University of New South Wales Faculty of Medicine Centre for Vascular Research Regulatory Mechanisms in Vascular Injury and Repa

University of New South Wales Faculty of Medicine Centre for Vascular Research

Regulatory Mechanisms in Vascular Injury and Repair

A thesis presented for the degree of Doctor of Philosophy By

Fernando Santos Santiago

2012

Originality Statement

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’

Signed ………………………………………… Fernando Santos Santiago

Date …………………………………………

‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right the use in future works (such as articles or books) all or part of thesis or dissertation.

I authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International.

I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.’

Signed ………………………………………… Fernando Santos Santiago

Date …………………………………………

Acknowledgments

I would like to thank the following people without whom this part-time studentship will not be possible:

Professor Levon M. Khachigian for his untiring supervision, encouragement and support; Professor Colin Chesterman for all the support as a co-supervisor; Dr. Hideto Ishii for his animal works and pull-down experiments; Drs. Peter Kanellakis and Alex Bobik for the rat injury experiments; Drs. Mark Raftery and Joel Mackay for the mass spectrometry and protein-protein interaction studies, respectively; Dr. Lionel Lourenco-Dias for the help in proof-reading, and formatting of the thesis; past and present staff and students of Transcription and Gene Targeting Laboratory, Centre for Vascular Research for making this journey a memorable one; my parents, brothers and sisters and their families for all the understanding, moral support and love; and to my mother, Antonia Santos Santiago (RIP) for the motherly unconditional love, memories and sacrifices unselfishly extended to me in my younger years.

3 Publication

Santiago FS, Ishii H, Shafi S, Khurana R, Kanellakis P, Bhindi R, Ramirez MJ, Bobik A, Martin JF, Chesterman CN, Zachary IC and Khachigian LM. Yin yang-1 inhibits vascular smooth muscle cell growth and intimal thickening by repressing p21WAF-1/Cip1 transcription and p21WAF1/Cip1-cdk4-cyclin D1 assembly. Circ Res. 2007;101:146-155

Prizes Australia Vascular Biology Society Young Investigator/Travel Award 14th International Vascular Biology Meeting, June 6-10, 2006, Noordwijkerhout, The Netherlands Dean’s List Award. School of Medical Sciences, Faculty of Medicine, UNSW. 2008

Conference Poster Presentation Abstract Injury-inducible Yin Yang-1 Inhibits Vascular Smooth Muscle Cell Growth and Intimal Thickening by Repressing p21WAF1/Cip1 Transcription and Perturbing Cyclin D1-Cdk4-p21WAF1/Cip1 Assembly. Santiago, F.S., Ishii, H., Hulett, M.D., Bobik, A. Martin, J.F., Chesterman, C.N., Zachary, I.C., Khachigian, L.M. 16th International Vascular Biology Meeting, June 6-10, 2006, Noordwijkerhout, The Netherlands

4 Abstract Proliferation of SMC after vascular injury accounts for clinical conditions in transplant vasculopathy, in-stent restenosis and vein bypass graft failure. Vascular injury upregulates the expression of many transcription factors, two important ones are Yin yang-1 (YY-1) and early growth response-1 (Egr-1). The aims of this thesis were (1) to examine a possible mechanism by which a transcriptional repressor YY-1 inhibits SMC proliferation and (2) the development of a phospho-specifc antibody to a transcription activator, Egr-1 found to be a positive regulator of SMC proliferation. The results show that YY-1 inhibits p21WAF1/Cip1 transcription that perturbs the formation of p21WAF1/Cip1/cdk4/cyclin D1 complex thus blocking the downstream pRBSer249/Thr252 phosphorylation and expression of PCNA and TK-1. This inhibition was observed only in SMCs and not in ECs. Moreover, inhibition of endogenous YY-1 was performed to show the gain- and loss-of function of this transcription factor. YY-1 binds with Sp1 and prevents its occupancy of a Sp1 binding element in the p21WAF1/Cip1 promoter without YY-1 itself binding to the promoter. YY- 1 suppression of p21WAF1/Cip1 also involves p53 ubiquitination and proteasomal degradation. Further studies showed that overexpression of the first two-zinc finger region of YY-1 can inhibit smooth muscle cell proliferation and not ECs. This cell-type specific effect of YY-1 could be a potential tool in controlling SMC proliferation in drug eluting stent.

The second part of this dissertation is the generation of phospho- specific antibody to Egr-1. Egr-1 controls a variety of genes implicated to SMC proliferation. Phosphorylation of Egr-1 can induce or repress the expression of its target gene depending on what type of kinase is involved. Preliminary data show that a phospho-specific antibody to Egr-1, pS26, can detect the Egr-1 phosphorylated protein from cell extract. Specificity of pS26 was determined also using slot blots of synthetic peptides and recombinant proteins, peptide blocking and phosphatase treatment. Further validation is needed to

5 fully confirm the specificity of this new phospho-specific antibody to Egr-1. A phospho-specific antibody to Egr-1 will serve as a tool to dissect mechanism by which this immediate early gene product exerts it control on fibroproliferative vasculopathies.

The results generated by this thesis have added a new layer on the understanding of mechanism on inhibition of SMC proliferation and a generation of potential tool to dissect the mechanism of phosphorylation of a transcription factor implicated to SMC proliferative vasculopathy. .

6 Abbreviations ACE angiotensin-converting enzyme AD Alzheimer disease ADP adenosine diphosphate ADPase adenosine diphosphatase ANOVA analysis of variance ANT1 adenine nucleotide translocator 1 AP-1 activator protein-1 AP-2 activator protein-2 AR androgen receptor ASD autism spectrum disorder ATII angiotensin II AVV adeno-associated virus BACE beta-site APP-cleaving enzyme 1 BAP1/HCF-1 BRCA1associated protein-1/host cell factor 1 BDKRB1 bradykinin B1 receptor bFGF basic fibroblast growth factor or FGF-2 BNP B-type natriuretic peptide bp base pair BSA bovine serum albumin CABG coronary artery bypass graft CASM coronary artery smooth muscle cDNA complementary DNA CDX4 caudal type homeobox 4 CEBPD CCAAT/enhancer binding protein delta CF-1 common factor-1 CFTR cystic fibrosis transmembrane conductance regulator ChIP chromatin immunoprecipitation ChM-1 chondromodulin-1 CHOP-10 C/EBP homologous protein-10 CMV cytomegalovirus c-myc cellular myelocytomatosis COX-2 cytochrome c oxidase cox7c cytochrome c oxidase subunit VIIc

7 CREB cAMP responsive element binding protein CsCl cesium chloride CuZn SOD copper/zinc superoxide dismutase CVD cardiovascular disease CXCR4 chemokine (C-X-C motif) receptor 4 DAB diaminobenzidine DES drug eluting stent DMEM Dulbecco's modified eagle medium DMR differentially methylated region DNA deoxyribonucleic acid DNAzyme deoxyribonucleic acid enzyme DR5 death receptor 5 DTT dithiothreitol E2F E2 transcription factor EC endothelial cell ECM extracellular matrix EDTA ethylenediamine tetraacetic acid EGFR epidermal growth factor receptor Egr-1 early growth response-1 EMSA electrophoretic mobility shift assay ERBB2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 ERCC5 excision repair cross-complementing rodent repair deficiency, complementation group 5 ERGIC-53 ER-Golgi intermediate compartment-53 Erk-1 extracellular regulated kinase-1 FBS fetal bovine serum FGF-2 fibroblast growth factor-2 GAPDH glyceraldehyde 3-phosphate dehydrogenase GATA3 GATA binding protein3 GDAP1 ganglioside-induced differentiation-associated protein 1 GEO Gene Expression Omnibus GLAST sodium-dependent glutamate/aspartate transporter GNA12 guanine nucleotide binding protein 12

8 GST glutathione S-transferase GTP guanosine triphosphate HAT histone acetyltransferase HDAC2 histone deacetylase 2 HDAC3 histone deacetylase 3 Hg mercury HIF-2alpha hypoxia inducible factor 1, alpha subunit HLJ1 human liver DnaJ-like protein HO-1 heme oxygenase-1 HOXA11 homeobox A11 HOXB13 homeobox B13 HP1-alpha heterochromatin protein 1- alpha ICAM-1 inter-cellular adhesion molecule IEL internal elastic lamina IFN beta interferon beta IGF-II insulin growth factor II ip intraperitoneal ITRs inverted terminal repeats iv intravenous kDa kilodalton kg kilogram LDL low density lipid LEF-1 lymphoid enhancer-binding factor-1 MCP-1 macrophage chemoattractant-1 MDM2 murine double minute 2 Mecp-1 methyl CpG binding protein microRNA micro ribonucleic acid min minutes miR-29 microRNA29 mm millimeter MMP matrix metalloproteins MMP-9 matrix metallo-peptidase 9 MOI multiplicity of infection MOR mu opiod receptor

9 mRNP messenger ribonucleoprotein particles mRPD3 mouse reduced potassium dependency-3 N1IC Notch 1 receptor intracellular domain NF-E1 nuclear factor erythroid 1 NF-kappaB nuclear factor of kappa light polypeptide gene enhancer in B-cells NFR1 nuclear respiratory factor 1 NMP1 nuclear matrix protein1 NO nitric oxide Nrf2 NF-E2-related factor-2 Otx2 orthodenticle homeobox 2 p53 tumour protein 53 p73 tumour protein 73 PAGE polyacrylamidegel electrophoresis Pax7 paired box 7 PBS phosphate buffered saline PC3 prostate cancer cells 3 PCFT proton-coupled folate transporter PcG polycomb group PCNA proliferating cell nuclear antigen PCR polymerase chain reaction PDGF platelet derived growth factor Peg3 paternally expressed 3 pfu plaque forming unit PHO pleiohomoetic PMSF phenylmethanesulfonylfluoride PPAR delta peroxisome proliferator-activated receptor delta PRC2 polycomb repressive complex 2 PREP-C2ORF34 prolyl endopeptidase-like-chromosome 2 ORF 34 PSA prostate-specific antigen PTCA percutaneous transluminal coronary angioplasty PTEN phosphatase and tensin homolog QTL quantitative trail locus Rb retinoblastoma

10 RBD receptor binding domain REST RE1-silencing transcription factor RGS16 regulator of G-protein signaling 16 RIPA radioimmunoprecipitation assay RIZ1 retinoblastoma protein-interacting zinc finger RNAi Ribonucleic acid interference RT-PCR reverse transcription polymerase chain reaction RYBP Ring1-YY-1 binding protein SAA serum amyloid A SAP30 Sin3A-associated protein SDS sodium dodecyl sulphate sec seconds sem standard error of mean Sin3A SIN3 homolog A siRNA short interfering ribonucleic acid SMC smooth muscle cell SMEs smooth muscle elements SNAI1 snail homolog 1 SNAP synaptosomal-associated protein SNP single nucleotide polymorphism Sp1 specificity protein 1 SRF serum response factor SRY sex determining region StAR steroidogenic acute regulatory protein SUMO small ubiquitin-like modifier SUZ12 suppressor of zeste 12 homolog TCF4 transcription factor 4 TF tissue factor TGF-beta1 transforming growth factor beta 1 TK-1 thymidine kinase-1 U6 pol III U6 snRNA UCRBP upstream conserved region binding protein VCAM-1 vascular cell adhesion molecule-1 VECs vascular endothelial cells

11 VEGF vascular endothelial growth factor A VSMCs vascular smooth muscle cells YAF-2 YY-1-associated factor 2 YY-1 Yin Yang-1

12 Table of Contents

Originality Statement 1 Copyright and DAI Statement 2 Acknowledgements 3 Publication 4 Prizes 4 Conference Poster Presentation 4 Abstract 5 Abbreviations 7 Chapter 1 Vascular Smooth Muscle Cells Proliferation; Yin yang-1 and Early Growth Response-1 15 Cardiovascular disease 16 Anatomy of normal blood vessel 16 Hypotheses of atherosclerosis 18 Endothelial cells 19 Vascular smooth muscle cells 19 Gene therapy 26 Yin yang-1 30 Structure and features of YY-1 30 Regulation of YY-1 expression 32 Post-translational modifications of YY-1 33 Transcriptional mechanisms of YY-1 34 Genes regulated by YY-1 and biological functions 40 YY-1 in disease 47 YY-1 in CVD 47 Early growth response-1 50 Structure and regulation of Egr-1 51 Egr-1 in disease 53 Post-translational modifications of Egr-1 56 Mitogen-activated protein kinase (MAPK) cascade 56 Aims 60

Chapter 2 Materials and Methods 61 Construction of adenoviral YY-1 62 Cesium chloride banding of adenovirus 64 Adenoviral titer assay 64 Cell culture and plasmid transfection 65 Cell proliferation assays 65

13 Carotid artery injury and adenovirus delivery 66 Immunohistochemical staining 67 Transient transfection and reporter gene analysis 67 Western blot analysis 68 Immunoprecipitation analysis 68 RT-PCR 69 EMSA analysis using nuclear extracts and recombinant proteins 70 Chromatin immunoprecipitation (ChIP) analysis 70 Generation of truncated YY-1 constructs 71 In vitro phosphorylation 73 Mass spectrometry 73 Generation of phospho-specific antibody 73 Slot blot and immunoblotting 74 Animal ethics and statistics 74

Chapter 3 Results 75 YY-1 represses p21WAF1/CIP1 expression in SMC 76 YY-1 inhibits p21WAF1/CIP1 transcription by preventing Sp1 occupancy p21WAF1/CIP1 promoter 83 YY-1 siRNA stimulates SMC growth and intimal thickening 91 YY-1 regulates thymidine kinase-1 expression 91 YY-1 first two-zinc finger region alone can inhibit SMC proliferation 99 Amino acids 33-341 in YY-1 are critical in inhibiting SMC proliferation 99 Serine26 is highly phosphorykated by Erk-1/2 104 Initial validation tests of phospho-specific antibody proved to be specific for Ser26 111

Chapter 4 General Discussion and Future Directions 119

References 128

Chapter 1

Vascular Smooth Muscle Cell Proliferation; Yin Yang-1 and Early Growth Response-1

15 Cardiovascular Disease Cardiovascular diseases (CVD) is responsible for more than one-third of mortality in the United States of America with estimated direct and indirect cost for the year 2009 of US$475.3 billion (1). In Australia, CVD was responsible for 34% of all deaths in 2006 and cost the health system an estimated $7.6 billion in 2004 (2).

CVD refers to the disease of heart or blood vessels but actually, the term refers to any disease that affects the cardiovascular system. Atherosclerosis is the process of progressive thickening and hardening of the arterial walls leading to narrowing or stenosis of the arterial lumen. It is a factor in several conditions like coronary heart disease, myocardial infarction, cerebral vascular disease, thrombotic stroke, transient ischemic attacks, claudication or insufficient blood supply to lower limbs, organ damage and vascular complications of diabetes. Risk factors for atherosclerosis include high level of “bad” cholesterol, high blood pressure, smoking, diabetes and a genetic family history of atherosclerotic disease.

Anatomy of the normal blood vessel The anatomy of the normal artery consists of three morphologically distinct layers (Fig. 1.1). The intima is sandwiched by a monolayer of endothelial cells on the luminal side and by internal elastic lamina on the peripheral side. The media is mostly composed of smooth muscle cells and connective matrices such as collagens I and III, vitronectin, laminin and elastin fibrils (3). The outer layer, adventitia, consists of interspersed fibroblasts, smooth muscle cells, nerve cells, collagens and elastin fibrils. Separating the media and adventitia is the external elastic lamina (3).

Fig. 1.1. Anatomy of a normal artery. Longitudinal cross section of normal artery indicating different layers of cells (http://www.vacularsurgeon.co.uk).

Hypotheses of atherosclerosis Several schools of thought emerge on the origin of the atherosclerosis based on the accumulations of observations and deductions. In an organized-thrombus hypothesis, it is claimed that thrombogenic factors like platelet or fibrin deposits may trigger the initial process of atherosclerosis (4). Reports have shown that small platelet thrombi were associated with early macrophage foam cell lesion (5). On the other hand in monoclonal growth theory, smooth muscle elements of an atherosclerotic plaque is thought to be of monoclonal origin, thus lesions would be analogous to neoplastic alteration of the vasculature (6). The infection-inflammation hypothesis has been speculated due to observations that some inflammatory cells are always present within the plaque and atherosclerotic lesions are reminiscent of scar following any type of tissue damage. Several investigations identified inflammation as the key regulatory process that links multiple risk factors for atherosclerosis (7). Evidence also exists that viral, Coxsackie, (8) and Herpes simplex bacterial (9), Helicobacter pylori (10) and parasite, Chlamydia pneumoniae (11) may be associated with atherosclerosis as reviewed by Ismail et al (12).

Atherosclerosis is generally believed to result to a response to injury initiated by circulating factors and modulated by the local anatomy and haemodynamics (13), (14), (15). It is generally accepted that endothelial dysfunction with up-regulation of adhesion molecules, adherence of monocytes and platelets, and increased permeability to lipids facilitate the development of lesion. Proliferation of smooth muscle cells emigrating from the media contributes to growth of the plaque. The most recent hypothesis on the initiation of atherosclerosis is the “vasa vasorum hypoxia” claiming that the most vulnerable site for hypoxia is the final end of the vasa vasorum artery (16).

Endothelial Cells Vascular endothelial cells (VECs) are recognized to participate in the regulation of vascular tone, nutrient delivery, waste removal, inflammation, thrombosis and coagulation. In quiescent state, VECs release anti-platelet aggregation factors like heparan sulfate, prostacyclin, nitric oxide and adenosine diphosphatase (ADPase) (17). VECs role in modulating vasoactivity, is by the production of autocrine and paracrine mediators including nitric oxide (NO), prostaglandins, endothelium-derived hyperpolarizing factors, endothelin and angiotensin II. Among these mediators, NO is the most characterised (18) and the most potent vasodilator (19). In subjects with atherosclerosis, endothelial vasodilator dysfunction arises from variation in blood flow. This dysfunction can be demonstrated in patients with risk factor for atherosclerosis in the absence of atherosclerosis itself (20) (21). The clinical relevance of endothelial dysfunction has been borne out in two studies: the presence of endothelial dysfunction predicts the presence of significant coronary artery disease (22) and prognostic information about the likelihood of events in patients with coronary artery disease (23). Data show that vascular stem/progenitor cells might be the source of cells for endothelial repair during development of atherosclerosis (24-26). In case of severe endothelial damage in hyperlipidemia, stem cell repair in atherosclerosis has been proposed (27).

