm after Smad3 is exported from the nucleus to terminate Smad3 transcriptional activity
(Fukuchi et al, 2001).
1.4.4.4 Regulating the level and activity of the Co-Smad, Smad4
The common TGFβ signaling effector Smad4 has also been shown to be target for proteasomal degradation (Moren et al, 2003; Saha et al, 2001; Wan et al, 2002). Wan et al demonstrated that Jab1 antagonizes TGFβ signaling by inducing the ubiquitylation and degradation of Smad4 via the 26S proteasome (Wan et al, 2002). Saha et al showed Ras- 42 induced decrease in Smad4 expression via the MEK/ERK/MAPK pathway, which is prevented by an inhibitor of the MEK/ERK/MAPK pathway (Saha et al, 2001). Studies on Smad4 mutants derived from human cancers with missense mutation in the MH1 domain showed that all mutants exhibit enhanced polyubiquitination and proteasomal degradation (Moren et al, 2003), providing further support for the role of ubiquitination- proteasome pathway in regulating the Smad4 level in the cells. However, in all cases, the mechanism of Smad4 ubiquitination currently is not clear and remains to be determined.
In addition to regulating the level of Smad4, the regulation of Smad4 activity has been reported as well. Moren et al reported that Smad4 is mono-ubiquitinated at lysine
507 and mono- or oligo-ubiquitinated Smad4 exhibits enhanced ability to oligomerize with R-Smads and enhanced transcriptional activity (Moren et al, 2003). Recent studies show that Smad4 can also be modified by sumoylation (Lee et al, 2003; Lin et al, 2003;
Long et al, 2003 and 2004). Sumoylation, by addition of the small ubiquitin-related modifier (SUMO) to proteins, is another way to regulate protein function, like protein- protein interaction, subnuclear localization, protein-DNA interaction and enzymatic activity, but not their degradation (Schwartz and Hochstrasser, 2003). Lee et al and Lin et al reported that sumoylation of Smad4 by the SUMO E3 ligase PIASy (protein inhibitor of activated STATy) promoted the nuclear accumulation of Smad4 and enhanced Smad4 stability and Smad4-dependent transcriptional activity in mammalian cells (HeLa cells and MDA-MB-468 cells) and Xenopus embryos (Lee et al, 2003; Lin et al, 2003). However, Long et al reported the opposite effects of Smad4 SUMO modification by PIASy, a repression of Smad4 transcriptional activity in Mv1Lu cells 43 (Long et al, 2003 and 2004). Thus, it appears that the net effect of sumoylation of Smad4 may enhance or repress its transcriptional activity, depending on the target promoter analyzed and the cell type used, and perhaps the activation state of other signaling pathways.
Furthermore, TRAP1 (TβRI-associated protein-1) has been identified to interact with Smad4 in a ligand-dependent fashion and deletion constructs of TRAP1 have been shown to inhibit TGFβ signaling and diminish the interaction of Smad4 with Smad2, thus suggesting a role for TRAP1 as a specific molecular chaperone for Smad4 facilitating the
Smad2/3-Smad4 complex formation in the vicinity of the TGFβ receptors (Charng et al,
1998; Wurthner et al, 2001).
1.4.4.5 Regulating the subcellular compartmentalization of Smad2/3
Exciting new findings in a variety of cellular and developmental systems supports the proposal that intracellular trafficking and subcellular compartmentalization of signaling components can play a more direct and active role in signal propagation and amplification, and disruption of their normal trafficking to specific subcellular compartment may lead to accumulation of such signaling components at the wrong place and have devastating consequences, like cancer (Bache et al, 2004; Penheiter, 2002;
Szymkiewicz et al, 2004).
