Jiliang Zhou

Repression of Versican Expression by MicroRNA-143

Smooth muscle cells (SMCs) retain remarkable plasticity to undergo phenotypic modulation in which the expression of smooth muscle markers is markedly attenuated while conversely expression of extracellular matrix (ECM) is dramatically up-regulated. Myocardin is perhaps the most potent transcription factor for stimulating expression of smooth muscle-specific genes; little is known, however, about whether myocardin can orchestrate ECM expression to act in concert with smooth muscle differentiation program. In this study, we demonstrated myocardin coordinate smooth muscle differentiation by inducing transcription of microRNA-143 (miR-143), which attenuates ECM versican protein expression. Previous studies have shown that versican is a chondroitin sulfate proteoglycan of the ECM that is produced by synthetic SMCs and promotes SMC migration and proliferation. Our data demonstrated that myocardin significantly represses versican expression in multiple cell lines, and this occurs through the induction of miR-143 by myocardin. By a modified reverse transcribed PCR, we found that miR-143 specifically binds to the 3′-untranslated region of versican mRNA. Reporter assays validated that miR-143 targets versican 3′-untranslated region through an evolutionarily conserved miR-143 binding site. Furthermore, overexpression of miR-143 significantly represses versican expression, whereas conversely, depletion of endogenous miR-143 results in up-regulation of versican expression. In addition, we demonstrated that myocardin represses versican through induction of miR-143. Finally, we found that the regulation of versican by miR-143 is involved in platelet-derived growth factor BB-induced SMC migration. This study provides the first evidence that myocardin, in addition to activating smooth muscle-specific genes, regulates ECM expression through induction of microRNAs during smooth muscle differentiation.

Myocardin-Related Transcription Factor A Mediates OxLDL-Induced Endothelial Injury

Rationale: Atherosclerosis proceeds through a multistep reaction that begins with endothelial injury caused by a host of stress signals, among which oxidized low-density lipoprotein (oxLDL) plays a critical role. OxLDL disrupts normal functionality of the endothelium by upregulating adhesion molecules (eg, ICAM-1) and concomitantly downregulating endothelial nitric oxide synthase (eNOS) expression. The transcriptional modulator that mediates the cellular response to oxLDL remains largely obscure. Objective: Our goal was to determine whether myocardin-related transcription factor (MRTF)-A, a key protein involved in the transcriptional regulation of smooth muscle cell phenotype, is responsible for the endothelial injury by oxLDL, and, if so, how MRTF-A promotes the proatherogenic agenda initiated by oxLDL. Methods and Results: OxLDL stimulated the expression of MRTF-A in endothelial cells as evidenced by Western blotting and immunofluorescence. Overexpression of MRTF-A synergistically enhanced the induction of ICAM-1 and suppression of eNOS by oxLDL. In contrast, disruption of MRTF-A, either by small interfering RNA or dominant negative mutation, abrogated the pathogenic program triggered by oxLDL. Finally, chromatin immunoprecipitation assays indicate that oxLDL preferentially augmented MRTF-A binding to ICAM-1 and eNOS promoters and that MRTF-A drove differential epigenetic alterations taking place on these promoters in response to oxLDL. Conclusions: Therefore, our data provide the first demonstration that MRTF-A is critically linked to pivotal pathophysiological events in the vascular endothelium.

The SWI/SNF Chromatin Remodeling Complex Regulates Myocardin-Induced Smooth Muscle–Specific Gene Expression

Objective—Regulatory complexes comprising myocardin and serum response factor (SRF) are critical for the transcriptional regulation of many smooth muscle–specific genes. However, little is known about the epigenetic mechanisms that regulate the activity of these complexes. In the current study, we investigated the role of SWI/SNF ATP-dependent chromatin remodeling enzymes in regulating the myogenic activity of myocardin. Methods and Results—We found that both Brg1 and Brm are required for maintaining expression of several smooth muscle–specific genes in primary cultures of aortic smooth muscle cells. Furthermore, the ability of myocardin to induce expression of smooth muscle–specific genes is abrogated in cells expressing dominant negative Brg1. In SW13 cells, which lack endogenous Brg1 and Brm1, myocardin is unable to induce expression of smooth muscle–specific genes. Whereas, reconstitution of wild-type, or bromodomain mutant forms Brg1 or Brm1, into SW13 cells restored their responsiveness to myocardin. SWI/SNF complexes were found to be required for myocardin to increase SRF binding to the promoters of smooth muscle–specific genes. Brg1 and Brm directly bind to the N terminus of myocardin, in vitro, through their ATPase domains and Brg1 forms a complex with SRF and myocardin in vivo in smooth muscle cells. Conclusion—These data demonstrate that the ability of myocardin to induce smooth muscle–specific gene expression is dependent on its interaction with SWI/SNF ATP-dependent chromatin remodeling complexes.