Vascular smooth muscle cells Vascular smooth muscle cells (VSMCs) are heterogenous population of cells in normal vascular tissue. Spindle-shaped differentiated VSMCs show low frequency of proliferation, with contractile properties whereas the rhomboid-shape dedifferentiated show a high degree of protein synthesis, proliferation and migration. After endothelial injury, VSMCs in the arterial media are modified from a contractile to a synthetic phenotype. Differential expression of around 80 genes separates the intimal and medial smooth muscle cells (28). The origin of these different SMC phenotypes is a controversial one. The sources of the difference in SMC phenotypes had been extensively investigated after vascular interventions.

19 It has been inferred that possible sources for SMCs heterogeneity can contribute to vascular remodelling include migration of the cells from adventitia (29, 30), in situ differentiation and expansion (31) or accumulation from the distant sources like the bone marrow (26), (31), (32).

Many factors have been shown to influence SMC phenotype but the molecular mechanisms involved in its regulation and switching are not well established. Several different signaling cascades have been implicated in VSMC regulation. SMC differentiation and phenotypic modulation/switching are dependent on the complex interaction of a profusion of genes that are specific or selective for vascular SMC (33). Known factors to influence SMC phenotype are mechanical forces (34, 35), contact agonist (36, 37), reactive oxygen species (38), endothelial- SMC interaction (39, 40), thrombin (41), neuronal factors (42, 43), transforming growth factor-beta1 (TGF-b1) (44), and extracellular matrix components (45, 46).

Although it is well recognized the important role of SMCs in the repair of vascular wall injury, very few factors and pathways have been identified in regulating its dedifferentiation and phenotypic switching. Different signals triggering different pathways and signals from one cascade inhibit a parallel cascade (Fig 1.2). For example, insulin growth factor 1 (IGF-1) causes the expressions of genes implicated with the contractile phenotype through the PI3Kakt (phosphatidylinositol 3-kinase-protein kinase B) pathway and at the same time, blocks the Ras/MAPK pathway with the IRS-I/SHP2 (insulin receptor substrate-1/src homology protein 2) complex (47). PDGF-BB is one of the few growth factors implicated in SMC phenotype switching by stimulating MAPK (48) as well as cleaving the IPS/SHP2 complex. Other growth factors like basic FGF and EGF (49) can induce IPS/SHP2 complex triggering the activation of the MEK-ERK1/2 and MAPK kinase 6 (MKK6-p38MAPK pathways, mediated by the Ras activation and Grb2/Sos (growth factor receptor-bound protein/son of sevenless) complex.

Fig. 1.2. Regulation of SMC differentiation and phenotypic switching. From Muto and others (59)

21 The same pathways involved in VSMC differentiation and/or phenotypic switching also play a major role in VSMC activation after vascular wall injury. Vascular wall injury can induce humoral, autocrine and paracrine growth factors such as PDGF-BB and FGF-2 (Fig. 1.3) that in turn can stimulate the Ras-MAPK and PI3K-Akt pathways leading to cell proliferation (50). Akt recruits PI(3,4,5)P3 on the cell membrane and the 3- phosphatidylinositol-dependent kinase (PDK)1/2 then phosphorylates Akt (51). Phosphorylation of Akt activates the target of rapamcyin (mTOR)- raptor complex triggering transcription of growth genes leading to cell cycle progression (52). As previously mentioned, several vascular diseases involve SMC proliferation as the major pathophysiologic cause. Vascular SMC proliferation is considered the key element (14), (13), (53) in the process of atherosclerosis (Fig. 1.4) (54)). Initiation of atherosclerosis involves the response of ECs to pathogenic stimuli. The critical step in the early stage of atherosclerosis is the early response of ECs to induce adhesion molecules such as selectin and vascular cell adhesion molecule-1 (VCAM-1) (55). Oxidative modified lipoproteins can also confer expression of adhesion and cytokines in early atherogenesis (56, 57). Nitric oxide (NO) production is significantly changed with the subsequent reduction of the atherosclerotic function of the ECs (58).

Adhesion molecules can recruit mononuclear leucocytes into the surface of the ECs and once adhered, chemo-attractant chemokines like macrophage chemo-attractant-1 (MCP-1) facilitates the entry of the leucocytes into the artery wall. Mononuclear leucocytes, once inside the intima, become macrophage foam cells (60). These macrophages express platelet derived growth factors (PDGF) and other genes that encodes other growth factors such as heparin-binding epidermal growth factor fibroblast growth factor and insulin-like growth factors (60). A myriad of cytokines and growth factors can initiate both medial proliferation and migration of VSMCs. This is followed by the synthesis of the extracellular

Fig. 1.3. Proliferative signaling in VSMC as a response to injury. From Muto and others (59)

Fig. 1.4. Different stages of atherosclerosis showing the role of smooth muscle cells (SMCs). LDL, low-density lipoprotein; MCP, monocyte chemoattractant; VCAM, vascular cell adhesion molecule; PDGF-BB, platelet-derived growth factor (BB, b-chain homodimer); TNF, tumour necrosis factor; TGF, transforming growth factor; IL, interleukin 1; IGF, insulin-like growth factor; bFGF, basic fibroblast growth factor; Ang II, angiotensin II; EGF, epidermal growth factor; IFN, interferon. From Dzau and others (54).

25 matrix (ECM) proteins by the VSMCs that contributes to the formation of the fibrous cap of the newly formed plaque. VSMCs can also stimulate the production of matrix metalloproteinases (MMPs) involved in vascular modelling (55). Foam cells or lipid-laden macrophages and extracellular lipid droplets form the core region of the plaque. The presence of mast cells promotes plaque instability by promoting apoptosis of SMCs (61). Mast cells also up-regulates MMPs expressions that degrade ECM thus further destabilizing the plaque. Activated mast cells, however, have the ability to prevent platelet aggregation (62) at the site of EC injury by its intra-granular heparin. At the shoulder regions of the plaque, accumulation of T-lymphocytes can also be activated and can elaborate inflammatory cytokines such as gamma-interferon and tumour necrosis factor-beta (TNF-B) that in turn can stimulate macrophages, ECs and SMCs (63). Thinning of the fibrous cap can weaken the plaque making it more susceptible to rupture. Rupture of the plaque results to the contact between blood and thrombogenic material such as tissue factor, phospholipids and platelet-adhesive matrix molecules and as a result, leads to the formation of thrombus (64).

Gene Therapy Different therapeutic strategies have been employed to halt the progression of atherosclerosis. Since inflammation plays a key role in the development of this disease, powerful immuno-suppressants or anti- inflammatory compounds are used to block immune and inflammatory mechanisms that initiate and activate atherogenesis (64), (7), (63). However, non-specific anti-inflammatory therapies such as non-steroidal anti-inflammatory drugs (NSAIDs) have proven to be not beneficial in terms of cardiovascular outcome (65). The use of NSAIDs selective for cyclooxygenase-2 seems to increase the risk of thrombotic complications (65). Induction of protective immunity to atherosclerosis has been done in animals (66). Vaccination with oxidized LDL (67), bacteria containing modified phospholipid (68) or heat shock protein 65 (69) reduced atherosclerosis.

26 Although this approach looks promising, many mechanistic questions remains before this can be tested in humans. Clinical conditions like vein graft failure, in-stent restenosis and transplant vasculopathy are consequences of vascular proliferation. Prior to the use of drug-eluting stents, about 30-40% of patients who undergo percutaneous balloon angioplasty are likely to develop restenosis within the first six months (70). Drug-eluting stents reduce the restenosis but with associated increased risk of stent thrombosis (71). Vein graft failure occurs among 30-50% patients within 10 years (72). Due to SMC fibroproliferative vasculopathies, many investigations have targeted specific genes to inhibit proliferative response of SMCs to vascular injury (73) (74, 75). Gene therapy can target either the activator or suppressor of the cell cycle.

Cellular proliferation involves cell-cycle progression, and many genes controlling the cell-entry and progression have been investigated. These genes include the cyclin-dependent protein kinase inhibitors, p21 (76), p27 (77) and p53 (78). Gene transfer of retinoblastoma protein (Rb) (79) reduces SMC proliferation due to halting of cell-cycle progression in rat carotid artery. Gene transfer targeting several growth factors (80) cytokines (81) and chemokines (82) has also been employed. It has been reported that vascular endothelial growth factor (VEGF) reduces restenosis after arterial injury in animals (83) and similar effect was observed using VEGF gene-eluting stents (84). However, liposomal gene transfer of VEGF using human subjects (85) gave no significant improvement in the inhibition of restenosis. Other growth factors like basic fibroblast growth factor (bFGF) (86) and transforming growth factor-beta (TGF-B) (87) were used and showed a significant reduction in neointimal formation in rats. The question remains whether the results in animals can be sustained in the human.

Recent studies have demonstrated that microRNAs (miRNAs) are involved in the regulation of proliferation and differentiation of vascular SMCs after vascular injury (88). Over-expression miR-145 or miR-143 promotes differentiation and inhibits proliferation of SMC (89). PDGF can

27 transcriptionally induce the expression of miR-221 and miR 222 implicated in vascular SMC differentiation (90). Knockdown of miR-221 increased the expression of SMC differentiation markers and blocked the PDGF-induced proliferation and migration (91). In response to vascular injury, miR-21 showed a 5-fold increase compared to that of the control and using antisense oligonucleotide against miR-21, there was inhibition of neointimal formation in rat carotid artery after angioplasty (92). Although targeting miRNAs may have the potential as therapeutic strategy, more work is needed to establish its specificity as thousands of human genes are targeted by miRNAs (93).

Targeting transcription factors by gene therapy is also a strategy to abrogate SMC proliferation. These factors are upregulated or down regulated by different stimuli that initiate cell proliferation or impair cell cycle. Different nucleic acid based strategies (75, 94, 95) have been utilised as potential therapeutic tools by controlling the expression of these transcription factors. Antisense oligonucleotides (ODNs) are single- stranded complementary sequences that target specific sites in messenger RNA thus blocking the translation process. ODNs targeting c- myb (96), c-myc (97) and NF-kB p65 (98) resulted to inhibition of neointimal formation. Despite the success of antisense therapy, several limitations have been recognized. The clinical application of ODN technology is limited by a relative lack of specificity, slow uptake and fast intracellular degradation of the molecule (99). Decoys are synthetic double-stranded DNA bearing cis-acting elements that bind to transcription factor thus blocking the interaction of the nuclear protein with elements in the authentic promoter (100). Decoy was utilised to target E2F (101) reducing mRNA levels of PCNA and cdc2, inhibiting proliferation and abrogation of the neointimal formation in injured rat arteries. An important concern in the use of decoys is their potential inhibition of normal physiological response (102) given the ubiquity of transcription factors in the promoters. Catalytic RNA and DNA molecules like ribozymes (103) and DNAzyme (104) increase the rate of cleaving the mRNA substrate thus offering more potency and sustainability in reducing gene expression.

28 Ribozymes were employed to target c-myb (105) and TGF-B (87) (106) (107) to inhibit vascular smooth muscle cell proliferation both in vitro and in vivo. ED5, a DNAzyme targeting the Egr-1, inhibited Egr-1 induction and neointimal formation after balloon injury to the rat carotid artery wall (108) and the inhibitory effect of ED5 on the formation of neointima was later confirmed (109). A DNAzyme targeting human Egr-1, DzF selectively inhibited neointimal thickening after stenting pig coronary arteries (110). Although DNAzyme proved to be more resistant to degradation compared to that of ribozyme, efficient delivery is still a problem.

One of the aims of The Transcription and Gene Targeting Laboratory at the Centre for Vascular Research was to inhibit or suppress the SMC proliferation by looking into different transcription factors which may play a role on its pathobiology in vascular diseases and elucidate its mechanism(s).

Transcription factors play a fundamental role in the cell growth control, development and differentiation. Their critical role has been undoubtedly established in the activation, repression, and/or modification of gene regulation that are necessary and required for normal biologic processes. Regulation of transcription factors involved the cells’ response to inter- and intra-cellular signals. It is generally accepted that dysfunctional activation and/or repression of these proteins lead to cellular aberration and instability. Different transcription factors have been very well implicated in the control of vascular smooth muscle cells. Using cDNA array analysis, a transcription factor, Yin yang-1 (YY-1) was found to be immediately upregulated after vascular injury and over-expression of YY-1 inhibited vascular smooth muscle cell proliferation but not endothelial cells (111). Inhibiting smooth muscle cells without affecting endothelial cell proliferation could be useful in inhibiting neointimal thickening without affecting re-endothelialization and re-establishment of non-thrombogenic surface. However, the mechanism by which YY-1 exerts its effect on smooth muscle cells and not on endothelial cell is unknown.

29 Yin Yang-1 Yin Yang-1 (YY-1), also known as NF-E1, UCRBP, CF1 and delta () nuclear factor, is a ubiquitously expressed zinc-finger transcription factor. Initial investigation by Chang and colleagues identified two elements in the adeno-associated virus (AAV) P5 promoter that had negative effect in the absence of the E1A protein but acted as activators in the presence of the E1A protein (112). One of the elements “R1-R2 region” (P5-60 site), a tandem repeat of 10 bases, was later identified by Shi and others to be the binding site for a protein named as Yin Yang 1, because of its dual transcriptional activity (113). In the same year, Park and Atchison identified the same protein termed NF-E1 based on its ability to bind with the Igk 3’ enhancer (114). Also, Hariharan and others identified the same protein binding to the sequence elements downstream of the transcriptional start sites in the ribosomal protein L30 and L32 genes and designated the protein as delta () (115). Subsequently, YY-1 has been identified by other groups and named it as Upstream Conserved Region Binding Protein (UCRBP) (116), nuclear matrix protein NMP1 (117) and common factor 1, CF1 (118).

Structure and features of YY-1 The YY-1 gene has been mapped on the human chromosome 14q32.2 (119) and its amino acid composition is highly homologous with mouse, 95% (119) and with rat, 97.8% (120). YY-1 consists of five highly conserved exons and encodes a protein of 414 amino acids with the calculated molecular weight of 44 kDa but migrates on SDS gels as a 65- 68 kDa probably due to the structure of the protein (121). YY-1 is a phosphoprotein with a half-life of 3.5 hours (122) and ubiquitously expressed in different tissues including brain, heart, limb, and immune system (123) (124) (125) (126) (127) (128). . The YY-1 protein contains four C2H2-type zinc-finger motifs (amino acid 298-397) (Fig. 1.5), the specific DNA-binding domain (115) and later identified as the repression

Fig. 1.5 Schematic representation of the different domains of YY-1. Transcriptional activation and repression domains of YY-1 together with their relative amino acid positions. His, histidine–rich region; GK, glycine- lysine-rich domain; PHR, pleiohomoetic homology region; Zn, zinc. From He and Casaccia-Bonnefil (134)

31 domain as well (113) (130) (131) (132) (133). The second repression domain has been identified and mapped to amino acid 170 and 200 of YY- 1 (134). Mapping the activation domain(s) was somewhat controversial. The activation domain has been pinpointed at the N-terminus region (amino acid 43-53) (121) (135). Other studies showed that the first 90 (131) or 69 (136) amino acids of YY-1 as the activating domain. Furthermore, study by Bushmeyer and colleagues divided the activation domains into two regions, aa 16-29 and aa 80-100 (133). A glycine-rich domain and 11 consecutive histidine residues (aa 70-80) downstream of the activation domain were noted whose function is still unknown. A central domain, pleiohomoetic (137) region, aa 205-226, mediates the interaction with the homeobox Hox proteins (138) (139) (140). YY-1 consensus DNA recognition sequence or motif was first identified by PCR- based binding site selection technique and the consensus sequence reads 5’-(C/g/a)(G/t)(C/t/a)(CATN(T/a)(T/g/c)-3’ (141). Shorter YY-1 binding motifs with high affinity, CCAT and ACAT, were later identified by Yant and others (142). Compilation and analysis for over-represented motifs from a set of 723 human core promoter sequences revealed that YY-1 DNA recognition sequence mostly reside immediately downstream from the transcriptional start site (143). Also, YY-1 DNA recognition sequence overlapped with the translational start sites suggesting that the YY-1 motif plays a dual role in both transcription and translation initiation (143).

Regulation of YY-1 expression Little is known about the mechanism of transcriptional regulation of YY-1. YY-1 is involved in the transcriptional control of a large number of genes, estimated to be around 10% of the total mammalian gene set (144) and since it involved in so many pathways, the expression level of this transcription factor must be tightly monitored for the survival of cells and organisms (129). High levels of YY-1 proteins are often observed in normal and cancer cells (145). It has been reported that the YY-1 locus of all vertebrates contains a cluster of its own DNA-binding sites within the first intron implying that YY-1 is auto-regulated through its own DNA- binding sites (146). In SMCs, YY-1 expressions in both mRNA and protein

32 are increased by vascular injury via the autocrine and paracrine effect of basic fibroblast growth factor (bFGF or FGF-2) (111). The level of YY-1 transcripts is relatively constant in the organism but it varies during development. Higher levels of YY-1 were detected in undifferentiated keratinocytes compared to that of differentiated cells (147).

Post-translational modifications of YY-1 After its discovery, numerous studies have been performed with YY-1 in different cell types and at different developmental stages resulting to conflicting roles of YY-1. One possible explanation is that YY-1 can be modulated by multiple post-translational modifications (144). Reported post-translational modifications of YY-1 include phosphorylation, acetylation, deacetylation, O-linked glycosylation, caspase-dependent cleavage, sumoylation, poly(ADP-ribosy)ation and S-nitrosation (145). Eight consensus phosphorylation sites are found in the zinc finger regions of YY-1 and it was suggested that phosphorylation decreases the ability of YY-1 to modulate transcription (148). However with adeno-associated viral p5 promoter, treatment with phosphatase did not affect YY-1 binding (121). YY-1 activity is regulated by intricate mechanisms involving negative feedback loops, histone deacetylation (149) and the recognition of the cognate DNA sequences by acetylation and deacetylation of the YY-1 protein. Exposure to glucose increases the O-linked beta-N- acetylglucosamine (O-GlcNAc) modification of YY-1 disrupting YY-1- retinoblastoma protein (Rb) complex formation (150). In HeLa cells, fragmented YY-1 caused by caspase-dependent cleavage still able to bind to the DNA although YY-1 is no longer able to stimulate transcription (151). YY-1 can also bind with a SUMO E3 ligase, PIASy, making YY-1 more stable but sumoylation abrogates its transcriptional activity (152). YY-1 is accessible to poly(ADP-ribosylation) only when unbound and prevents YY-1 binding to DNA (153). However, poly(ADP-ribosyl) transferase can not attach poly(ADP-ribose) chain when YY-1 is already bound to DNA. S-nitrosation, on the other hand, inhibits YY-1 activity as demonstrated by the up-regulation of Fas expression after NO treatment resulting to prostate cancer sensitisation to Fas-induced apoptosis (154).