It has been demonstrated that endocytosis of TGFβ receptors and recruitment of
Smad2/3 onto EEA1-positive early endosomal compartments is required for the propagation of the TGFβ signal through the Smad proteins (Anders et al, 1997 and 1998; 44 Di Guglielmo et al, 2003; Felici et al, 2003; Hayes et al, 2002; Mitchell et al, 2004;
Runyan et al, 2005). However, the exact mechanism is not yet fully understood. Di
Guglielmo et al show that TGFβ receptors are internalized into two distinct endocytic compartments, caveolin-positive vesicles and EEA1-positive vesicles, respectively (Di
Guglielmo et al, 2003). As shown in Fig. 2, the two pathways play different roles in
TGFβ signaling through Smad2/3: the internalization into caveolin-positive vesicles promoting signaling, while the internalization into EEA1-positive vesicles dampening it
(Di Guglielmo et al, 2003).
Many adaptor proteins have been identified for Smad2/3 and have been shown to interact with Smad2/3 and to bring Smad2/3 into association with the endosomal compartments within the cell. These adaptors include SARA (Smad Anchor for Receptor
Activation) (Hu et al, 2002; Itoh et al, 2002; Tsukazaki et al, 1998), Hrs (Hepatocyte growth factor-Regulated tyrosine kinase Substrate) (Miura et al, 2000), cPML (the cytoplasmic form of the ProMyelocytic Leukemia tumor suppressor protein) (Lin et al,
2004), Dab2 (Disabled 2) (Hocevar et al, 2001 and 2005; Mishra et al, 2002), TLP
(TRAP-1 Like Protein) (Felici et al, 2003), and β-spectrin ELF (Embryonic Liver Fodrin)
(Tang et al, 2003), etc. However, so many different proteins acting as adaptor proteins for
Smad2/3 highly suggests there is functional redundancy among them, therefore, they may exhibit cell-type differences in their functions, or the presence of additional adaptor proteins for more specific interaction regulations. For example, in agreement with this, both SARA and Hrs contain a FYVE domain that binds to phosphatidylinositol 3- phosphate (PI3P) and help to bring SARA and Hrs onto EEA1-positive early endosomal 45 TGFβ Plasma Clathrin- membrane Caveolae coated pit
Caveolin-1 Smurf2 P P Smad7 P P P P P P TβRII TβRI P SARA Smad2/3
Caveolin-1 positive vesicle Early endosome
Smurf2 P P Smad7 P P P SARA P Smad2/3 P
T R degradation β TGFβ signaling
Fig. 2. TβR internalization by clathrin- and lipid-raft-mediated endocytosis. At the cell plasma membrane, the tetrameric TβR complex is internalized by two distinct endocytic pathways. The TβR complex is composed of two TβRIs and two TβRIIs. In the clathrin-mediated endocytic pathway, receptors are directly towards the early endosomes. Here, the receptors interact with SARA, which is associated with Smad2. From the early endosomes, the receptors are able to signal through Smad2 phosphorylation. In the lipid raft/caveolae-mediated endocytic pathway, TβRs associate with Smad7-Smurf2, and are internalized into caveolin-1 positive vesicles. This leads to the ubiquitin-mediated degradation of the receptors. 46 compartments, which have shown to be sites of TGFβ receptor accumulation to propagate the TGFβ signaling within the cell (Hayes et al, 2002; Hu et al, 2002; Itoh et al, 2002; Lin et al, 2004; Miura et al, 2000; Tsukazaki et al, 1998). They also both contain a Smad-binding domain (SBD) to augment TGFβ-stimulated transcriptional activity, and they seem to have a cooperative effect to enhance TGFβ signaling (Miura et al, 2000). But, Goto et al and Lu et al reported that interaction between Smad2 or 3 and
SARA is not essential for Smad2- or Smad3-dependent responses (Goto et al, 2001; Lu et al, 2002), and Miura et al also reported that the FVYE domain of Hrs is dispensable for the increased induction of TGFβ-mediated transcription. Lin et al demonstrated that cPML is required for TGFβ signaling by interacting with Smad2/3 and facilitating their recruitment onto the EEA1-positive early endosomal compartments (Lin et al, 2004).
TGFβ responses can be restored by putting back PML into the cPml-null cells or removal of the PML–RAR oncoprotein by degradation induced by retinoic acid or As2O3 treatment. However, PML is dispensable for embryogenesis, suggesting the presence of other redundant genes, or a very restricted regulatory role for cPML in the regulation of
TGFβ signaling during embryogenesis.