Transcriptional regulation mechanisms of YY-1 Many mechanisms have been proposed on how YY-1 activates or represses gene expression. Multiple mechanisms on repression by YY-1 have been extensively reviewed (121). Briefly, YY-1 mediated repression is possible by activator displacement (Fig. 1.6), interference with activator function by binding at the promoter (Fig. 1.7) or interference by directly to the activator (Fig. 1.8) or by recruitment of a co-repressor (Fig. 1.9). Activator displacement by YY-1 is possible when YY-1 site overlaps that of an activator or it competes with binding to the promoter. In human interferon gamma promoter, YY-1 site overlaps with AP-1 site and competes for binding thus AP1 enhancer activity was suppressed (155). Displacement mechanisms were also been responsible for the skeletal and smooth muscle alpha actin muscle regulatory elements (MREs) and muscle creatine kinase CArG (121).

Different models for the YY-1 as an activator have been proposed (156) (121). YY-1 can directly bind to the promoter with close interaction with other transcription factor (Fig. 1.10). It can also recruit a co-activator to facilitate its enhancement of regulation of gene (Fig. 1.11). YY-1 being a direct activator can possibly due to its two acidic activation domains (124). Physical interactions with other proteins like E1A can convert YY-1 from a repressor to an activator as mediated by p300 (136). YY-1 can direct and initiate transcription in vitro when bound to initiator (Inr) elements (157). Suggestion was made that YY-1 to be an Inr element-binding protein (158) since it can function like the TATA box-binding protein. The third model proposes the masking and the unmasking of the repression (C-terminus) and activation (N-terminus) domains, respectively (Fig.1.12). YY-1 interaction with co-factors can undergo structural alterations in the C- terminus thereby masking the YY-1 repression.

Fig. 1.6. Model of YY-1 mediated repression. YY-1 interferes with an activator by binding itself to the same promoter. YY-1 overriding the effect of the activator. Modified from Gordon et al (145)

Fig. 1.7. Model of YY-1 mediated repression. YY-1 repression via displacement of an activator occupying an overlapping YY-1 binding site in a promoter. Modified from Gordon et al (145)

Fig. 1.8. Model of YY-1 mediated repression. YY-1 can repress a gene without binding to its promoter; it can interact with the activator to silence its activity. Modified from Gordon et al (145)

Fig. 1.9. Model of YY-1 mediated repression. YY-1 can recruit a co-repressor and can either bind directly to the promoter or bind to the activator to exert its repressive activity. Modified from Gordon et al (145)

Fig. 1.10. Model of YY-1 mediated activation. Activation of transcription by YY-1 direct interaction with a transcription factor. Double arrow denotes activation. Modified from Gordon et al (145)

Fig. 1.11. Model of YY-1 mediated activation. YY-1 recruits a co- activator that modifies or interacts with other transcription factors. Double arrow denotes activation. Modified from Gordon et al (145)

Fig. 1.12. Model of YY-1 mediated activation. YY-1 indirect activation by YY-1 interacting with a repressor by masking and/or unmasking of the C-terminus and/or of the N-terminus of YY-1. C- terminus of YY-1 is the designated repression domain whereas the N-terminus is the activation domain. Modified from Gordon et al (145)

39 Also, it was proposed that the C-terminus could unmask the N-terminus leading to a significant increase in the transcriptional activation of YY-1. Recruitment of co-activators is another model being proposed. YY-1 can interact with co-activators with HAT activity such as CBP and p300 (159).

Genes regulated by YY-1 and biological functions Since YY-1 discovery, there was a surge of reports of different promoters and genes regulated by this enigmatic transcription factor. Shi and colleagues first published a comprehensive list of the genes controlled by YY-1 including identified elements relevant to its activating or repression or initiation activity and the relative position of YY-1 sites in the promoters (121). Gordon and colleagues reviewed the therapeutic implications of YY-1 in cancer (145) and recently, gene expression analysis of the YY-1 in different human tumour types was performed using computational analysis on 36 publicly available Gene Expression Omnibus (GEO) datasets (160). Genes regulated by YY-1 can be divided into several groups according to known or implicated biological functions: development, differentiation, cellular proliferation, invasion, apoptosis, tumourigenesis and viral gene regulation. Deng and others (152) classified the YY-1-associated proteins into four categories; namely, tumour suppressors, oncogene products, post-translational associated products and transcriptional and chromatin remodelling proteins. Table 1 lists some of the promoters/genes regulated by YY-1 and its associated proteins published from 2006 till present. No general rule can be generated regarding the “specific” biological role of YY-1 since its effect varies from cell type to cell type, cellular environment, stage of development and over-all YY-1 binding pattern. For example in human erythroleukemia K562

Table 1. Promoters/genes regulated by YY-1. List of genes/promoters controlled by YY-1, its associated protein and its effect. ND denotes not determined. Please see the Abbreviation Section for the meaning of the other acronyms.

41 Associated Promoter/Gene Protein Effect References

P73 E2F1 activation (161) mitochondrial genes PGC-1alpha activation (162)

MOR Sp1 activation (163)

ERGIC-53 ND activation (164)

HLJ1 AP-1 activation (165)

Otx2 HAT activation (166) c-myc H3 enhancer activation (167)

COX-2 ND activation (168)

REST ND activation (169)

Peg3 ND activation (170)

EGFR Sp1 activation (171) c-myc N1IC activation (172)

RIZ1 ND activation (173) beta- cdx4-hox arrestin1 activation (174)

BNP HDAC2 activation (175)

HO-1 ND activation (176) cox7c BAP1/HCF-1 activation (177)

U6 promoter SNAP activation (178)

BACE1 ND activation (179)

GDAP1 ND activator (180)

PSA AR activator (181)

PCFT ND activator (182)

42 PREPL-C2ORF34 NRF-2 activator (183)

HP1 alpha ND activator (184)

Occludin Sp3 activator (185)

SNAI1 NA activator (186) miR-206 AP1 (c-Jun) repression (187)

RGS16 ND repression (188)

PPAR delta ND repression (189) Laneosterol synthase HDAC3 repression (190)

ERCC5 E2F1 repression (191)

StAR HDAC1 repression (127)

Tcf4 HDAC1 repression (128)

VEGF ND repression (192)

TGF-beta1 ND repression (192)

DR5 ND repression (193)

GLAST ND repression (194)

CuZn SOD ND repression (195)

PcG ND repression (196) a3b1 integrin CREB repression (197)

Fas ND repression (198)

CXCR4 ND repression (199)

GLAST ND repression (200)

IFN-beta SAP30/Sin3A repression (201)

CEBPD SUZ12 repression (202)

CHOP-10 ND repression (203)

43 c-myc p300/HDAC3 repression (204)

PrP (Prion) ND repression (205)

HOXB13 HDAC4 repression (206)

Troponin ND repression (207) miR-29 NF-kappaB repression (208)

MMP-9 HDAC3 repression (209)

CFTR Nrf2 repression (210)

ChM-I HDAC repression (211)

LEF-1 ND repression (212)

ANT1 Mecp-2 repression (213)

Pax7 PRC2 repression (214)

Hoxa11 HDAC2 repression (140)

Amelogenin ND repression (215)

ERBB2 AP-2 repression (216) p16(INK4a) HDAC3/4 repression (217)

HIF-2alpha ND repressor (218)

P97 promoter ND repressor (219)

44 cells activated Notch 1 receptor intracellular domain (N1IC) can bind with YY-1 to activate c-myc in a CBF-1 independent manner. In quiescent human mammary epithelial cells MCF-10A, YY-1 binds to p300 cooperating with HDAC3 to repress c-myc transcription (204). Cellular environment determines the post-translational modification of YY such as phosphorylation and thus, affecting its mode of action. Fentanyl-induced beta-arrestin-mediated ERK phosphorylation led to YY-1 phosphorylation impairing its association with the Talin2 promoter leading to decrease transcription of talin2 (220). In contrast, morphine induces ERK phosphorylation but cannot induce phosphorylation of YY-1 via protein kinase C pathway and has no effect on the transcription level of talin2 (220). Also, YY-1 can stimulate Rex1 (zfp42) in normal human prostate epithelial cells but it has no effect in prostate cancer cells (PC-3) (221). A single nucleotide polymorphism in a promoter of nitric oxide synthase dramatically affect YY-1’s action and has been associated with hypertension in a Chinese Han population (222). This effect of polymorphisms on YY-1 activity is explicitly emphasize by the preferential binding of YY-1 to the -18483C allele as compared to that of -18483A allele of the FcepsilonRIalpha gene (223). YY-1 can also be an activator or repressor depending on the location of its DNA binding motif and/or the time of viral infection (224). Binding of YY-1 to murine beta interferon promoter can either activate or repress its expression by recruiting CBP or HDAC respectively as dictated by the position of YY-1 occupancy after infection (225). Its been reported that YY-1 can inhibit transcription in undifferentiated cardiomyocyte cells, H9C2 but it can be an activator when these cells are differentiated (226). This bifunctional activity of YY-1 is due to the localization of HDAC5 whereby in differentiated cells, HDAC5 is in the nucleus and inter-acting with YY-1 (226). In undifferentiated cells, HDAC5 is localized in the cytoplasm and cannot cooperate with YY-1 (226). Cytoplasmic YY-1 in Xenopus oocytes had been shown to associate with a variety of RNA in complexes termed as messenger ribonucleoprotein particles (mRNPs) (227). These complex particles are shown to be important in the storage of maternally derived transcripts that are selectively utilized in the development stages (227). YY-1 is also able

45 to associate with large RNA molecules independent of the 5’ cap structure and YY-1-RNA interaction is very stable that YY-1 possesses the ability to interact with structurally divergent RNA substrates. The functional significance of this interaction of YY-1 with mRNPs is yet to be fully established.

Genetic ablation of yy-1 in mice resulted in lethality shortly after implantation showing the important role of YY-1 in development (125). YY-1-deficient lymphoid cells showed slow growth implying the possible pro-proliferative function of YY-1 (228) and this was further supported by the high level of YY-1 in human prostate cancer tissue (145). This proproliferative function of YY-1 is controversial since other studies showed the opposite. In human breast cancer cells (229) and SMC (111). YY-1 is classified as an inhibitor of proliferation. To make things more complicated, a study reported that YY-1 function is independent of the cell cycle (231) in conditional deletion of YY-1 in oligodendrocyte lineage.

Knockdown studies on YY-1 showed higher polyploidy and chromosomal aberrations and established that YY-1-INO80 complex regulates genomic stability by homologous recombination-based repair (232). YY-1 can also facilitate the binding of small nuclear RNA activating protein complex (SNAPc) to the proximal sequence element (PSE) on both polymerase I and II promoters (178). Clustered YY-1 binding sites have been identified in imprinting control regions (ICRs) (233). These genomic regions usually control the imprinted genes clustered in specific regions of chromosomes. YY-1 binds to tandem repeat sequence structure of the paternal expressed gene 3- differentially methylated region (Peg3-DMR) and possibly suggests a significant role of YY-1 in mammalian genomic imprinting (234). The same group later reported that the most likely role of YY-1 in oogenesis is the de novo DNA methylation of the DMRs of Peg3 and the maintenance of unmethylation of these DMRs during spermatogenesis (235).

46 YY-1 in disease The role of YY-1 in different diseases and the genes controlled by YY-1 has been reviewed accordingly. Potential role(s) of YY-1 in the nervous system has been appraised (129) (236) (237) (238) (179) (138). The precise function of YY-1 and its mechanism of action in the nervous system are still yet to be thoroughly investigated (129). YY-1 has been implicated in cancer development and progression (145), (239), (240) (160). Overexpression of YY-1 was reported in many types of cancer such as prostate (241), ovarian (242), breast (216), osteosarcoma (199), myeloid leukaemia (243), Hodgkin lymphoma (137) and Non-Hodgkin lymphoma (244). Recent work by Ishii and others showed that overexpression of YY-1 inhibits human breast carcinoma and glioblastoma cells (245).

YY-1 in CVD The role of YY-1 in the development of cardiovascular disease has also been investigated. Induction of serum amyloid A (SAA) by pro- inflammatory stimuli lead to analysis of its regulation and YY-1 is one of the transcription factors identified to be involved in SAA regulation (124). The molecular impact of SAA has been substantially linked to lipid metabolism/transport, induction of extracellular matrix-degrading enzymes and recruitment of inflammatory cells to sites of inflammation reflecting typical scenarios in the early stages of atherosclerosis. In rat, YY-1 represses SAA1 promoter (246) and YY-1 can be displaced by NF-kB. Competition between YY-1 and other transcription factors is also exemplified with a positive acting-SRF and a cardiogenic homeodomain factor, Nkx-2.5 in regulating cardiac -actin promoter activity (247). The repressive effect of YY-1 can be reversed by co-expression of Nkx-2.5 and SRF. Alpha myosin heavy chain expression is very much implicated to cardiac hypertrophy since during heart failure, a significant reduction of this muscle protein was reported (248), (249) (250). YY-1 binds to the alpha myosin heavy chain promoter and represses its expression in neonatal cardiac myocytes but an activator of the same promoter in non- cardiac cells (251). They showed later that the YY-1 repressive ability is

47 greater when it interacts with Ku proteins (252). The upregulation of YY-1 may be an anti-hypertrophic factor as YY-1 also interacts with HDAC5 (253) Their data suggest that YY-1 is needed for normal myocyte phenotype by down regulating the “fetal gene program” thus preventing myocyte hypertrophy (254). However, in a study of heart failure from chronic volume overload, there was no change in the protein expression of YY-1 between the sham and cardiac overloaded hearts suggesting that YY-1 is not playing a role in alpha-myosin heavy chain repression (255).

In an effort to dissect the genetic factors associated with neointimal formation, Yuan and colleagues (256) crossed C57BL/6 and C3H/HeJ apolipoprotein E-deficient mice. The former strain of mice shows dramatic differences in neointimal formation as compared to the latter when deficient in apolipoprotein E (ApoE-/-) and fed a Western diet (257). They identified a significant quantitative trail locus (QTL) named Nih1 and one of the probable candidate genes is YY-1. The level of YY-1 mRNA and proteins in uninjured arterial walls were higher in C3H than in BL/6 thus a greater inhibition of neointimal formation in C3H. Moreover, the high level of YY-1 led them to hypothesize that the strong YY-1 expression of proliferating neointima might also regulate its growth. A 10-bp deletion and 5 short nucleotide polymorphisms were identified in the YY-1 promoter that might be responsible for the higher baseline expression in C3H (257). As previously mentioned, even a single-nucleotide polymorphism (SNP) in the YY-1 DNA-binding motif can significantly change its effect. A change of nucleotide from C to A in the -1026 site of the nitric oxide synthase can result to higher transcriptional activity and this functional SNP can be correlated with hypertension in a Chinese Han population (222).

Previous studies on SNP on various promoters whose genes have been implicated to high blood pressure have been done and failed to directly link YY-1. SNP in the GNAI2 gene promoter has been associated with high blood pressure in a Caucasian population in Italy (258). Inspection of the promoter of GNAI2 revealed potential binding site for Sp1 and YY-1

48 but using supershift technique of the HeLa nuclear proteins, they showed that Sp1 binds specifically to the site but not YY-1. A disrupted motif for sex determining region Y (SRY) and YY-1 in the –A-261T of the Chromogranin B promoter was noted to effect blood pressure (259). Analysis of different variants showed that only SRY might contribute to the sexual dimorphism of blood pressure in the population.

Petrova and co-workers showed that in growth-arrested human coronary artery smooth muscle cells (CASM), overexpression of YY-1 activates DNA synthesis (260). However, when retinoblastoma protein (Rb) was coexpressed with YY-1, Rb abrogates YY-1 effect. Rb interacts with YY-1 to form a complex thus inhibiting YY-1-dependent transcription in vitro and YY-1 may serve as a checkpoint in cell cycle transition of differentiated cells (260). YY-1 can also compete with SRF in binding to DNA element CC(A/T)6GG or CArG motif of the smooth-muscle-specific myosin heavy chain promoter. Using nuclear extracts from rat aortic SMC, exogenous nitric oxide (NO) restores the platelet-derived growth factor BB-induced suppression (261) as a result of inhibiting YY-1 and favoring the binding of SRF to the CArG element. Using the murine smooth muscle 22 alpha (SM22a) promoter, binding of SRF to CArG box is necessary but not sufficient to restrict transgene expression to SMCs in vivo (262). Of the six smooth muscle elements (SMEs) identified, SME4 contains the CArG boxes where SRF and YY-1 can potentially bind. Two other unidentified nucleoprotein complexes were detected by electrophoretic mobility shift assay (EMSA) and these two complexes where enriched by UV cross- linking (262). Experiments on transgenic mice with LacZ under the control of the SM22a wherein the c-fos SRE has been substituted for the CArG boxes showed that binding of SRF to CArG box is not sufficient to activate transcription in arterial SMCs (262). Since YY-1 is an inhibitor of muscle cell differentiation and expression of muscle-specific genes, a temporal localization of YY-1 in vivo and in vitro in porcine pulmonary arteries was determined as an effect of actin polymerization (263). Their results show that actin polymerisation controls YY-1 subcellular localization. In the absence of actin de-polymerisation, cytoplasmic YY-1 can increase

49 smooth muscle-specific gene expression and may contribute to the pathobiology of pulmonary hypertension (263). YY-1 was also implicated with the regulation of bradykinin B1 receptor (BDKRB1) by interacting with TATA-box-binding protein and transcription factor IIB (264). BDKRB1 expression is rapidly induced after different tissue injury or inflammation under the effects of cytokines and growth factors (265).

The induction of YY-1 expression after vascular injury in SMCs has been shown for the first time to be under the autocrine/paracrine control of the endogenous basic fibroblast growth factor or FGF-2 (111). Overexpression of YY-1 selectively inhibited SMCs proliferation but not ECs. YY-1 is induced by either injury or FGF-2 and is differentially expressed in normal and diseased human arteries. The mechanism by which YY-1 selectively inhibits SMC proliferation is unknown.

In Transcription and Gene Targeting Laboratory at the Centre for Vascular Research, other transcription factors are being examined aside from YY-1. One particular transcription factor is the Egr-1 whose dependent genes are very well implicated to SMC proliferation. To date, no interplay study between YY-1 and Egr-1 has been shown. Although extensive work was performed on Egr-1 since the late 1980s and studies implicated it in disease and ERK-activation, no commercially available phospho-specific antibody to Egr-1 is available at present.

Early Growth Response-1 Egr-1 is an immediate early gene also known as nerve growth factor inducible-A (NGFI-A) (266), Zif268 (267), Krox24 (268), Tis8 (269) and ZENK (avian homologue of Zif268, Egr-1, NGFI-A, Kox-24) (270). This transcription factor belongs to the Cys2His2 class of zinc finger proteins and other members of this group include Egr-2 (271), Egr-3 (272), Egr-4 (273), Egr- (274) and the Wilm tumour gene (275). Members of Egr family recognize the GSG or EBS (Egr-1 binding site) motif, GCG(G/T)GGGCG (276) (277) and once bound to the DNA, these transcription factors alters gene transcription through mechanisms

50 dependent on both co-activators and co-repressors. Egr-1 can either activate or repress target genes by its interaction with the EBS. For example, Egr-1 can activate the cardiac a-myosin heavy chain and acetylcholinesterase genes (278) (279) but can repress the expression of adenosine deaminase (ADA) (280).

Structure and regulation of Egr-1 Human Egr-1 is located on chromosome 5q23-q31(281) with two exons and one intron spanning approximately 3.6 kb (282). This gene encodes 533 amino acids and this nuclear phosphoprotein has an observed molecular weight between 75-82 kDa and a half-life of 2 hours (283). Egr- 1 gene is composed of functionally independent domains (Fig. 1.13, upper panel) consisting of N-terminal (rich in serine and threonine) and C- terminal regions (serine, threonine and proline rich). Egr-1 contains four activation regions (A1, amino acids 16-41; A2, amino acids 89-148; A3, amino acids 226-267; A4, amino acids 420-536) (284). Egr-1 activation domain is a controversial one since other studies mapped it from amino acids 3- 281(279) (285). Another study showed that deletion of activation domain between amino acid 174 to 270 inhibits Egr-1-induced transcription (286). The three zinc finger motifs of Egr-1 spanning from amino acids 331 to 419 is the DNA-binding domain (DBD) and structured as an antiparallel beta-pleated sheet and an alpha helix. The repression domain sandwiched between the activation domain and the DBD serves as the binding site for the transcriptional co-repressor proteins NAB1 and NAB2 (NGFI-A binding proteins 1 and 2) (287) (288). The bipartite nuclear localization signal (NLS) of Egr-1 is located within the DBD from animo acids 361 to 419 (289) and the specific region that promotes nuclear localization of Egr-1 includes the flanking sequence together with zinc finger two or three but not zinc finger one (279). The functional elements in the promoter region of Egr-1 control its transcriptional expression (Fig. 1.13, lower panel). Egr-1 promoter contains five serum response elements (SRE) (290), calcium response element (CRE)-like sites, CCAAT box, five binding sites for ternary complex factors (Ets) and Egr-1 binding site (EBS) (291). A canonical NF-kB binding site in the Egr-1 promoter

Fig. 1.13. Schematic representation of Egr-1 gene and promoter. A. Upper panel. Structural and function regions of the Egr-1 protein indicating the activation and repression domains together with the three zinc finger region. From Thiel and Cibelli (295). B. Lower panel. Promoter of Egr-1 showing different control elements and their relative positions. From Thiel et al (296)

52 was also identified (292). Full promoter analysis of the human Egr-1 identified a fifth functional SRE and recently, two activating transcription factor 5 (ATF5) had been identified in the Egr-1 promoter (293). Two transcription factors are required for SRE mediated activity, first is the serum response factor (SRF) and second, the ternary complex factor that forms a ternary complex with the SRF protein. Ternary complex factor can be Elk-1, Sap1 or Sap2 that contact DNA and also have to bind to SRF in order to exhibit biological activity. It has been shown that Elk-1 connects the activated Erk1/Erk2 signaling pathway with enhanced transcription via the SRE as demonstrated for Egr-1 (294).

Egr-1 is weakly expressed basally but is rapidly and transiently expressed in many different cell types in response to a plethora of extracellular stimuli like growth factors (297) (298) (299), hypoxia (300) (301) (302), cytokines (303) and injurious stimuli (304) (305). Injurious stimuli that stimulate Egr- 1 expression include fluid shear stress (306), mechanical stress (307), ischemia/reperfusion (308) and acute tissue injury (304).

Egr-1 controls the expression of a wide variety of genes implicated to proliferation, differentiation, inflammation associated with atherosclerosis, cardiac and pulmonary fibrosis and a variety of physiological roles in the central nervous system. Biological roles attributed to Egr-1 include female reproductive capacity (309), wound repair (304), controlling synaptic plasticity (310), neurite outgrowth (311) and apoptosis (312). Egr-1 knockout animals appeared normal with infertility observed in homozygous null females (309) and it was implied that the physiologic roles of Egr-1 might only be elaborated in response to environmental challenges or stimuli. Using microarray and/or chromatin immunoprecipitation, target genes of Egr-1 in different biological settings have been identified (313), (314) (315) (316).

Egr-1 in disease Egr-1 expression is induced under diverse mitogenic signals on different cell types. Its genetic expression does not require de novo protein

53 synthesis that implies it acts as an immediate response mediator between cell surface receptor signalling and gene expression regulation. Egr-1 can modulate the immune response by means of induction of differentiation of lymphocytes precursors (317). Several studies have focused attention on the role of Egr-1 in activation of tissue factor (TF) and several growth factors such as PDGF-A, PDGF-B, TGF-b1, IGF-II and bFGF (318) (319) (320) (321). In some tumours, Egr-1 has been described as a tumour suppressor gene since its expression is poorly expressed and can act as growth and transformation suppressor when overexpressed (292). Egr-1 has been proposed to be a pro-apoptotic protein since it can directly activate p53 promoter (322), binds to the transcription factor c-Jun augmenting its pro-apoptotic activity (323) and transactivating the PTEN gene (324). Induction of Egr-1 protein was also noted in the central nervous system as a result of brain injury, ischaemia and nerve transection (325). Studies on central nervous system neurons have implicated Egr-1 as a candidate gene for synaptic plasticity (326) (310). A wide spectrum of Egr-1 inducible genes (Fig. 1.14) implicated to atherosclerosis has been extensively reviewed (297) (327) (328). Egr-1 plays an important role in the development and progression of atherosclerosis based on its role in the regulation of tissue factor (TF) (329) (330), plasminogen activator inhibitor (PAI-1) (331), SMC mitogens (PDGFs, bFGF, TGFb) (332), adhesion molecules (ICAM-1, CD-44, VCAM-1) (333), chemokines MCP-1 (334) and oxidative stress via superoxide dismutase 1 (335). Egr-1 is an important mediator of SMC growth and intimal thickening in the reparative response to vascular injury since inhibition of Egr-1 expression by antisense oligonucleotide and DNAzyme significantly reduced SMC proliferation and intimal thickening (298) (108) (336).

Fig. 1.14. Schematic representation of different cardiovascular disorder under the control of Egr-1. Different stimuli can trigger Egr-1 transcription via the MAP kinases thus creating a cascade of activation of Egr-1-dependent genes very much implicated to various cardiovascular pathological processes. From Khachigian (297)

Post-translational modification of Egr-1 The majority of all proteins undergo post-translational modifications altering their physical and chemical properties, folding, conformation stability, activity and consequently, its function. Several potential N- and O-linked glycosylation sites are present in the predicted Egr-1 protein (266) and later, Egr-1 proteins were shown to contain N- acetylgalactosamine or N-acetylglucosamine moieties in vKr24.4-infected HeLa cells (337). Cellular redox state may also control the DNA-binding activity of Egr-1 by excluding zinc from the protein and rendering the oxidized form inactive (338). Two forms of Egr-1 were found, one form residing in the nucleus and the other one in the cytoplasm (339). The cytoplasmic form of Egr-1 is truncated before the zinc finger region that contains the nuclear localization signal (289). Egr-1 transcriptional activity can also be regulated through the interaction of other transcription factors at the site of the DNA-protein as exemplified by the interaction of Egr-1 and Sp1 proteins (280) (340). Egr-1 can undergo SUMOylation (small ubiquitin-related modifier protein phosphorylation) mediated by alternate reading frame protein (ARF), a tumour suppressor, that directly transactivates PTEN (phosphatase and tensin homologue, deleted on chromosome 10) (341). Another post-translational modification of Egr-1 is phosphorylation.

Mitogen-activated protein kinase (MAPK) cascade Mitogen-activated protein kinases are a family of serine/threonine kinases that activate transcription factors in response to any stimulatory cue that modulate gene transcription. MAPKs control key cellular functions including proliferation, differentiation, migration and apoptosis. In humans, there are at least 11 members of the MAPK superfamily that can be divided into six groups based on sequence similarity (342). The major ones are extracellular signal-regulated kinases (ERKs), c-Jun N terminal kinases (JNKs) and p38s. Each group can be stimulated by a different protein kinase cascade that include the sequential activation of MAPK kinase kinase (MAPKKK) and a MAPK kinase (MPKK) which in turn

56 phosphorylates and activates the downstream MAPKs (343). MAPKs activate the ternary complex factors with different affinities and display specificity for transcription factors (344). Among the MAPK-mediated pathways, the RAS-MEK1/2-ERK1/2 pathway has been the most extensively characterized (Fig. 1.15). Briefly, growth factor binds to its receptor tyrosine kinase receptor (RTK) triggering receptor dimerization with the subsequent autophosphorylation of the internal tyrosine residues of the receptor. Tyrosine phosphorylation allows the binding of RTK with sequence homology 2 (SH2) domains of adaptor protein such as growth factor receptor-bound protein 2, GRB2. The complex brings the cytosolic protein, son of sevenless (SOS) into close proximity with RAS and converting it into an active GTP-bound RAS. The active RAS initiates the signalling cascade by phosphorylating RAF MKKK, which in turn, phosphorylates MEK MKKs (MEK1 and MEK2). Activated MEKs phosphorylate the extracellular signal-regulated kinases ERK (ERK1 and ERK2) translocating them into the nucleus and phosphorylate transcription factors or other specific substrates. Erk1/2 does not possess a nuclear localization signal but it binds with the c-Met-binding domain of growth factor receptor-bound protein 2 (Grb2) –associated binder (Gab1) and their nuclear translocation play a crucial role in Egr-1 nuclear accumulation (345). Direct recruitment of ERK cascade components to Egr-1 has been recently reported and proposed a model wherein Pol II transcription-driven recruitment of heterogenous ribonucleoprotein (hnRNP) K along the EGR- 1 locus compartmentalizes activation of the ERK cascade that regulate synthesis of mature mRNA (346).

Fig. 1.15. MAPK-signalling pathway. Stimuli like growth factor can bind to its respective receptor creating a complex formation that phosphorylate itself and activates RAS. Activation of RAS initiates the signalling cascade by phosphorylating RAF MKKK, which in turn phosphorylates the MEK MKKs. Activated MEKs phosphorylate the ERK1/2 translocating them into the nucleus and phosphorylate transcription factors. From Kabbarah and Chin (347).

P42/p44 mitogen-activated protein kinase, also known as ERK1/2 is preferentially activated by growth factors with the cascade leading to the Egr-1 promoter. Different stimuli can activate distinct signalling cascades promoting Egr-1 transcription. For example, fluid shear stress increased Egr-1 mRNA via ERK1/2 (348) but JNK and p38 isoforms can be responsive to mechanical, oxidative or environmental stress (349).

Phosphorylation of Egr-1 can either induce or suppress the expression of target gene depending on what kinase phosphorylates Egr-1. Casein kinase II (CKII) -dependent phosphorylation of Egr-1 in NH3T3 cells had a negative effect on its DNA binding and transcription activities (350). Another study relating to CKII in NIH3T3 cells, phosphorylated Egr-1 resulted to less binding to Sp1 thus higher levels of Sp1 stimulated transactivation of macrophage colony stimulating factor (MCSF) was observed (351). Induction of Egr-1 by Angiotensin II (352) and FGF-2 (353) has shown to be mediated in SMCs in the MEK/ERK pathway independent of JNK activity. Studies also showed that Egr-1 in vascular SMCs was phosphorylated via Erk1/2 upon treatment of PMA (354), TNF- a (355), TGF-b (356), triglyceride-rich lipoprotreins (357), hemin (358), low density lipid (359), b-D-endogluconidase heparase (360), sulfated oligosaccharide PI-88 (361), endogenous FGF-2 released after vascular injury (298) and even its autoregulation (362). Exogenous application of FGF-2 in several in vivo models (363) (364) (365) showed enhanced peripheral and myocardial collateral flow and MEK/ERK signal transduction cascade was activated during arteriogenesis (366) arguing for a FGF/MEK/ERK/Egr-1 pathway in vivo.

Potential phosphorylation site in Egr-1 was first reported by Milbrandt (266) at Thr/Ser-Gly-Arg residue. Three polyclonal antisera recognizing Egr-1 were obtained and by phosphoamino acid content analysis, Egr-1 was phosphorylated on a serine residue (337). Phosphorylation of Egr-1 in BALB/c3T3 cells was transiently increased by the treatment of serum

59 (367) and treatment of these cells with okadeic acid, an inhibitor of serine/threonine phosphatases, increased the Egr-1 phosphorylation and its DNA-binding activity (340). In other studies, phosphorylation of Egr-1 decreased its DNA binding and transcriptional activities (350) (351). The phosphorylation state of Egr-1 was also proposed to mediate its interaction with some inhibitory factor(s) and in turn reduced its capacity to transactivate but not affecting its DNA binding activity (284).

The ability to detect the phosphorylation state of a protein is of great value to fully establish function. To date, there is no commercially available phospho-specific antibody to Egr-1. Phosphatase treatments and/or use of phosphatase inhibitors are widely used to establish the phosphorylation state of Egr-1. Commercially available antibodies directed against phosphorylated tyrosine, serine or threonine are also employed. However, these techniques do not necessarily indicate whether Egr-1 has been phosphorylated at key regulatory site(s).

AIMS The main objectives of this thesis were: 1. To elucidate the possible mechanism(s) by which YY-1 can selectively inhibit SMC proliferation and not EC, and 2. To identify phosphorylated amino acids in Egr-1 by Erk-1 and generate a site-specific, phosphorylation state-specific antibody to Egr-1.

Chapter 2

Materials and Methods

61 Construction of adenoviral YY-1. YY-1 cDNA was produced by restriction of pCB6-YY-1 with BglII with NEB buffer 3 (50 mM Tris-HCl, 10 mM MgCl, 100 mM NaCl, 1 mM DTT, pH 7.9) at 37C for one hour. The linearized plasmid was loaded into 0.8% Ultrapure agarose (Invitrogen, MA) in 1X TAE buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.5) and was purified with Wizard SV gel and PCR Clean Up System (Promega, Madison WI). Briefly, the extracted gel with the DNA was transferred into a sterile 1.5 ml tube and membrane-binding solution was added at the ratio of 10 μL of solution to 10 μg of gel. The mixture was heated at 55C until gel slice was completely dissolved. The solution was loaded into the column and incubated at room temperature to allow maximum binding of DNA to the membrane. The column was centrifuged and the flowthrough was discarded. The column was washed twice with the provided Wash solution prior to elution of DNA with sterile nuclease-free water. The plasmid was further digested with KpnI in NEB buffer 1 (10 mM bis Tris propane-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.0) with BSA (100 μg/ml) at 37C for 2hours and the 1.5 kb YY-1 cDNA was extracted with the Wizard System. The 1.5 kb YY-1 cDNA was ligated into the adenovirus shuttle vector, pShuttle-CMV (AdEasy, Stratagene) at a site between the CMV promoter and the SV40 polyadenylation using T4 DNA ligase (NEB, MA) in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM DTT, 1 mM ATP at 16C for 1 hour. The ligated mixture was transformed in XL-Gold with kanamycin (50 μg/ml) selection. Transformants were isolated and grown overnight in LB/kanamycin broth. Plasmid DNA was prepared using Wizard Plus SV DNA purification System. Briefly, one ml overnight culture was pelleted for 5min and resuspended with 250 μL Cell Resuspension solution. Cell lysis solution was added to each sample and mixed by inverting. Alkaline protease was added to and the mixture was incubated for 5 min at room temperature. Neutralization solution was added and centrifuged for 10 min at room temperature. The cleared lysate was poured into the column and spun for 1 min at top speed at room temperature. The flowthrough was discarded and the column was washed with wash solution. Washing step of the column was repeated and DNA was eluted with nuclease-free water. The plasmid DNA was digested with

62 restriction enzymes BglII and KpnI to confirm the insert and sequenced to confirm the junctions of the clone. The shuttle vector was then linearized with PmeI in 20 mM Tris acetate, 10 mM Magnesium acetate, 50 mM potassium acetate, 1 mM DTT, pH7.9 at 37C for 1 hour. The linearized plasmid was purified by agarose electrophoresis and extracted using the Wizard SV gel and PCR Clean Up System. The linearized plasmid was transfected into BJ5183-AD-1 competent cells pretransformed with adenobackbone plasmid to produce a recombinant Ad plasmid. Briefly, pre-chilled electrophoration cuvette (BioRad, CA) was loaded with 40 μL of BJ5183-AD-1 competent cells and 1 μg of linearized shuttle vector. The sample was pulsed using the Gene Pulser Xcell Electroporation System (BioRad, CA) set at 200 ohms, 2.5 kV and 25 μF. Controls, linearized pShuttle-CMV-lacZ vector and transformation control plasmid from the kit were also pulsed separately with BJ5183-AD-1 competent cells. The pulsed-cell suspension was immediately removed from the cuvette and 1 ml of sterile LB broth was added. The cell suspension was transferred to a sterile 14-ml BD Falcon polypropylene and incubated with shaking at 37C for one hour. The cell suspension was plated on LB-kanamycin agar plates. Transformants were selected for kanamycin resistance, and recombinants were identified by restriction digestion then produced in bulk quantities using the recombination deficient XL10-GOLD strain. Purified recombinant adenoplasmid DNA was digested with PacI in NEB buffer 1

(10 mM bis Tris propane-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.0) with BSA (100 μg/ml) at 37C for 2hours to expose its inverted terminal repeats (ITRs). The PacI digested recombinant Ad plasmid DNA was transfected into AD293 cells using the Stratagene Vira pack transgfection Kit (Agilent Technologies, CA). Briefly, the AD293 cells were plated at 7-8 x 105 cells per 60-mm tissue culture dish. The cells were washed twice with phosphate-buffered saline (PBS) and 4 ml of MBS (DMEM with 7% (v/v modified bovine serum and 25 μM Chloroquine)-containing medium was added in each 60-mm plate. The PacI Ad plasmid DNA was added in a 5 ml BD Falcon polystyrene tube containing water such that the final volume was 225 μL. Twenty five μL of Solution I and 250 μL of Solution II from the Viral Kit was added into the tube with DNA. The DNA mixture was

63 incubated at room temperature for 10 minutes. The DNA mixture was added dropwise into the AD-293 cells in MBS-containing medium. After incubating the cultures in the 37C incubator, medium from the plates were replaced with growth medium with 25 μM chloroquine and further incubation in 37C incubator was done. After 6-7 hour of incubation, the growth medium containing chloroquine was replaced with growth medium without chloroquine. The culture plates were incubated at 37C for 6-7 days and AD-293 cells were harvested when starting to detach. Pelleted cells were washed with PBS and harvested with 0.5 ml PBS. The cells were immediately subjected to freeze/thaw in dry ice-methanol bath and 37C water bath with vigorous mixing after each thaw. Cellular debris was collected by centrifugation at 12,000x g for 10 minutes at room temperature. The primary virus stock (supernatant) was transferred into a screw-cap microcentrifuge cap and stored at -80C.