Growing evidence show that Smad2 and Smad3 may drive distinct TGFβ downstream signaling pathways and responses (Flanders, 2004; Ju et al, 2006; Kim et al,
2005; Kretschmer et al, 2003; Levy and Hill, 2005; Roberts et al, 2006). Consistent with these studies, Axin (Furuhashi et al, 2001), TLP (Felici et al, 2003), and β-spectrin ELF
(Tang et al, 2003) have been identified spefically as Smad3 adaptor proteins. It has been demonstrated that Axin localizes in a punctate pattern within the cell similar to that of 47 SARA, both Axin and β-spectrin ELF interact with Smad3 and their appropriate subcellular localization plays an essential role in determining their functions as Smad3 adapters facilitating its specific subcellular localization and activation by TβRI for efficient TGFβ signaling (Furuhashi et al, 2001; Tang et al, 2003). Axin mutant
(Furuhashi et al, 2001) or β-spectrin ELF deficiency (Tang et al, 2003) results in mislocalization of Smad3 and loss of the TGFβ-dependent transcriptional responses.
However, TLP interacts predominantly with TβRII, and over-expression of TLP interferes with Smad3–Smad4 complex formation and blocks the Smad3-dependent transcriptional response, while it potentiates the Smad2 response, thus suggesting a negative role of TLP in regulating the subcellular location of Smad3 (Felici et al, 2003).
However, once the TGFβ receptors are internalized, how the receptors and the signaling components (Smad2/3 and others) are directed to the appropriate subcellular localizations, another important step of regulation, is just beginning to be studied.
Previously, it has been reported that Smad2/3 are sequestered through their interaction with microtubules (MT), and TGFβ stimulation leads to their activation and release to translocate into the nucleus (Dong et al, 2000). Recently, a TGFβ receptor-interacting protein km23 was identified in Dr. Mulder’s laboratory and demonstrated to be a light chain of the motor protein dynein (Tang et al, 2002). Cytoplasmic dynein is a motor complex that transports membrane vesicles and diverse cargoes along MT in a retrograde manner (Hirokawa, 1998; King et al, 2002; Vale, 2003; Vallee et al, 2004). It plays a wide variety of functions, such as mitotic spindle assembly and orientation, positioning of the Golgi apparatus, and transport of various intracellular organelles, including 48 endosomes and lysosomes (Hirokawa, 1998; Kamal and Goldstein, 2002; King et al,
2002; Mallik and Gross, 2004; Vale, 2003; Vallee et al, 2004). The dynein motor complex is a large multimeric complex, generally consisting of heavy chains (HC), intermediate chains (IC), light-intermediate chains (LIC), light chains (LC), and other adaptor or accessory proteins, such as dynactin. Three distinct families of dynein light chains (DLCs) have been identified in mammals, including the DYNLL, DYNLT, and
DYNLRB (Pfister et al, 2005; Vale, 2003; Williams et al, 2005; Wu et al, 2005). In addition to binding the dynein intermediate chain (DIC) at distinct regions (Susalka et al,
2002), DLCs have also been shown to interact with a number of cargoes to exert diverse functions (Hirokawa, 1998; King et al, 2002; Vale, 2003; Vallee et al, 2004). km23 was cloned as a TGFβ receptor-interacting protein, and was identified also as a dynein light chain now commonly termed DYNLRB1 (Pfister et al, 2005; Tang et al, 2002).
DYNLRB1 has been shown to interact preferentially with Smad2, and is required for
Smad2-dependent signaling (Ding and Mulder, 2004; Jin et al, in revision), suggesting that TGFβ signaling components like Smad2 may be targeted by dynein motor for efficient transport along the MT for their appropriate subcellular localization, thus maximizing the efficiency of signal propagation and maintaining signal specificity.
However, the exact mechanism needs to be further investigated.
1.4.5 Transcriptional regulation of target gene expression in the nucleus
In the basal state, Smad2 and 3 are predominantly localized in the cytoplasm (Shi and Massaue, 2003). Receptor-mediated phosphorylation of Smad2 and 3 (at the C- terminal two serine residues in the SXS motif) drives their activation and induces their 49 translocation into the nucleus. Smad3 translocates into the nucleus through importinβ1 and Ran-depdendent manner (Kurisaki et al, 2001; Xiao et al, 2000), while Smad2 enters the nucleus by a different mechanism, through direct association with components of the nuclear pore complex (the nucleoporins CAN/Nup214 and Nup153) (Xu et al, 2002).
Smad4 accumulates in the nucleus by association with activated Smad2 and 3 (Kawabata et al, 1999; Liu et al, 1997; Shi and Massague, 2003). After translocation into the nucleus, in conjunction with other nuclear cofactors, Smad2 and 3 regulate the transcription of target genes.
The Smad2, 3 and 4 proteins contain two conserved structural domains, the N- terminal MH1 domain and the C-terminal MH2 domain (Shi and Massague, 2003). Both the MH1 and MH2 domains interact with a large number of proteins in the nucleus, which may modulate the function of Smad2 and 3 for transcriptional regulation. As mentioned above, Smad2 and 3 may drive distinct pathways and TGFβ responses.
Although the mechanism involved is not completely clear, recent studies have showed that it is at least partially due to their differential DNA binding capacity and unique transcriptional activation effect. As shown in Fig. 3, the major difference between
Smad2 and Smad3 is the N-terminal MH1 domain where Smad2 contains two additional stretches of amino acids whereas Smad3 does not (Yagi et al, 1999; Dennler et al, 1999).
Crystal structural studies demonstrated that Smad3 MH1 domain bound to DNA is immediately before the DNA-binding β-hairpin of the MH1 domain, while the insertion of a 30-residue stretch in the Smad2 MH1 domain disrupts the conformation of the DNA binding hairpin and precludes Smad2 from binding directly to DNA (Shi and Massague, 50
Smad3 Linker
Smad2 Exon3 Linker
MH1 domain MH2 domain
DNA binding Protein-protein interaction Smad3 MH1 binds to SBE TGFβ receptors (Smad binding element), Smad adaptors but Smad2 MH1 cannot. Transcriptional cofactors
Fig. 3. Comparison of Smad2 and Smad3 51 2003). Therefore, Smad3, but not Smad2, binds to Smad-Binding Elements (SBEs)
(termed the CAGA box), which was found in target gene promoters (including plasminogen activator inhibitor type I and Smad7 promoters) and responsible for their
TGFβ responsiveness (Dennler et al, 1998; Jonk et al, 1998). Thus, Smad3 is a transcriptional factor by itself (Dennler et al, 1998; Zawel et al, 1998), while Smad2 may function as a coactivator for other transcriptional factors like FAST-1 (Chen et al, 1996).
A broad array of transcription factors has been identified as Smad partners (Feng and Derynck, 2005). It is now generally accepted that TGFβ-mediated transcriptional regulation of target genes requires Smad2 and 3 to recruit transcriptional coactivators or corepressors to such gene promoters to elicit specific transcriptional responses
(Massague, 2000; Massague et al, 2005). Some of these factors may be ubiquitous and mediate the same response in all cell types, while other transcription factors may be cell- type specific and responsible for distinct responses (Massague et al, 2005). Through such interaction with various transcription factors, Smad proteins regulate a broad spectrum of cellular processes in a large variety of cell types, including cell proliferation, differentiation, apoptosis, and embryogenesis (Massague et al, 2000; ten Dijke et al,
2002). The MAPK pathway and other signaling pathways also transduce signals initiated by TGFβ (Moustakas and Heldin, 2005; Yue and Mulder, 2001; Derynck and Zhang,
2003), for PKC and PKA pathways (Choi et al, 1999; Hirota et al, 2000; Sylvia et al,
2000; Yakymovych et al, 2001), PI3K (Bakin et al, 2000; Runyan et al, 2004), and NF-
κB (Arsura et al, 1996; Kon et al, 1999; Bitzer et al, 2000). Although their mechanisms of activation by TGFβ are not well understood, recent studies suggest that activation of 52 these signaling pathways may phosphorylate the linker regions between the MH1 and
MH2 domains of Smad2 and 3, and allow specific crosstalk between these signaling pathways and the Smad pathway. Therefore, the Smad pathway appears to have an important function to integrate the multiple stimuli a cell receives and multiple pathways to generate specific responses.