Cesium chloride banding of adenovirus. Two cesium chloride gradients were banded in 14x89 Beckman ultracentrifuge. The adenomaxiprep that undergone freezing/thawing was transferred to a 50 ml conical tube and spun at 3K rpm to pellet cell debris. The supernatant was carefully layered onto the two gradients and immediately overlaid with mineral oil. The samples were loaded into the SW41 swinging buckets and centrifuged at 35K rpm for 1 hour at 15C. The lower band (virus) was collected using a 20G needle and a 3 ml syringe and carefully layered in another cesium chloride gradient. The gradient was covered with mineral oil prior to centrifugation for 16-20 hours at 35K rpm at 15C. The lower band was collected and desalted by chromatography on PD-10 columns in storage buffer (10 mM Tris, pH 7.4, 1 mM MgCl2, 10% glycerol).

Adenoviral Titering. AdEasy Viral titer kit was used to determine the adenoviral titers. The kit is a simple enzyme-linked immunoassay for adenoviral capsid protein, hexon. Briefly, Ad-293 cells were plated at a density of 2.2 x 105 cells per well of 24-well culture plate. Ten-fold serial dilution of the viral stock with DMEM was done. Fifty μL of each viral dilution was added dropwise into at least two wells. The cells were

64 incubated at 37C in a 5% CO2 humidified incubator for 24-48 hours. The medium was removed by aspiration and let the cells to dry for 5-10 minutes. Cells were fixed with 0.5 ml ice-cold 100% methanol. The plate was incubated at -20C for 10 minutes. The methanol was aspirated and washed twice with 1 X PBS containing 1% BSA (1xPBS/1%BSA). The diluted mouse anti-hexon antibody was added into the well and incubated for 1 hour at 37C. The anti-hexon antibody was aspirated and the cells were washed twice with 1xPBS/1% BSA. The diluted horseradish peroxidase (HRP) -conjugated goat anti-mouse antibody was added into the cells and incubation was done for 1 hour at 37C. The secondary antibody was removed and cells were incubated with 1X diaminobenzidine (DAB) substrate solution after washing twice with 1XPBS/1%BSA. The cells were incubated at room temperature until brown staining color developed. The number of adenoviral titer was calculated accordingly.

Cell culture and plasmid transfection. Primary human and rat aortic SMCs and bovine aortic ECs were purchased as frozen stocks of passage 2-3 from Cell Applications, Inc (San Diego, CA) and confirmed by CD31+ and SM -actin+ staining. SMCs and ECs were grown in Waymouth’s (Invitrogen) and DMEM (Invitrogen), pH. 7.4, respectively, containing 10% fetal bovine serum, 10 U/ml of penicillin and 10 μg/ml of streptomycin at o 37 C in a humidified atmosphere of 5% CO2. Cells were used between passages 4-8. Plasmid transfections were performed at 60-70% confluency using FuGENE6 (Roche) in a 1:3 ratio (1 μg DNA: 3 μL Fugene). The DNA/Fugene mixture was incubated at room temperature for at least 15 minutes prior to addition into the cell cultures. Cell density seeded varies depending on the format of the tissue culture plate. For a 96-well plate, 3,000 cells were seeded whereas 1 x 104 cells for a 6-well plate. One million cells were seeded into the 100mm petri dish.

Cell proliferation assays. Cells in 96-well plates were transfected with the indicated amounts of plasmid and after 3 days, the cells were trypsinized (0.05% Trypsin/0.53 mM EDTA in 1xPBS) and suspended in Isoton II (Beckman Coulter) and quantitated using a Coulter counter.

65 Where antisense oligonucleotides were used, growth-quiescent SMCs in 96-well plates were transfected with 3 μg of pCB6-YY-1 or pCB6+ together with 0.5 μM of antisense p21WAF1/Cip1 (p21AS, 5’GAC ATC ACC AGG ATC GGA CAT3’) or control (p21CL, 5’TGG ATC CGA CAT GTC AGA3’). Cell numbers were quantitated after 3 days. Where adenovirus was used, cells in 96-well plates were transduced with the indicated amounts of adenoviral vector (MOI as indicated) in 10% FBS/Waymouth's (Sigma Aldrich) and after 3 days, the cells were trypsinized and quantitated using a Coulter counter. Where siRNA was used, growth-quiescent SMCs in 96- well plates were transfected with 0.25 μM of YY-1 siRNA (rat, 5'r(GAG GUG AUU CUG GUG CAG A)dTdT)3' or siRNAns (5'r(UAG GCU UGA AGA GGU CGAU)dTdT)3' and grown in 5% FBS/Waymouth's (transfected 6 hour after arrest and again at the time of serum stimulation). After 3 days, the cells were trypsinized and quantitated using a Coulter counter.

Carotid artery injury and adenovirus delivery. Balloon catheter injury to the carotid artery of adult Sprague Dawley rats (450-550g) and adenovirus delivery was carried out in Professor Alex Bobik’s Laboratory (Baker IDI, Melbourne). Briefly, after anesthetizing rats with ketamine (80 μg/kg i.p.) and xylazine (20 μg/kg ip) two incisions were made, the first a midline leg incision to expose the femoral artery and the second a midline neck incision to expose the carotid bifurcation. A 2F Fogarty (Baxter) arterial embolectomy catheter was passed through an arteriotomy in the femoral artery and advanced into the left common carotid artery to its bifurcation. The balloon was inflated with saline and withdrawn to the aortic arch. The procedure was repeated three times, then the catheter was removed, the femoral artery ligated and the incision closed. A fine catheter was then inserted through an arteriotomy in the external carotid artery and passed into the common carotid artery. Temporary ligatures were then placed around the catheter before flushing the vessel with adenovirus containing solution and placing a second ligature on the vessel towards the aortic arch. One hundred μL of PBS containing 5x1010 pfu adenovirus or the vehicle itself was infused into the ligated segment for 20 min at 100mm Hg. Alternatively, 100 μL of PBS solution containing 10 μL FuGENE6 and

66 50 μg siRNA or the vehicle itself was infused into the ligated segment for 20 min at 100mm Hg. The ligatures and catheter were removed, the external carotid ligated and the incision neck closed. The rats were sacrificed 14 days later. Tissue processing and morphometry were then carried out. Briefly, after administering an overdose of pentobarbitone (i.p.) to the rats, the chest was opened and an 18G i.v. catheter was inserted into the heart and perfused with saline solution at 140mm Hg for 2 min. Then the vessels were fixed by perfusion with 10% formalin at the same pressure for approximately 7 min. The carotid arteries were then removed, cleaned and the ligated segment divided into three sections for embedding into paraffin, before cutting 6μm thick cross-sections. Cross-sectional areas of the media, the neointima and overall vessel size were measured using a computer-interfaced imaging system (Optimus Bioscan2, Thomas Optical Measurement system, Inc). 5-7 rats were used in each group.

Immunohistochemical staining. Staining was performed with antibody to p53 (1:200; Santa Cruz Biotechnology) on consecutive paraffin sections of formalin-fixed carotid arteries. Prior to staining, deparaffinized sections were boiled in citrate buffer, pH 6.0, to retrieve antigenicity, and treated with 3% hydrogen peroxide. After washing with PBS, pH 7.4, sections were incubated with primary antibody for 1 h, followed by incubation with the secondary antibody, horse anti-mouse for 1 h and finally with avidin- biotin complex (Elite ABC kit; PK-6100, Vector). Immunogenicity was visualized by treatment with 3,3’-diaminobenzidine (DAB) solution for 2 min, which produced brown coloration. Sections were counterstained with Mayer’s hematoxylin.

Transient transfection and reporter gene analysis. Cells grown in 6- well plates were transfected with indicated amounts of Firefly luciferase plasmid together with 0.5 μg of pRL-TK (Renilla luciferase driven by the thymidine kinase promoter). Constructs 3*6 and 7*3 contain 1975 and 1270 bp of human p21WAF1/Cip1 promoter, respectively, upstream of Firefly luciferase in pGL3-basic 5. Constructs Sp1-7-pGL3-prom and mSp1-7-

67 pGL3-prom were created by subcloning Oligo p21WAF1/Cip1 (-1387/-1358 in the p21WAF1/Cip1 promoter): 5’AAA GAA GCC TGT CCT CCC CGA GGT CAG CTG3’ (sense strand, putative Sp1 binding site underlined) or the mutant 5’AAA GAA GCC TGT TTG TTT CGA GGT CAG CTG3’ (sense strand), respectively, into the SmaI site of pGL3-prom (Promega). Construct 3*6mSp1-7 was created using the QuickChange mutagenesis kit (Stratagene). Luciferase activity in the lysates was measured 24 hour after transfection using the Dual Luciferase Assay System (Promega). Firefly luciferase activity was normalized with Renilla luciferase to normalize for transfection efficiency.

Western blot analysis. Samples were resolved by electrophoresis using denaturing SDS-polyacrylamide gels for 2 hour at 100V. Proteins were transferred to Immobilon P nylon membranes (Millipore) prior to incubation with non-fat skim milk to block nonspecific binding sites. Membranes were incubated with the indicated antibodies (Santa Cruz Biotechnology) at dilution of 1:1000 (1:30000 for -actin, Sigma). Detection was achieved with HRP-linked secondary antibodies and chemiluminescence (Perkin- Elmer). Coomassie blue-stained gels were destained and photographed to confirm equal protein loading. Where E2F siRNA was used, SMCs in 100mm petri dish were transfected overnight with 20 μg pCB6+ or pCB6- YY-1 with or without siRNA to E2F at a 0.4 μM final concentration. The sense strand sequence of the E2F siRNA was 5'r(CGG AGG CUG GAU CUG GAA A)dTdT3' (sense strand).

Immunoprecipitation analysis. Cells were transfected with indicated plasmid constructs, and after 24 hour, harvested in 1xRIPA (radioimmunoprecipitation assay) buffer (20 mM Tris-HCl (pH 7.5) 150 mM

NaCl, 1 mM Na2EDTA 1 mM EGTA 1% NP-40 1% sodium deoxycholate

2.5 mM sodium pyrophosphate 1 mM -glycerophosphate 1 mM Na3VO4 1 μg/ml leupeptin) and pre-cleared with pre-washed protein G-Sepharose beads for 1 hour prior to incubation with the indicated primary antibody overnight at 4oC with gentle shaking. Pre-washed Sepharose beads were incubated with the lysate/antibody mixture for a further 2 hours. Beads

68 were washed several times with 1xRIPA followed by a final wash with 200 mM NaCl/1xRIPA. Proteins were resolved by 12.5% SDS-PAGE and immunodetected by Western blot analysis. Using recombinant proteins, human Sp1 (0.4 μg) (Promega) was incubated with GST-tagged full-length YY-1 protein for 1 hour gently mixing at 4oC then pre-cleared with equal mixture of Protein A and G Sepharose-4-Fast Flow (Amersham Biosciences). The protein mixture was then divided into three equal parts. Goat polyclonal anti-GST antibody (Amersham Biosciences) or goat polyclonal GATA-1 antibody (Santa Cruz Biotechnology) was added and incubated for a further 1 hour. An equal volume of fresh Protein A and G suspension was added and complex formation was allowed to proceed for another 4 hour at 4oC. After sequential washing, the beads were resuspended in loading buffer, boiled and the supernatant was loaded onto denaturing 10% SDS-polyacrylamide gels for Western blot analysis.

RT-PCR. Total RNA was prepared from human SMCs with TRIzol in accordance with the manufacturer’s instruction (Invitrogen, Life Technologies, Inc.). RNA was reversed transcribed to cDNA using oligo(dT) primers and Superscript (Invitrogen, Life Technologies, Inc.). PCR was performed using Platinum Taq DNA polymerase (Invitrogen, Life Technologies, Inc.) with the following amplification conditions: 20 mM Tris-

HCl, pH 8.4, 50 mM KCl, 1 mM MgCl2, 250 μM dNTP, 0.5 μM primers, 1μL of cDNA, and 1 unit of Platinum Taq DNA polymerase. For YY-1 PCR, cycling conditions were 94oC for 30 sec, 30 cycles of 95oC for 10 sec, 53oC for 30 sec, and 72oC for 1 min. For p21 and GAPDH PCR, the same cycling conditions were used except for the annealing temperature of 55oC and 58oC, respectively with only 25 cycles for GAPDH. Primer sequences for YY-1 were YY-1a5 (5’GAA AAC ATC TGC ACA CCC ACG GTC C3’) and YY-1a3 (5’CAC TGC TTG TTT TTG GCC TTA GA3’) whereas those of p21 were Frp21 (5’CCA GGA GGC CCG AGA ACG G3’) and Rrp21 (5’CGAGGGGAGGGGGCAGGC3’). GAPDH primers sequences were GAP5 (5’ACC ACA GTC CAT GCC ATC AC3’) and GAP3 (5'TCC ACC ACC CTG TTG CTG TA3’).

69 EMSA analysis using nuclear extracts and recombinant proteins. SMC nuclear extracts were prepared essentially as previously described (111) and incubated with the indicated 32P-labeled double-stranded oligonucleotide probes (150,000 cpm) in 10 mM Tris-HCl, pH 8, 50 mM

MgCl2, 1 mM EDTA, 1mM DTT, 5% glycerol, 1 μg salmon sperm DNA, 5% sucrose, 1 μg of poly(dI.dC) and 1 mM PMSF. The mixture was incubated for 30 min at 25oC. In nucleoprotein complex antibody supershift or elimination experiments, nuclear extract were incubated with 2 μg of antibody for 10 min prior to the addition of the probe. Samples were loaded onto 6% non-denaturing polyacrylamide gels and binding activity was visualized by autoradiography overnight. Recombinant proteins were incubated with the indicated radiolabeled probes in 10mM Tris-HCl, pH 7.5, 50mM NaCl, 1mM EDTA, 5% glycerol, 1 ug/ml BSA, 0.2% NP-40 and 1 mM DTT at 4oC for 30 min. The probes used for EMSA were Oligo p21WAF1/Cip1 (-1387/-1358): 5’AAA GAA GCC TGT CCT CCC CGA GGT CAG CTG3’ (sense strand) and Oligo MVYY-1: 5’TGC CTT GCA AAA TGG CGT TAC TGC AG3’ (sense strand). Where indicated, an equivalent amount of BSA was used in binding reactions for negative control purposes. GST-Sp1(263-619) was prepared by cloning the corresponding PCR product into the EcoRI site of pGEX-5X with primers 5EcoRISp1263-619 (5’GGC CCT GGA ATT CAA CAT CAC CTT G3’) and 3EcoRISp1263-619 (5’TTT CTT TTT GAA TTC ATC CCC CGA G3’).

Chromatin immunoprecipitation (ChIP) analysis. Human SMCs grown in 100-mm dishes were transfected overnight with 30 μg of the indicated plasmid(s). pcDNA-GSTYY-1 was generated by digesting pGEX-2TK-

GST-YY-1 with HincII in NEB 3 (50 mm Tris-HCl, 10 mm MgCl2, 100 mm NaCl, 1 mm DTT, pH 7.9 at 37C and ligating the blunt-ended GST-YY-1 fragment into EcoRI-digested pcDNA3.1 that had also been blunt-ended. pcDNA-GST-YY-1ZNF was generated by amplifying YY-1 cDNA omitting the zinc finger region and cloning the product into pcDNA3.1. Cells were washed with cold PBS, pH 7.4 prior to ChIP using the antibodies indicated. Briefly, cells were fixed with 1% formaldehyde and quenched with 0.1 M glycine. After sequential washings, cells were lysed in buffer and

70 sonicated. The supernatant was added to dilution buffer and pre-cleared with Protein A and G Sepharose slurry. The supernatant was equally divided into four microcentrifuge tubes and appropriate antibodies were added with appropriate controls; namely, total input and no antibody. After sequential washing, samples were heated at 65oC for 6 hour prior to Proteinase K treatment. DNA was extracted with phenol:chloroform:isoamyl and precipitated with ethanol. DNA pellet was resuspended with MilliQ water and 1μL aliquot was used in the PCR. Primers were designed to amplify the region –1643/-798 of the human p21WAF1/Cip1 promoter spanning the Sp1-7 element. PCR was performed in

1 mM MgCl2, 0.1 mM dNTP, 0.1 μM primers and 1 U Platinum Taq polymerase (Invitrogen). Cycling conditions were as follows: 94oC for 2 min; 35 cycles of 94oC for 30sec; 58oC for 10 sec and 72oC for 1 min, with an extension time of 4 min. Human p21WAF1/Cip1 promoter was amplified using primers hwafF6 5’AAG GCA GTG GGA GAA GGT G3’ and hwafR6 5’GGG AGG ATT TGA CGA GTG AG3’ (upstream amplicon), or hwafsp1F 5’GCT GGC CTG CTG GAA CTC3’ and hwafsp1R 5’GGA CAC GCA GGG ACA CAC3’. Cycling conditions were the same as above except with 2 mM MgCl and 40 cycles. PDGF-A ChIPs was performed with primers humPDGFAprom-1492 5’CTT CTT CCT CGG TGC GTT C3’ and humPDGFAprom-716 5’CGG GGC TTT GAT GGA TTT AG3’ with 2 mM o MgCl2, 54 C annealing temperature and 40 cycles.