Thus, through both positive and negative regulation at multiple levels, the multifunctional TGFβ family ligands exert diverse functions in various cellular processes, thus maintaining tissue homeostasis under normal physiological conditions. 53
Chapter 2
Requirement of DYNLRB1 for TGFβ-mediated induction of fibronectin
Dyneins are molecular motors in the cells that play a wide variety of important functions including mitotic spindle assembly and orientation, and transport of various intracellular cargoes, including organelles like endosomes. We have demonstrated previously that dynein light chain DYNLRB1 is a novel TGFβ receptor-interacting protein. Here it is demonstrated that TGFβ stimulates the phosphorylation of DYNLRB1 in Cos-1 cells and TβRII kinase is required for this phosphorylation. It is also demonstrated that this phosphorylation occurs only on serine residues of DYNLRB1, implicating DYNLRB1 as signaling intermediate downstream of TGFβ receptors. It is showed that DYNLRB1 is a cytoplasmic protein with a DYNLRB1 antibody. It has been further been demonstrated that DYNLRB1 expression knockdown using small interfering
RNA (siRNA) significantly impaired TGFβ-induced fibronectin expression in Madin
Darby canine kidney (MDCK) epithelial cells. Here, TGFβ has also been demonstrated to stimulate the recruitment of DYNLRB1 to the dynein motor complex through the DIC, which requires the TGFβ-stimulated phosphorylation of DYNLRB1, suggesting a role for
DYNLRB1 to bring specific cargo to the dynein motor. Therefore, these results indicate that DYNLRB1 is a TGFβ signaling intermediate, and is required for the induction of fibronectin expression by TGFβ. 54 2.1 INTRODUCTION
TGFβ is the prototype for a family of highly conserved ubiquitous peptides that show a remarkable diversity in the biological actions they mediate.
These biological responses include effects on cell growth, cell death, cell differentiation, and the extracellular matrix (ECM) (Derynck and Zhang, 2003;
Shi and Massague, 2003, Mehra and Wrana, 2002).
TGFβ initiates its signals by inducing a heterotetrameric receptor complex composed of TβRI and TβRII serine/threonine kinase receptors. After TGFβ binds to
TβRII, it transphosphorylates, and thereby activates TβRI. The active receptor complex then propagates signals to downstream cellular components and regulatory proteins
(Derynck and Zhang, 2003; Shi and Massague, 2003, Mehra and Wrana, 2003). Many, if not all, of the biological effects of TGF-ß are considered to be Smad-dependent, through transcriptional regulation of extracellular matrix, adhesion, and growth regulatory genes.
But, the mechanisms of these complex effects are not well understood yet. Thus, identification of additional TGFβ signaling pathways and components will assist in our understanding of the mechanisms by which alterations in these pathways contribute to human disease.
Fibronectin, a major component of the ECM, plays important roles in cell adhesion, migration, growth and differentiation (Danen and Yamada, 2001;
Pankov and Yamada, 2002). TGFβ is one of the most potent stimulators of the
ECM, and it has been shown to play a significant role in the accumulation of 55 specific ECM components such as fibronectin and collagen (Yue and Mulder,
2001; Massague et al, 2000; Roberts et al, 2003). Despite the suggestion that
Smads play a critical role in TGFβ-mediated responses, the signaling mechanisms leading to TGFβ-mediated accumulation of ECM proteins are unclear. For example, Hocevar et al have shown that TGFβ can induce fibronectin synthesis through a Jun N-terminal kinase (JNK)-dependent pathway, but Smad4 was not involved (Hocevar et al, 1999). In addition, Gooch et al reported that calcineurin was involved in TGFβ-mediated regulation of ECM accumulation (Gooch et al,
2004). It is likely that other novel TGFβ signaling intermediates are required for mediating the effects of TGFβ on the synthesis of ECM components such as fibronectin.