Generation of truncated YY-1 constructs. Different truncated regions of the YY-1 gene (Fig. 2.1) were generated by PCR using primers with BamHI linkers having a translation start site at the 5’ primer and a stop site at the 3’ primer. PCR products were digested with BamHI in NEB 3 buffer

(50 mm Tris-HCl, 10 mm MgCl2, 100 mm NaCl, 1 mm DTT, pH 7.9) at 37C prior to ligation to pcDNA3.1+ previously digested with BamHI and dephosphorylated with calf intestine phosphatase (NEB, MA). The clones were sequenced accordingly to confirm junctions and in-frame cloning.

Fig. 2.1. Schematic of the YY-1 structure showing the activation and repression domain together with its four zinc finger regions. Different truncated constructs of YY-1 were generated and cloned into pcDNA3. YY-1B, YY-1C and YY-1D contained the first two, the first three and the four zinc fingers, respectively. YY-1F/L denotes the full length YY-1 construct. Primers to generate the fragments; 1321F, 5’-AGA ATG AAG GGA TCC AAA ATT AAA ATG GAT GAT- 3’; 1572R, 5’-AAA GCG TTT GGA TCC GCC TTC GAA CTA GCA CTG-3’; 1656R, 5’-CTT ATT AGA GGA TCC GAA GGG GCA TCA ATA GGG CCT G-3’; 1749R, 5-CGA GAA GGG GGATCC CTC TTC TTT TCA CTG GTT GTT-3’. Bold and underlined sequences indicate the BamHI site, underlined and italized sequences indicate the translational start site and bold sequences denote the termination site.

72 pcDNA3.1+-based construct plasmids were used in the transient transfection in proliferation assays.

In vitro phosphorylation. Synthetic peptides or recombinant proteins were incubated in 1x Erk-1 buffer (20 mM MOPS, pH 7.2, 20 mM - glycophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 10 mM MgCl2 and 1 mM DTT), 25 ng of Erk-1 enzyme, 10uCi gamma-ATP to a total volume of 50 μL at 30C for 30min. The sample was loaded into Millipore Microcon YM-30 and spun for 10min at 3000 rpm to remove unincorporated radionucleotide. The sample was spotted into a filter paper and dried prior to washing twice with 1% acetic acid and water. The filter paper was counted for radioactivity in a Beckman scintillation counter.

Mass spectrometry. The in vitro kinase reaction was carried out with the recombinant protein as described above in a total volume of 30 μL and cold ATP was used instead of the radionucleotide. Unincorporated ATP was removed using the Millipore Microcon YM-30 and 2 μL of sample was diluted with 18 μL of 30% acetonitrile/0.1% formic acid and briefly centrifuged at 14,000 rpm. Five μL of the supernatant was loaded into a Pico Tip Emitter (New Objective, Woburn, MA, USA) and analyzed by static nanoelectrospray mass spectrometry using an Applied Biosystems 4000Q-Trap. The presence of phosphorylation was detected by conducting neutral loss scans of +98m/z or +49m/z. The peptide sequence responsible for producing the diagnostic phospho-ion was MS/MS verified.

Generation of phospho-specific antibody. Phospho-specific antibodies against Egr-1 were prepared by immunizing rabbit and mouse with peptide containing phosphorylated Ser26 (pS26):FGSFPH(pS)PTMDNYC coupled to keyhole limpet hemocyanin (EzBiolab Inc., Carmel, IN). Briefly, it involved the immunization of animals for 5-6 weeks followed by the fusion of the spleen cells into SP2/0-Ag14 myeloma cells using polyethylene glycol. Supernatants were tested in the ELISA screening assay and

73 hybridomas were selected prior to cloning. Ascites from each animal were collected and the sera were purified by peptide affinity chromatography with subsequent dialysis against PBS prior to lyophilization.

Slot blot and immunoblotting. Recombinant proteins and synthetic peptides were diluted with 1x PBS and serial dilutions were made prior to blotting into Millipore Immobilon PVDF membrane in Bio-dot SF microfiltration trap (Bio-Rad) connected to a vacuum pump. Membrane was dried and blocked overnight with 5% skim milk. Membranes were incubated with the indicated antibodies and detection was achieved with HRP-linked secondary antibodies and chemilluminescence (Perkin-Elmer).

Animal ethics and statistics. Experiments were conducted in accordance with the Animal Care and Ethics guidelines at the University of New South Wales (Sydney) and Baker IDI Medical Research Institute (Melbourne). Values are expressed as the mean + S.E.M. In vitro experiments were performed in duplicate or triplicate on 2 or 3 independent occasions. Differences between groups were tested for statistical significance using Student's (unpaired) t-test or ANOVA and considered to be significant at p<0.05.

Chapter 3

Results

75 YY-1 represses p21WAF1/Cip1 expression in SMCs. It was reported previously that vascular injury in SMC activated YY-1 expression and this upregulation was due to the rapid release of FGF-2 (111). Overexpression of YY-1 inhibited SMC proliferation but not EC. To elucidate the possible molecular mechanism by which YY-1 inhibits SMC proliferation, genes involved in cell cycle machinery were investigated. Previous study (126) showed the inverse relationship between YY-1 and p21WAF1/Cip1 in hypomorphic mice expressing 25% of normal YY-1 level. Transient transfection of YY-1 inhibits the p21WAF1/Cip1 in a dose-dependent as indicated by the Firefly luciferase reporter activity driven by 1975 bp of the p21WAF1/Cip1 promoter (177) (Fig. 3.1). Control plasmid, pCB6+ did not show any change in luciferase activity even with increased amount of plasmid. Inhibition of p21WAF1/Cip1 protein was confirmed by Western analysis when YY-1 was over expressed and exogenous YY-1 protein accumulates in the nucleus.

To fully establish the role of p21WAF1/Cip1 in the SMC proliferation, rescue experiment (Fig.3.2) was done by overexpressing p21WAF1/Cip1 (pCEP- WAF1). Total number of SMC was reduced when pCB6+YY-1 and pCEP were overexpressed, as expected with concomitant reduction in p21WAF1/Cip1 protein (Fig.3.2, inset). When both pCB6+ YY-1 and pCEP- WAF1 were overexpressed, exogenous p21WAF1/Cip1 abrogated the YY-1’s ability to inhibit SMC proliferation and the levels of p21WAF1/Cip1 protein across the samples correlated to the corresponding total number of SMC cells. This forced expression of p21WAF1/Cip1 confirmed its involvement in the YY-1 inhibition of SMC proliferation.

Using an antisense to p21WAF1/Cip1, further confirmation on the role of YY-1 and p21WAF1/Cip1 on SMC proliferation was done. Combinational treatment of YY-1 overexpression and antisense to p21WAF1/Cip1 (p21AS) was performed (Fig. 3.3). YY-1 alone can reduced the proliferation with or without the presence of p21AS. Depletion of p21 by antisense rendered YY-1 unable to further inhibit SMC proliferation. The serum inducible SMC

Fig. 3.1. Effect of YY-1 on p21. Upper left panel. Effect of YY-1 on p21WAF1/Cip1 promoter-dependent reporter gene expression with increasing amounts of pCB6+ or pCB6-YY-1 (0.5, 1 and 2 μg) promoter construct 3*6. Firefly luciferase activity was assessed after 24 hour. Lower left panel. Localization of YY-1 protein in SMCs. NE denotes nuclear extract whereas Cyto denotes cytosolic extracts. Numbers on the right indicates the protein molecular weight markers in kDa. Right panel. Effect of YY-1 on p21WAF1/Cip1. SMCs were transfected with 30 μg of pCB6+ or pCB6-YY- 1 in 100-mm plates and Western blot analysis was performed after 24 hour in the presence of serum. - actin was use as the normalizing protein. Error bar denotes SEM. *denotes p<0.05 compared to control p<0.05. .

Fig. 3.2. Effect of exogenous p21WAF1/Cip1 on YY-1 inhibition of SMC proliferation. SMCs in 96-well plates were transfected with pCEP-WAF1 (0.75 μg), pCEP (0.75 μg), pCB6-YY-1 (3 μg) or pCB6+ (3 μg) and cell numbers were counted after 48 hour in the presence of serum. Inset. Western of p21WAF1/Cip1. Cells were transfected in 100-mm plates and total proteins were extracted after 24 hour. Number on the right side indicates the protein molecular weight marker in kDa. Error bar is SEM. * denotes p<0.05 compared to control.

Fig. 3.3. Effect of YY-1 overexpression with antisense to p21WAF1/Cip1. SMCs were seeded in 96-well plates and transfected with 0.3mmole/L of p21AS (antisense to p21) or p21CL (control) (369) together with 3 μg of pCB6-YY-1 or pCB6+. Cells were counted after 72 hour in the presence of serum. Alternatively, cells were transfected in 100-mm dishes and total protein extracted after 24 hour. ODN denotes oligonucleotides. *denotes p<0.05 compared with control.

79 proliferation control (No plasmid, No ODN) was comparable to that of control for YY-1 (pCB6+, No ODN). As expected the corresponding antisense control, p21CL had no effect. Western blotting for p21WAF1/Cip1 protein (Fig.3.3, inset) was also done to confirm the effect of YY-1 and antisense molecule in conjunction with the proliferation study.

To further explore the role of YY-1 in the cell cycle in SMCs and ECs, cyclin D1-cdk4-p21WAF1/Cip1 complex formation was investigated by immunoprecipitation (IP) (Fig. 3.4). When YY-1 was exogenously expressed in SMC and immunoprecipitated with p21WAF1/Cip1 antibody, both Cdk4 and cyclin D1 proteins were decreased indicating a perturbation in the cyclin D1-cdk4-p21WAF1/Cip1 complex formation. Using the same samples but without pulling down with the p21WAF1/Cip1 antibody, Cdk4 (Wst) and Cyclin D1 (Wst) showed no change in protein levels compared to the SMC transfected with the backbone alone, pCB6+. In EC, overexpression of YY-1 did not affect the cyclin D1-cdk4-p21WAF1/Cip1 complex formation as indicated by the comparable proteins levels between pCB6+ and pCB6+ YY-1 after IP with p21WAF1/Cip1 antibody. As expected, no protein band was obtained when a species-matched immunoglobulin (No p21WAF1/Cip1 antibody) was used in the IP. Reverse IPs were also done to confirm this abrogation of cyclin D1-cdk4-p21WAF1/Cip1 complex formation when YY-1 was overexpressed in SMC. Since cyclin D1-cdk4- p21WAF1/Cip1 complex formation mediates the phosphorylation of pRbSer249/Thr252 (370), Western blotting was performed for p- pRbSer249/Thr252 in both SMC and EC where YY-1 was overexpressed. YY-1 inhibited the phosphorylation of pRbSer249/Thr252 in SMC but not in EC. The overexpression of YY-1 in SMC and EC were confirmed by RT-PCR and by Western in EC (Fig. 3.5). With the exogenous YY-1, p21WAF1/Cip1 mRNA was inhibited in SMC but not in EC. Taken together, YY-1 perturbs the cyclin D1-cdk4-p21WAF1/Cip1 complex formation in SMC with subsequent inhibition of phosphorylation of pRbSer249/Thr252 halting the cell cycle progression.

Fig. 3.4. Co-immunoprecipitation and Western blotting. SMCs or ECs were grown in 100-mm plates and total proteins were extracted after transfection with 30 μg pCB6-YY-1 or pCB6+. Extracts were immunoprecipitated (IP) with indicated antibodies followed by Western blotting with indicated antibodies. Total extracts were Western blotted (Wst) for cdk4 or cyclin D1 antibodies. Where indicated, the primary antibody was omitted as control. The reverse co-immunoprecipitation and Western blotting were also done. Number on the left side of the membrane indicates the protein molecular weight markers. Courtesy of Hideto Ishii.

Fig. 3.5. Effect of YY-1 on p21WAF1/Cip1 expression. Bovine aortic ECs were transfected with 30 μg of pCB6-YY-1 or pCB6+ in 100-mm plates for 24 hour and Western blotting (left panel) was performed for YY-1, p21WAF1/Cip1 or -actin. Reverse transcription was performed in ECs and SMCs for YY-1, p21WAF1/Cip1 and GAPDH 24 hour after transfection.

82 YY-1 inhibits p21WAF1/Cip1 transcription by preventing Sp1 occupancy of the p21WAF1/Cip1 promoter. In search of possible YY-1 binding motif in the p21WAF1/Cip1 promoter, different computer programs (Match or P-Match or AliBaba 2.1) were used and revealed that p21WAF1/Cip1 does not contain consensus recognition elements for YY-1 (5’-NNNNNCCATNTWNNNWN- 3’ or 5’-NNNCGGCCATCTTGNCTSNW-3’, where it acts as a repressor or activator, respectively) (371). 5’ deletion constructs were made and transient transfection analysis were done to determine the region mediating YY-1 repression of the p21WAF1/Cip1. Luciferase reporter system revealed that repressive region can be located at -1975/-1270 (Fig. 3.6, upper panel) and this region bears atypical Sp1 binding site denoted as Sp1-7. This atypical Sp1 motif had been previously shown to support Sp1 interaction in other promoters (372) (373). Mutation of this element from 5’-CCTCCC-3’ to 5’-TTGTTT-3’ in construct 3*6 (bearing the 2kb of the p21WAF1/Cip1 promoter) ablated YY-1 repression (Fig. 3.6, lower left panel). Sp1-7 element was also cloned in front of the SV40 promoter in pGL-3 prom and as expected, the wild type Sp1-7 induced the luciferase activity whereas the mutant did not (Fig. 3.6, lower right panel).

Interaction of the Sp1 with this region in p21WAF1/Cip1 promoter was established by electrophoretic mobility shift assay (EMSA) using SMC nuclear extract. A nucleoproteins complex was formed which was supershifted by the Sp1 antibody but not the YY-1 antibody (Fig. 3.7, left panel) implying that Sp1 interacted to the promoter region of p21WAF1/Cip1 whereas YY-1 did not. To check the integrity of the YY-1 antibody as well as the YY-1 protein in the nuclear extract, EMSA was done (Fig. 3.7, right panel) using a positive supershift probe, MVVYY-1, bearing a consensus YY-1 binding element (111). Results of both EMSA implied that YY-1 indirectly represses p21WAF1/Cip1 transcription by interacting with Sp1. This is the first report of such interaction of YY-1 and Sp1 on the p21WAF1/Cip1 promoter.

Fig. 3.6. Deletion analysis to determine the YY-1 repression domain in p21 promoter. SMCs in 6-well plates were transfected with 10 μg of construct 3*6, 7*3 or 3*6mSp1-7 and 3 μg of pCB6-YY- 1 or pCB6+. Firefly luciferase activity was determined in the lysate after 24 hour and normalized to the backbone plasmid, pCB6+ which was set at 100% (left bottom panel). Alternatively, the cells were transfected with 10 μg of Sp1-7-pGL3-prom, mSp1-7-pGL3-prom or pGL3-prom and 3 μg of CMV-Sp1 or CMV-gutless (lower right panel). Error bar denotes SEM and * indicates p<0.05.

Fig. 3.7. Electrophoretic mobility shift assay (EMSA). EMSA was performed with SMC nuclear extracts (NE) and 32P-Oligo p21WAF1/Cip1(- 1387/-1358) or 32P-Oligo MVYY-1. Supershift-grade antibody (2 μg) was added 10 min before addition of the 32P-labeled probe. Arrow indicates the nucleoprotein complex.

85 Chromatin immunoprecipitation (ChIP) was also performed to validate this indirect repression of p21WAF1/Cip1. Overexpression of YY-1 inhibited the interaction of Sp1 (Fig. 3.8, top panel) whereas vector alone, pCB6+ showed the interaction of Sp1 with p21WAF1/Cip1 as indicated by the formation of the 845 bp amplicon. Previously, reports showed that Sp1 and YY-1 interact via the zinc finger region of YY-1 (374, 375); thus, GST- tagged full length YY-1 and GST-tagged YY-1 without the zinc-finger region (DZNF) were constructed. GST-tagged full length YY-1 and GST- YY-1 without the zinc finger region (DZNF) constructs were overexpressed and ChIP indicated that the interaction of YY-1 and Sp1 was via the zinc finger region of YY-1 (Fig.3.8, lower panel).

Pulldown experiment on recombinant GST-YY-1 and Sp1 proteins with either GST or GATA-1 antibody was performed to further show YY-1 and Sp1 interaction (Fig. 3.9, left panel). GATA. The samples were probed for Sp1 immunoreactivity and nuclear extract from rat was included as a positive control. Whereby a species-matched antibody, GATA-1 antibody was used as a negative control. pCDNA-GST-YY-1 and its back bone control, pCDNA were overexpressed in both SMC and EC with the aim of establishing that Sp1 and YY-1 interacts in the former and not on the latter cell type. Immunoprecipitation analysis with the GST antibody confirmed that GST-YY-1 and Sp1 physically interact (Fig. 3.9, right panel) in SMC but not in EC. To show that Sp1 but not YY-1 binds to the atypical Sp1 binding element (Sp1-7) of the p21WAF1/Cip1 (Fig. 3.10, top panel), EMSA was done with recombinant Sp1 and GST-YY-1 proteins. Formation of a nucleoprotein complex was obtained with Sp1 alone whereas the no complex was formed with the GST-YY-1 alone. When Sp1 and GST-YY-1 were combined, the binding of Sp1 was greatly reduced as indicated by the reduced nucleoprotein complex formation. The presence of BSA (same amount of protein to that of YY-1) did not interfere with the capacity of Sp1 to the atypical Sp1 binding element. Again, the integrity of the GST- YY-1 protein was confirmed using Oligo-MVYY-1 (Fig. 3.10, middle panel) and the mutation of this element abolished Sp1 binding (Fig. 3.10, lower panel).

Fig. 3.8. Chromatin immunoprecipitation (ChIP) analysis to determine the effect of YY-1 overexpression on DNA binding in p21WAF1/Cip1 promoter. Human SMCs in 100-mm plates were transfected with 30 μg of pCB6-YY-1, pCB6+, pcDNA3.1-GST-YY-1 or pcDNA3.1-GST-YY-1DZNF or pcDNA3.1 before chromatin immunoprecipitation and PCR. The 845 bp PCR product was sequenced and its identity was confirmed. No Ab indicates no antibody added.

Fig. 3.9. YY-1 and Sp1 physical interaction. GST-YY-1 and Sp1 were incubated before immunoprecipitation with GST or GATA-1 antibodies and Western blotting for Sp1 (left panel). Where indicated, rat SMC nuclear extracts were probed for Sp1. Alternatively, SMCs or ECs were transfected with 30 μg of pCB6-YY- 1 or pCB6+ and after 12 hour, cell extract were immunoprecipitated with YY-1 antibody before Western blotting for Sp1 (right panel). Arrow indicates Sp1 immunoreactivity.

Fig. 3.10. Recombinant protein EMSA. EMSA was performed with 32P-Oligo p21WAF1/Cip1(-1387/-1358), 32P-Oligo MVYY-1 or 32P-Oligo p21WAF1/Cip1mSp1-7(-1387/-1358), together with 0.5 μg of Sp1, YY-1 or BSA, added alone or in combinations as indicated

89 Previously, it has been shown that Sp1 and YY-1 interact and the interaction occurs in the first one and haft zinc finger region of YY-1 and the zinger finger region plus activation domain D of Sp1 (374, 375). Three truncated Sp1 proteins (Sp1-Q(11-262), Sp1(263-619, Sp1-ZnF/D(620-778) were employed in EMSA (Fig. 3.11). Full- length recombinant YY-1 and Sp1 proteins were used as negative and positive controls, respectively. The truncated Sp1 proteins with N-terminal glutamine-rich activation domain (amino acid residues 11- 262) and with amino acid residues 263 to 778 did not bind to the atypical Sp1-7 binding element. The Sp1 protein with zinc finger region and activation domain D (amino acid 620-778) distinctly formed the nucleoprotein complex and in the presence of YY- 1 protein, less complex was formed due to interaction of YY-1 and Sp1. Sp1 zinc finger region plus activation domain D was confirmed to be the binding region of the YY-1 by EMSA These results are consistent with the previously published reports (374) (375) based on deletion and protein-protein interaction studies.

To further determine how specific is YY-1 inhibition in the context of its interaction with Sp1, ChIP was also done on a well-established Sp1-dependent gene, platelet-derived growth factor A-chain (PDGF- A) (321) (Fig. 3.12, upper panel). Primers were designed to encompass the Sp1 binding elements, SBE generating a 777 bp amplicon. Overexpression of YY-1 did not affect Sp1s interaction on SBE. In p21WAF1/Cip1 promoter, six Sp1 binding elements (Sp-1, Sp-2, Sp13, Sp1-4, Sp1-5 and Sp1-6) are also present in the proximal region of p21WAF1/Cip1 (376) all of which are included in the 347 bp (Fig. 3.12, lower panel). Again, YY-1 overexpression did not affect the occupancy of Sp1 on SBE in p21WAF1/Cip1 promoter. These findings clearly demonstrated that YY-1 prevents Sp1 binding in this atypical Sp1 binding element in a site-selective manner.

Fig. 3.11. Domain of Sp1 interacting with YY-1. EMSA was performed with 32P-Oligo p21WAF1/Cip1(-1387/-1358) using 0.5 μg of Sp1, Sp1-Q1(1-262), Sp1(263-619), Sp1-ZnF/D(620-778), YY-1 or BSA, added alone or in combinations as indicated. Sp1Q1(1-262) represents the N-terminal glutamine-rich activation of Sp1; Sp1(263- 619) spans residues 263-619; ZnF/D(620-778) represents the three zinc fingers and activation domain D of Sp1.

Fig. 3.12. ChIPs for other Sp1 binding sites in the p21WAF1/Cip1 and platelet-derived growth factor-A (PDGF-A) promoters. Human SMCs in 100-mm plates were transfected with 30 μg of pCB6-YY-1 or pCB6+ before chromatin immunoprecipitation and PCR for the PDGF-A promoter (upper panel) or the proximal region of the p21WAF1/Cip1 promoter bearing Sp1 binding sites (lower panel). SBE denotes Sp1 binding elements. PCR products were sequenced and confirmed. No Ab indicates no antibody added.

92 YY-1 siRNA stimulates SMC growth and intimal thickening. By using siRNA targeting YY-1, a rescue result will further confirm the effect of YY-1 on SMC proliferation and neointimal formation. YY-1 siRNA reduced the serum-inducible YY-1 and increased p21WAF1/Cip1 mRNA expression without affecting the level of GAPDH (Fig. 3.13, right panel) whereas the control or nonsense (ns) siRNA had no affect. The same approach was done in serum-induced proliferation assay in SMCs. A significant increase of total cells was obtained with YY-1 siRNA compared to that of the controls (No ODN and siRNAns) (Fig. 3.13, left panel). Transfection experiments with siRNA to YY-1 increased the rat carotid artery intimal thickening 14 days after balloon injury beyond that of the nonsense or vehicle groups (Fig. 3.14). These two rescue experiments confirm that YY-1 inhibits SMC proliferation both in vitro and in vivo.

YY-1 regulates thymidine kinase-1 expression. YY-1 influence on events down-stream of pRbSer249/Thr252 phosphorylation and E2F- dependent thymidine kinase-1 (TK-1) were investigated. Luciferase TK-1 reporter plasmid with either backbone pCB6+ or pCB6+ YY-1 were transfected in SMC. YY-1 overexpression dramatically reduced TK-1 promoter activity (Fig. 3.15, upper panel) with no apparent change in Luciferase activity when backbone alone, pCB6+ was used. To further show the effect of YY-1 on TK-1 expression, SMC were transfected with YY-1 and siRNA to E2F. Western blotting showed that TK-1repression by YY-1 was blocked by E2F siRNA (Fig. 3.15, lower panel) indicating that YY-1 regulation is mediated through E2F. Previous study showed that p53 positively regulates p21WAF1/Cip1 and YY-1 may reduce p21WAF1/Cip1 via p53 (377). Cells were transfected with YY-1 and total protein extracts were pulled down with ubiquitin antibody prior to blotting with p53 (Fig. 3.16, top right hand panel). Consistent with Sui and coworkers (228), YY- 1 increased p53 ubiquitination. Western for p21 was also done to further show that p21WAF1/Cip1 protein levels was reduced when YY-1 was overexpressed. Taken together, YY-1 increased p53 ubiquitination with concomitant reduction in p21WAF1/Cip1 expression (Fig. 3.16, top right hand

Fig. 3.13. Rescue experiment with siRNA to YY-1. Growth-arrested SMCs in 96-well plates were transfected with 0.25 μM of YY-1 siRNA or siRNAns (nonsense or control) before exposure to 5% fetal bovine serum. Cells were counted after 3 days (left panel). Alternatively, SMCs were treated identically except that total RNA was extracted and RT-PCR (right panel) was performed.

94 .

Fig. 3.14. Effect of YY-1siRNA on neointimal formation in rat. Fifty micrograms of siRNA molecules were delivered transluminally in rat carotid arteries and neointimal formation was measured 14 days after balloon injury. Error bar represents SEM; *denotes p<0.05; n=5- 6 rats per cohort. Courtesy of Drs. Peter Kanellakis and Alex Bobik. Error bar represents SEM; *denotes p<0.05; number on the left side of RT-PCR indicates the DNA marker in base pair.

Fig. 3.15. YY-1 represses TK-1 expression. Transient transfection of YY-1 inhibits TK-1 expression as indicated by Renilla luciferase activity (upper panel) after 24 hour. SMCs in 6-well plate were transfected with pCB6-YY-1 or pCB6+ (0.5, 1, 2 and 3 μg together with 1 μg of construct pRL-TK. Luciferase activity was normalized with the backbone alone, pCB6+. Also, SMCs in 100-mm dishes were transfected with 30 μg of pCB6-YY-1 or pCB6+ (with or without E2FsiRNA, 0.4 μM) (lower panel) and after 24 hour, total proteins were extracted and subjected to Western blotting for TK-1 or -actin. Error bar represents SEM; *denotes p<0.05

96 panel). Ad-YY-1 was delivered to the rat arterial wall prior to injury and immmunostained for p53. (Fig. 3.16) to test the in vitro result in animal model. Immunohistorychemistry revealed that YY-1 abrogated p53 expression whereas AdLacZ, a control for Ad-YY-1 did not. YY-1 overexpression reduced p53 immunoreactivty in SMCs both in culture and in the arterial wall (Fig. 3.16) consistent with our previous observation of an inverse relationship between p53 and YY-1 expression (111). To determine whether proteasomal degration can also play a role in YY-1/p53 interplay, MG132 was added during the transfection of YY-1 in SMC. MG132 is a potent proteasome inhibitor thus reducing the degradation of ubiquitin-conjugated proteins in mammalian cells. Western blot indicated that the blockade of the proteasome prevented the YY-1 suppression of p53 protein level (Fig. 3.16, lower right panel). In contrast, p53 mRNA was not altered by YY-1, either in the absence or presence of MG132. These data demonstrate that YY-1 suppression of p21WAF1/Cip1 in SMC also involves p53 ubiquitination and proteasomal degration.

Fig. 3.16. YY-1 suppression of p21WAF1/Cip1 involves p53 ubiquitination and proteasomal degradation. Left panel. Ad-YY-1 overexpression decreases p53 immunoreactivity after 14 days after injury of rat artery wall. Courtesy of Hideto Ishii. Upper right panel. SMCs in 100-mm dishes were transfected with 30 μg pCB6-YY-1 or pCB6+ and after 24 hours, extracts were immunoprecipitated (IP) with ubiquitin antibody prior to Western blotting with p53 antibody. Alternatively, Western blot analysis was performed for p21WAF1/Cip1.

98 YY-1 first two zinc finger region alone can inhibit SMCs proliferation. Different truncated YY-1 constructs were generated (see Fig. 2.1) and overexpressed in SMCs with the main aim of determining the minimal YY- 1 that will inhibit proliferation. Minimal functional YY-1 can also have the advantage to be delivered more efficiently than the full-length. As expected, the full length YY-1 (YY-1FL) inhibited the serum-induced SMC proliferation (Fig. 3.17, top panel) at dose-dependent manner but not EC (Fig. 3.17, bottom panel). YY-1 first zinc finger region (YY-1B) significantly reduced proliferation compared to YY-1FL.

Amino acids 339-341 in YY-1 are critical in inhibiting SMC proliferation. To interrogate the critical amino acid residues in YY-1 responsible for the interaction with Sp1, a computer program, Receptor Binding Domain (RBD) Finder (378) was used to predict the amino acid residue(s) in YY-1 that might be involved in protein-protein interaction (Fig. 3.18). This fast method of predicting protein interaction sites using amino acid sequence only is based on the analysis of sequence hydrophobicity and detects hydrophilic domain. Different mutants of YY- 1B were overexpressed in SMC together with the wild type YY-1B. Single mutation at selected amino acid residue did not block SMC inhibition (Fig. 3.19) but a triple mutation at amino acids 339-341 ablated the YY-1B inhibitory effect significantly. The result of this mutational analysis in context of inhibiting SMC proliferation looks promising.

Fig. 3.17. Proliferation assay of SMCs and ECs. Various constructs were transfected into the cells at the indicated amounts of DNA. Upper panel, SMCs. Lower panel. ECs. Cells were seeded into a 96-well plate and grown to 70-80% confluency prior to serum arrest overnight. Plasmid DNA was transfected with Fugene overnight prior to stimulation with 5% FBS. Cells were harvested three days after stimulation and counted using the Coulter counter. Samples were done in triplicates and counted three times per well. n=9. Error bar denotes standard error of the mean.

100

Fig. 3.18. Receptor binding domain results. The RBD program predicts the secondary structure of the protein based on the amino acid sequence. Upper panel. Amino acids contained in the blue polygon are most likely to be the binding region and printed in red in the amino acid sequence. Lower panel. Probability plot of amino acids which are unusually far away from the rest of the points will be farther away from the diagonal line. Underlined amino acids denote the zinc finger region in YY-1.

101

Fig. 3.19. Proliferation assay of YY-1B and mutant forms in SMCs. YY-1B contains the first two zinc finger region of YY-1 cloned into the BamHI and different mutant forms were generated based on the result of the RBD Finder. YY-1BM308 (Arg > Ala); YY- 1BM339 (Lys > Ala); YY-1BM340 (Leu > Ala); YY-1BM341 (Lys > Ala); YY-1BM339-341 (Lys > Ala, Leu > Ala, Lys > Ala). A) Upper panel. Proliferation assay with 0.1μg DNA. B) Lower panel. Proliferation assay with 0.2μg DNA. Dunnett’s multiple comparison was done with YY-1B as control to compare the effect of mutation on proliferation. Error bar denotes standard error of the mean. n=9

102

103 To generate a phospho-specific antibody, a choice has to be made whether it is a polyclonal or monoclonal. Polyclonal antibody is cheaper and faster to generate compared to monoclonal. However, monoclonal has the advantage of recognizing only one epitope on an antigen thus it is more specific. Once hybridomas are made, monoclonal antibody offers more consistency as all batches will be identical. Monoclonal phospho- specific antibody offers more specificity in detecting antigens in the tissues and will often give significantly less background staining (379). To determine the antigen or peptide sequence to produce a phospho-specific antibody, thorough in vitro Ekr-1 phosphorylation assays were done.

Serine26 is highly phosphorylated by Erk-1/2. To identify the amino acid residues in Egr-1 that are phosphorylated by Erk-1/2, recombinant Egr-1 was phosphorylated prior to mass spectrometry. Mass spectrometry of the Erk-1 phosphorylated Egr-1 protein revealed three amino acid residues (Fig. 3.20), Serine 26, Threonine 28 and Threonine 285 to be highly phosphorylated compared to the rest of amino acids. In vitro Erk-1 phosphorylation was done with synthetic peptides with single and double mutations to determine which amino acid residue is critical. Serine 26 (Ser26) contributed more than 60% of the total phosphorylation (Fig. 3.21) and mutation of Ser26 to Alanine dramatically reduced the phosphorylation of the recombinant Egr-1 protein (Fig. 3.22). Both in vitro phosphorylation assays revealed that Ser26 was the heavily favoured amino acid to be phosphorylated by Erk-1/2. Since angiotensin II can activate ERK1/2 by RAS/PKCz/MEK pathway in vascular SMCs, in vitro phosphorylation of Egr-1 recombinant protein by PKCz was also done. No phoshorylation of Egr-1 protein by PKCz was detected (Fig. 3.23) and as expected, PKCz can autophosphorylate itself. This result implies how specific Erk-1/2 in phosphorylating Ser26. Alignment of Egr-1 amino acid sequences indicated that Ser26 is conserved in all animals indicated (Fig. 3.24) and this amino acid is specific in Egr-1 and not with other members of Egr protein family (Fig. 3.25).

104

Fig. 3.20. Mass spectrometry result after Erk-1 phosphorylation. Amino acid sequence of mouse Egr-1. Underlined amino acid, in red denotes the phosphorylated amino acid and underlined amino acid residues indicate the three-zinc finger region of Egr-1.

105

Fig. 3.21. In vitro phosphorylation of synthetic peptides. One hundred nanogram of synthetic peptide was used in the Erk-1 phosphorylation. Wild type peptide contains both Ser 26 and Thr28 and used as a reference control. Peptide preceded with letter M denotes mutant peptide at indicated amino acid. Error bar denotes standard error of the mean. n=3.

106

Fig. 3.22. In vitro phosphorylation of recombinant Egr-1 proteins. Wild type and mutant (Ser26>Ala26) Egr-1 proteins (5 μg) were phosphorylated accordingly. Blank control denotes no enzyme or protein whereas enzyme denotes the presence of Erk-1 enzyme but no protein. Bonferroni’s multiple comparison test was done and shown is the statistical comparison between wild type and mutant recombinant Egr-1 proteins. ** denotes significant difference at p<0.01. Error bar denotes standard error of the mean. n=6.

107

Fig. 3.23. Comparison of Erk-1 and PKC-zeta phosphorylation. Wild type and mutant (Ser26>Ala26) recombinant Egr-1 proteins (5 μg) were phoshorylated accordingly. Equal units of the kinase were used. Bonferroni’s multiple comparison test was done and show is the comparison between wild type and mutant recombinant Egr-1 proteins after Erk-1 phosphoryation. ** denotes significant difference at p<0.01. Error bar denotes standard error of mean. n=3.

108

Fig. 3.24. Alignment of Egr-1 amino acid sequences. Multi- alignment of Egr-1 amino acid sequences from different organisms Accession number; mouse (NP_031939.1), rat (EDL76249.1), human (NP_001955.1), Monkey (XL_001107731.2), Cow (NP_001039340.1), Pig (XP_003124022.1), Horse (XP_001502603.2), Rabbit (XP_002710285), and zebrafish (NP_571323.1). Arrow indicates Ser26.

109

Fig. 3.25. Alignment of amino acid sequences of mouse Egr family. Multi-alignment of mouse Egr-1 (NP_031939.1), mouse Egr-2 (P08152), mouse Egr-3 (P43300) and mouse Egr-4 (Q9WUF2). Black arrow indicates the position of mouse Egr-1 Serine26.

110 Alignment of the amino acids was done to determine possible cross- reactivity of the phospho-specific antibody to be produced. The peptide sequence, FGSFPHSPTMDNY, was then used by EZBiolab (Carmel, IN, USA) to generate the mouse monoclonal antibody.

Initial validation tests of the phospho-specific antibody proved to be specific for Ser26. To test the specificity of pS26, peptides with or without the phosphorylated Serine were blotted into the membrane at different serial dilution. Immunoblot showed that pS26 can discriminately detect the phosphorylated peptide at 3.9 to 62 ng. Specificity of pS26 antibody was established when synthetic peptides without phospho-Ser26 cannot be detected by pS26 (Fig. 3.26). Site directed mutagenesis was done at Serine26 and GST- tagged full-length Egr-1 proteins, both wild type and mutant (M26) were generated. Both wild type and mutant M26 Egr-1 proteins were blotted at different serial dilutions (Fig. 3.27). GST protein was also included as a control. pS26 did not cross react with the GST protein alone neither to the GST-Egr-1 protein with mutation at amino acid 26 (GST-Egr-1 M26). The effect of Ekr-1 inhibitor, PD98059 together with p38 and JNK inhibitors were assessed on injured SMC cell extract (Fig. 3.28). As expected, both PD98059 and SP600125 blocked the injury- inducible phosphorylated Egr-1 protein whereas SB202190 did not. Other cellular proteins were detected by PS26. It is possible that PS26 can also detect phosphorylated serine residues from other cellular proteins aside from phosphorylated Egr-1 proteins. The blot showed consistently that the 75 kDa Egr-1 protein was upregulated by injury as compared to that of the uninjured sample. To establish that the 75 kDa protein was indeed the phosphorylated Egr-1, peptide blocking was done. PS26 was mixed with the peptide at mass ratio of 1:3 (PS26:peptide) prior to incubation of the membrane (Fig. 3.29) The 75 kDa protein was abrogated across all the samples with peptide blocking (Fig. 3.29, lower panel). Without peptide blocking, pS26 consistently detected the 75 kDa phosphorylated Egr-1 (Fig. 3.29, upper panel). To establish further that the 75 kDa protein was indeed phosphorylated, phosphatase treatment of samples was performed. Sample treated with 1 unit of calf intestine phosphatase (CIP)

111

Fig. 3.26. Slot blot of synthetic peptide. Serial dilutions of synthetic peptides were blotted and Western immunoblotting was done with the phospho-specific mouse monoclonal antibody at 1:30K dilution.