Dynein is a molecular motor protein that mediates intracellular transport by conveying cargo along polarized microtubules (MTs) toward the minus ends
(Hirokawa, 1998; King et al, 2002; Vale, 2003; Vallee et al, 2004). It is a large multimeric complex, generally consisting of heavy chains (HC), intermediate chains (IC), light-intermediate chains (LIC), and light chains (LC), as well as other adaptor or accessory proteins, such as dynactin. Activation of a motor may occur by posttranslational modifications, local changes in the cellular environment, or chaperone binding (Hollenbeck, 2001). Since growth factors and cytokines are known to regulate such events, the receptors and signaling pathways for these polypeptides are potential regulators of motor protein activation and 56 organelle trafficking, events which ultimately determine the collective spatial organization of the signaling pathways within the cell.
Recruitment of DLCs to the dynein complex is important not only for specifying the cargo that it binds, but also for the regulation of intracellular transport itself (Karcher et al, 2002; Vaughan and Vallee, 1995). Three distinct families of DLCs have been identified in mammals, including the DYNLL (previously termed LC8), the DYNLT
(previously termed Tctex-1/rp3), and DYNLRB (previously termed LC7/robl) (Vale,
2003; William et al, 2005; Wu et al, 2005). DYNLRB1 is a member of the DYNLRB family DLCs. We have demonstrated previously that DYNLRB1 is also a novel TGFβ receptor-interacting protein (Tang et al, 2002). In this report, it is demonstrated that
TGFβ stimulated the phosphorylation of DYNLRB1 on serine residues, and its subsequent recruitment to the dynein complex. It is also demonstrated that TβRII kinase was required for DYNLRB1 phosphorylation and interaction with DIC stimulated by
TGFβ. Further, it is demonstrated that blockade of DYNLRB1 using siRNA decreased the induction of fibronectin expression by TGFβ in MDCK cells. The results in this
Chapter suggest a role for DYNLRB1 in TGFβ signaling, and its requirement for the induction of fibronectin expression by TGFβ.
2.2 MATERIALS AND METHODS
Reagents--The anti-Flag M2 (F3165) antibody (Ab) and mouse IgG were from Sigma-
Aldrich (St. Louis, MO). The anti-dynein intermediate chain (DIC) monoclonal Ab
(MAB1618) was from Chemicon (Temecula, CA). The rabbit IgG was from Santa Cruz 57 Biotechnology, Inc. (Santa Cruz, CA). The anti-fibronectin Ab (610078) was from BD
Biosciences Transduction Laboratories (Palo Alto, CA). Protein A or G agarose were purchased from Invitrogen (Carlsbad, CA). 32P-orthophosphate (NEX-053) was from
PerkinElmer Life Sciences (Boston, MA). TGFβ1 was purchased from R & D Systems
(Minneapolis, MN). Lipofectamine 2000 (11668-019) was from Invitrogen (Carlsbad,
CA).
Antibody production--The rabbit DYNLRB1 anti-serum was prepared against the following sequence: GIPIKSTMDNPTTTQYA (corresponding to amino acids 27-43) of human DYNLRB1 (hDYNLRB1) (Strategic BioSolutions, Newark, DE, or Covance
Reseach Products, Inc, Denver, PA). Each company also provided pre-immune serum.
Cell culture--293 (CRL-1573), and COS-1 (CRL-1650) cells were purchased from
American Type Culture Collection (Manassas, VA) and were grown in DMEM supplemented with 10% FBS. MDCK cells (CCL-34) were also obtained from ATCC and were grown in MEM-α supplemented with 10% FBS. Cells with passage 10-15 are usually used for experiments. Cultures were routinely screened for mycoplasma using
Hoechst 33258 staining (5µg/ml Hanks’ solution), diluted from stock (500µg/ml) frozen at -20°C.
Transient transfections, IP/blot, Westerns, and In vivo phosphorylation assays-- were performed essentially as described previously (Hocevar et al, 1999; Tang et al,
2002; Yue and Mulder, 2000). C e l l s w e r e p l a t e d i n 6 0 c m d i s h e p l a t e 2 4 h, p r i o r t o