112

Fig. 3.27. Slot blot of the recombinant Egr-1 proteins. Serial dilutions of the GST-tagged recombinant wild type and mutant Egr-1 proteins were blotted with GST protein alone as the control. Immunoblotting was done with the phospho-specific mouse monoclonal antibody at 1:30K dilution.

113

Fig. 3.28. Western blotting of injured human SMCs using mouse monoclonal phospho-specific antibodies. Cells were grown in 100mm petri dish and at 60-70% confluency, cells were serum- arrested overnight. Treatment of different kinase inhibitors was done 1hr prior to injury. PD denotes Erk-1/2 inhibitor, PD98059 at 10μM; SB denotes p38 inhibitor, SB202190 at 500nM; JNK denotes JNK inhibitor, SP600125 at 25 nM. Un denotes the uninjured cells. Injury to SMC seeded on a 100mm dish was done using a sterile comb of pins and “scratching” was done uniformly for each dish. Number on the left denotes the protein markers in kDa. Arrow indicates the position of the 75 kDa phosphorylated Egr-1 protein.

114

Fig. 3.29. Western immunoblotting with peptide blocking. Cell extracts (10 μg) were loaded into 10% SDS-PAGE gel and transferred into PVDP membrane. Membrane was blocked overnight with 5% skim milk prior to incubation with phospho-specific mouse monoclonal antibody (Upper panel) or with phospho-specific mouse monoclonal antibody mixed with peptide (1:3 mass ratio). Treatment of different kinase inhibitors was done 1hr prior to injury. PD denotes Erk-1/2 inhibitor, PD98059 at 10μM; SB denotes p38 inhibitor, SB202190 at 500nM; JNK denotes JNK inhibitor, SP600125 at 25nM. Arrow indicates the position of the 75 kDa phosphorylated Egr-1 protein.

115 reduced dramatically the signal of the 75 kDa protein with total abrogation at 10 units of CIP (Fig. 3.30, upper panel). Immunoblot of the samples was also done with the use of total Egr-1 antibody (Fig. 3.30, lower panel). Samples treated with CIP showed just a slight decrease of signal compared to that of the CIP-treated injured sample. This slight decrease of signal can be accounted to other phosphorylated amino acid residues in Egr-1 protein. Uniform loading of samples was indicated by the -actin blots. Taken all together, initial validity tests showed that mouse monoclonal antibody to Egr-1, PS26 is potentially a phospho-specific antibody.

To determine whether Erk-1 phosphorylation can increase or decrease its DNA binding capacity, EMSA was performed. Using the EGR binding element from plasminogen activator inhibitor-1 as a probe, unphosphorylated Egr-1 proteins formed a strong nucleoprotein complex whereas the phosphorylated samples showed weak nucleoprotein complex indicating that the phosphorylation decreases Egr-1 DNA binding capacity (Fig. 3.31). However, either total Egr-1 or pS26 did not supershifted the nucleoprotein complex. Further EMSA studies are needed to establish the stoichemistry of Egr-1 protein and the antibody.

116

Fig. 3.30. Phosphatase treatment of cell extract. Ten microgram total protein was incubated with calf intestine phosphatase (CIP) at indicated number of units at 37C for 30 min and loaded into 10% SDS-PAGE gel. pS26 denotes immunoblotting with phospho- specific antibody to Egr-1 and its corresponding -actin as loading control (Upper panel). Total Egr-1 and its corresponding -actin immunoblots (Lower panel). Numbers on the left side denotes the protein markers in kDa.

117

Fig. 3.32. EMSA with unphosphorylated and phosphorylated recombinant Egr-1. EGR (-151/-127) probe sequence,5’-GGG AGG GAG GGA GGG AGG GGG AGAG-3’; 5’-CTC TCC CCC TCC CTC CCT CCC TCCC-3’ (331). U and P denote unphosphorylated and Erk-1/2 phosphorylated Egr-1 proteins, respectively. Antibodies, Total Egr-1 and pS26 were mixed to Egr-1 proteins prior to addition of the EGR (151/-127) probe. Arrow indicates the nucleoprotein complex.

118

Chapter 4

General Discussion and Future Directions

119 Vascular SMCs exhibit varied responses such as phenotypic changes, migration, proliferation, protein synthesis and apoptosis after vascular injury and surgical interventions. Injury to blood vessel wall initiates a cascade of molecular events that leads to induction of transcriptional activators and repressors. The net balance of the activities of these regulators governs the phenotypic outcomes which includes the vascular disorders like atherosclerosis, bypass graft failure, hemodialysis access graft failure, restenosis after percutaneous coronary angioplasty and stenting, the pathogenesis of each of which involves the formation of a smooth muscle-rich intima. Understanding the mechanisms by which these factors exert their effects on SMC can give a glimpse on how to control its proliferative vasculopathy. One tool in understanding the mechanism of disease development is a phospho-specific antibody that may also open many opportunities in investigative and diagnostic pathology.

The aims of this thesis were (1) elucidate the possible mechanism(s) by which YY-1 can selectively inhibit SMC proliferation and not EC, and (2) to identify phosphorylated amino acids in Egr-1 by Erk-1 and generate a site- specific, phosphorylation state-specific antibody to Egr-1.

YY-1 contributes to various normal biological processes and it can regulate target genes by activating or repressing their transcription directly or indirectly. The multifunctional properties of YY-1 appear to depend on the cellular environment and cell type. YY-1 has been shown to be upregulated after vascular injury and its overexpression inhibits SMC proliferation but not EC. This cell type specific effect of YY-1 promises a great potential as a therapeutic strategy especially in drug eluting stent intervention of restenosis.

YY-1 induction was noted in SMCs within hours of injury in the blood vessel (111) and its overexpression inhibited SMC proliferation but not EC. Experiments were done to elucidate the possible mechanism(s) in which YY-1 exerts its selective inhibitory effect. This thesis established that YY-

120 1 inhibits p21WAF1/Cip1 transcription in SMCs by preventing the occupancy of Sp1 in the p21WAF1/Cip1 promoter (Fig. 4.1). The inhibition of p21WAF1/Cip1 subsequently leads to perturbation of the formation of the p21WAF1/Cip1- cdk4-cyclin D1 complex, thereby blocking the pRb phosphorylation. This study challenges the role for p21WAF1/Cip1 as a negative regulatory in cell cycle progression. Earlier reports showed that p21WAF1/Cip1 can function as a positive regulator of the cell cycle (370) (380) (381). Other experiments indicated that p21WAF1/Cip1 is required for PDGF-induced vascular proliferation (382) and a proatherogenic in apolipoprotein E-null mice possibly as a consequence of cell cycle-independent activities of p21WAF1/Cip1 (383). Many reports have also implied that p21WAF1/Cip1 plays an important permissive role in proliferation. Growth factors like FGF-2 and PDGF known to have a proproliferative effect, induced p21WAF1/Cip1 expression (384). It was also reported that p21WAF1/Cip1 level increased after balloon injury to the rat carotid artery wall (385). Induction of p21WAF1/Cip1 levels was noted after rat carotid angioplasty with parallel increase of cyclins, cdks and PCNA (380). Moreover, antisense ODN targeting p21WAF1/Cip1 inhibits SMC proliferation (369) (386). The role of YY-1 in inhibiting SMC proliferation was further supported by the work of Beck and others (176). YY-1 was found to mediate the antiproliferative effect of heme oxygenase-1 (HO-1) in SMC in vitro and neointimal formation in vivo.

This study also provides the gain- and loss-of-function approaches to show that YY-1 inhibited p21WAF1/Cip1 transcription and abrogated the formation of the cyclin D1-cdk4- p21WAF1/Cip1 complex. Consistent with the model, YY-1 had no effect on vascular endothelial cell proliferation nor did it influence cyclinD1-cdk4- p21WAF1/Cip1 complex assembly or pRbSer249/Thr252 phosphorylation. YY-1 suppression of p21WAF1/Cip1 in SMCs also involves p53 ubiquitination and proteasomal degradation. p53 positively regulates p21WAF1/Cip1 transcription (377) and YY-1 reduces p53 stability by stimulating Mdm2 (E3 ubiquitin ligase)-dependent p53 ubiquitination and proteasomal degradation (387) (388). YY-1 has been shown to interact with p300 (136), thus can function as an E4 ligase for

121

Fig. 41. YY-1 repression of p21WAF1/Cip1 transcription and p21WAF1/Cip1- cdk4-cyclin D1 complex formation. YY-1 binds to Sp1 preventing the latter to occupy the -1375CCTCCC-1370 element in the p21WAF1/Cip1 promoter. YY-1 also stimulates p53 ubiquitination and proteasomal degradation. Reduced level of p21WAF1/Cip1, in turn, perturbs assembly of the p21WAF1/Cip1-cdk4-cyclin D1 complex thereby inhibiting pRbSer249/Thr252 phosphorylation and proliferation. SBE denotes Sp1-binding elements; D1, cyclin D1; K4, cdk4.

122 p53 polyubiquitination (389), thus there is a possibility that YY-1 may recruit p300 to promote p53 ubiquitination (228).

YY-1’s ability to suppress SMCs proliferation and not ECs may also involve cell-type specific differences in YY-1-dependent cofactor interactions. YY-1 interacts with a large number of transcription factors like RYBP (Ring1- and YY-1-binding protein), YAF-2 (YY-1-associated factor 2) (390), AP-2 as well as general factors such as transcription factor IIB, TAFII55, p300, cAMP responsive element-binding protein (CPB)- binding protein, p300 (122) which maybe be present in SMC but not in ECs or vice versa. It would be interesting to identify these co-factors by two-hybrid system using the full length YY-1 and the truncated YY-1 (YY- 1B) as baits thereby adding another layer to the mechanism reported here.

Moreover, differences in chromatin structure and methylation state of the promoter may influence YY-1’s accessibility and capacity to alter transcription. The interference of YY-1 with a functional transcriptional activator is very much exemplified by the ability of YY-1 to disrupt activating transcription factor/cAMP responsive element-binding protein (ATF/CREB) complex. The protein-protein interaction between YY-1 and the bZIP region of CREB represses transcription of c-fos (391). YY-1 can also inter-act with known repressors like mouse Reduced Potassium Dependency-3 (mRPD3) with around 85% homology with human histone deacetylase 1 (134). Because of this interaction, the question of mechanism of YY-1 in recruiting chromatin modifying enzymes has been raised (156). Enzyme modifying histones, histone acetyltransferase (HAT) and histone deacetylase (HDAC) typically function as activator and repressor, respectively, by altering the chromatin structure. YY-1 can bind to HAT or HDAC and can locally direct to a promoter leading to either activation or repression of that particular promoter. YY-1 has been shown to recruit HDAC4 and GATA3 (GATA binding protein) to repress interluekin-5 transcription (392). YY-1 activity can also be modulated by indirectly recruiting HDAC1 via YY-1 interaction with Sin3-associated polypeptide p30 (SAP30) (393). Moreover, YY-1 has been shown to

123 recruit HDAC-1 to repress the transcription of the long terminal repeat (LTR) of the HIV-1 (394) (395). Indirect HDAC-1 recruitment by YY-1 via SAP30-YY-1 interaction has been confirmed both in vitro and in vivo (393). The interaction domains between YY-1 and SAP30 were mapped in the zinc-finger region of YY-1 and the C-terminal 91 amino acid region of SAP30 (393).

Although deletion study (375) showed that the first one and a half zinc finger region of YY-1 physically interact with Sp1, the full first two zinc finger regions was used since exposed amino acid residues on the half of the second zinc finger region might bind non-specifically with other proteins and/or might undergo conformational change. In this study, amino acids 339-341 in the first two zinc finger regions of YY-1 are critical in the protein-protein interaction with Sp1. Overexpression of the first two zinc finger regions inhibited SMC proliferation whereas the mutant did not and as expected, neither constructs affects EC proliferation. Another study has reported that the first two-zinc finger region of YY-1 was sufficient to bind with transcription factor late SV40 factor (LSF) and repress the transcription of the long terminal region (LTR) of HIV-1 (394).

The work on protein-protein interaction between Sp1-D (zinc-finger region plus activation domain D) and the first two zinc-finger of YY-1 (YY-1B) has started. Initial result by BIACORE analysis showed protein-protein interaction between these two truncated constructs. BIACORE analysis of the mutant YY-1B 339-341 will confirm whether those three amino acid residues are critical for Sp1-D and YY-1B interaction.

In this study, the effect of the YY-1B in inhibiting SMC and not EC looks promising. Work is ongoing in the laboratory on the adenoviral delivery of the first zinc finger region of YY-1. The use of this shorter fragment with high efficiency delivery may represent a significant future therapeutic tool in inhibiting neointimal thickening. Animal ethics applications are being prepared to interrogate the effect of short YY-1 versus full length YY-1 on neointima formation in different animal models. Long -term plan will be the

124 use of this YY-1B in drug eluting stents so the delivery will be local and at the same time assess its clinical value on in-stent restenosis.

The mechanism of YY-1 repression involving Sp1 displacement generated by this work has not been described before. Other possible mechanism of SMC inhibition by YY-1 is plausible due to the fact that YY-1 has multiple target genes. YY-1-based gene therapy offers an opportunity of inhibiting SMC where re-endothelialization is desirable typical of the restenosis and vein grafting. The use of a much smaller functional YY-1 may overcome the delivery problem due to size and may also improve the efficiency of the gene transfer.

Egr-1 activation is considered to be a “master switch” of many genes very well implicated in SMC proliferation. Although many studies showed that Egr-1 phosphorylation by Erk-1 activated almost all Egr-1-dependent genes, it is not well established whether upregulation of these dependent genes is due to increased Egr-1 DNA binding activity or its ability interact with other regulatory proteins or both. In this study, serine26 was highly phosphorylated by Erk1/2 and a mouse monoclonal phospho-specific Egr- 1 antibody, pS26 was generated. Initial validation of specificity of this antibody was done using peptide blocking and slot blots with synthetic peptides and recombinant proteins. Moreover, immunoblotting with total cells extracts showed the capacity of this antibody to detect the 75 kDa phosphorylated Egr-1 protein from SMCs after vascular injury. Treatment with phosphatase of the total cellular proteins indicated that the inducible 75 kDa protein is the phosphorylated Egr-1 protein. The identity of the protein will be confirmed by mass spectrometry. EMSA indicated that Erk- 1/2 phosphorylation of recombinant Egr-1 decreased its DNA binding capacity to the EGR binding element of PAI. This result has to be further investigated in cell extract as other cellular factors can affect the DNA binding capacity of Egr-1.

The clinical application of phospho-specific antibody has been an important tool in the investigative and diagnostic pathology. Studies have

125 shown that there was a significant correlation between the long-term clinical outcome in breast cancers and phosphorylation of tyrosine- phosphorylated erbB-2 (396) (397) and it was suggested that erbB-2 signaling is important in the early stages of breast tumorigenesis (398). The phosphorylation of epidermal growth factor receptor (EGFR) strongly correlates with the worse clinical outcomes of non-small cell lung cancer and assessment of EGFR phosphorylation can be used for therapeutic decision-making (399). To improve the prognosis significance of Akt activation in non-small cell lung cancer, phosphorylation of T308 was established by two studies to be the more reliable biomarker than Ser473 (400, 401). Cardiac troponin-1 (Ser24) mouse monoclonal antibody served as a cardiac-specific cell marker and indicator of myocardial damage via serum ELISA (402).

Phospho-specific antibody, 5-lipoxygenase (Phospho-Ser523) polyclonal antibody, has been used to implicate phosphorylation of Ser523 in the reduction of inflammation and fibrosis (403). The use of this antibody can be extended to study the role of 5-lipoxygenase in atherosclerosis since variant 5-lipoxygenase genotypes were identified in a subpopulation of 470 healthy men and women with increased atherosclerosis (404). The use of a phospho-specific antibody to p38 established that allograft inflammatory factor-1 (AIF-1) enhanced SMC proliferation but not migration (405).

Phospho-specific antibodies can also aid in the study of different downstream signaling involve with inhibitors to SMC cellular events. For example, different phospho-specific antibodies were used to elucidate the pathway by which a NAD(P)H oxidase inhibitor regulate PDGF-dependent SMC chemotaxis (406). New insights into the regulatory mechanisms controlling the PKC-zeta-phospho-Sp1 axis and angiotensin II- inducible gene expression in SMC were further established with the use of three phospho-specific antibodies to Sp1 (407).

Validation of a phospho-specific antibody entails several steps to confirm

126 its specificity. Steps in testing the specificity include Western blotting, immunocytochemical staining, phosphopeptide preincubation or peptide blocking, phosphatase treatment and site-directed mutagenesis of the phosho-acceptor amino acid within the protein of interest (408). Brumbaugh and others also recommended siRNA and knockout mice of the gene of interest, use of inhibitors of kinases and phosphatases as additional steps to validate the phospho-specific antibody (408).

Initial validation of pS26 offers some potential and further validation steps are needed to fully establish its specificity. Overexpression construct was made with a site-directed mutation to Ser26 and will be used together with the Wild Type in expression studies to further validate pS26.

Immunohistochemistry of rat arteries following balloon injury was done by Dr. Kristine Malabanan in the Laboratory and initial experiment showed that phosphorylated Egr-1 was detected in the neointima and not in the media. Further experiments are needed to confirm this observation. Also, siRNA knockdown of Egr-1 prior to balloon injury will further establish the specificity of the antibody as well as its spatial distribution. Immunohistorychemistry of different types of lesions or vascular injury will also aid in the validation.

This dissertation has provided in depth insights into the mechanisms by which YY-1 selectively exerts its inhibitory effect on SMCs and not in ECs. Other possible mechanisms that might be involved in SMC proliferation were discussed. Moreover, initial validation of the work provided the first phospho-specific antibody to Egr-1 and further validation tests are needed with pS26. The influence of Egr-1 phosphorylation on transcriptional activity and its role in pathobiology of SMCs proliferation warrant an investigation. A phospho-specific antibody to Egr-1 will aid in a better understanding of mechanism(s) of Egr-1-dependent gene expression at multiple levels such as protein-protein interaction, occupancy of gene promoter and possibly as a diagnostic tool of diseased tissues.

127

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