Proinflammatory Stimuli Engage Brahma Related Gene 1 and Brahma in Endothelial Injury

Rationale: Endothelial dysfunction inflicted by inflammation is found in a host of cardiovascular pathologies. One hallmark event in this process is the aggregation and adhesion of leukocyte to the vessel wall mediated by the upregulation of adhesion molecules (CAM) in endothelial cells at the transcriptional level. The epigenetic modulator(s) of CAM transactivation and its underlying pathophysiological relevance remain poorly defined. Objective: Our goal was to determine the involvement of Brahma related gene 1 (Brg1) and Brahma (Brm) in CAM transactivation and its relevance in the pathogenesis of atherosclerosis. Methods and Results: In the present study, we report that proinflammatory stimuli augmented the expression of Brg1 and Brm in vitro in cultured endothelial cells and in vivo in arteries isolated from rodents. Overexpression of Brg1 and Brm promoted while knockdown of Brg1 and Brm abrogated transactivation of adhesion molecules and leukocyte adhesion induced by inflammatory signals. Brg1 and Brm interacted with and were recruited to the CAM promoters by nuclear factor &kgr;B/p65. Conversely, depletion of Brg1 and Brm disrupted the kinetics of p65 binding on CAM promoters and crippled CAM activation. Silencing of Brg1 and Brm also altered key epigenetic changes associated with CAM transactivation. Of intrigue, 17&bgr;-estradiol antagonized both the expression and activity of Brg1/Brm. Most importantly, endothelial-targeted elimination of Brg1/Brm conferred atheroprotective effects to Apoe-/- mice on a Western diet. Conclusions: Our data suggest that Brg1 and Brm integrate various proinflammatory cues into CAM transactivation and endothelial malfunction and, as such, may serve as potential therapeutic targets in treating inflammation-related cardiovascular diseases.

Transforming Growth Factor-β1-induced Transcript 1 Protein, a Novel Marker for Smooth Muscle Contractile Phenotype, Is Regulated by Serum Response Factor/Myocardin Protein

Background: TGFB1I1 is involved in vascular injury, but the regulation of TGFB1I1 expression is unknown. Results: TGFB1I1 is specifically expressed in smooth muscle cells and regulated by SRF/myocardin. Conclusion: TGFB1I1 is a novel marker for the smooth muscle contractile phenotype. Significance: This study provides the first evidence that TGFB1I1 is an SRF/myocardin-regulated smooth muscle marker and critical for maintaining the smooth muscle contractile phenotype. Serum response factor (SRF) plays a central role in regulating expression of smooth muscle-specific genes partly by associating with the potent tissue-specific cofactor myocardin. Previous studies have shown that transforming growth factor-β1-induced transcript 1 (TGFB1I1, also known as Hic-5) is a TGF-β-responsive gene and is involved in the cellular response to vascular injury, but the regulation of TGFB1I1 expression remains elusive. In this report, we demonstrated that TGFB1I1 is a novel marker for the smooth muscle contractile phenotype and is regulated by SRF/myocardin. We found that TGFB1I1 is specifically expressed in smooth muscle cells (SMCs) and in smooth muscle-rich tissues. Furthermore, TGFB1I1 expression is significantly down-regulated in a variety of models for smooth muscle phenotypic modulation. The TGFB1I1 promoter contains an evolutionarily conserved CArG element, and this element is indispensible for myocardin-induced transactivation of TGFB1I1 promoter. By oligonucleotide pulldown and chromatin immunoprecipitation assays, we found that SRF binds to this CArG element in vitro and in vivo. Ectopic expression of myocardin is sufficient to induce endogenous TGFB1I1 expression in multiple cell lines whereas knocking-down myocardin or SRF significantly attenuated TGFB1I1 expression in SMCs. Furthermore, our data demonstrated that SRF is essential for TGF-β-mediated induction of TGFB1I1. Finally, silencing of TGFB1I1 expression significantly promotes SMC proliferation. Collectively, this study provides the first evidence that TGFB1I1 is not only an SRF/myocardin-regulated smooth muscle marker but also critical for maintaining smooth muscle contractile phenotype by inhibiting smooth muscle proliferation.

Pro-Inflammatory Stimuli Engage Brahma Related Gene 1 (Brg1) and Brahma (Brm) in Endothelial Injury

Rationale: Endothelial dysfunction inflicted by inflammation is found in a host of cardiovascular pathologies. One hallmark event in this process is the aggregation and adhesion of leukocyte to the vessel wall mediated by the upregulation of adhesion molecules (CAM) in endothelial cells at the transcriptional level. The epigenetic modulator(s) of CAM transactivation and its underlying pathophysiological relevance remain poorly defined. Objective: Our goal was to determine the involvement of Brahma related gene 1 (Brg1) and Brahma (Brm) in CAM transactivation and its relevance in the pathogenesis of atherosclerosis. Methods and Results: In the present study, we report that proinflammatory stimuli augmented the expression of Brg1 and Brm in vitro in cultured endothelial cells and in vivo in arteries isolated from rodents. Overexpression of Brg1 and Brm promoted while knockdown of Brg1 and Brm abrogated transactivation of adhesion molecules and leukocyte adhesion induced by inflammatory signals. Brg1 and Brm interacted with and were recruited to the CAM promoters by nuclear factor &kgr;B/p65. Conversely, depletion of Brg1 and Brm disrupted the kinetics of p65 binding on CAM promoters and crippled CAM activation. Silencing of Brg1 and Brm also altered key epigenetic changes associated with CAM transactivation. Of intrigue, 17&bgr;-estradiol antagonized both the expression and activity of Brg1/Brm. Most importantly, endothelial-targeted elimination of Brg1/Brm conferred atheroprotective effects to Apoe-/- mice on a Western diet. Conclusions: Our data suggest that Brg1 and Brm integrate various proinflammatory cues into CAM transactivation and endothelial malfunction and, as such, may serve as potential therapeutic targets in treating inflammation-related cardiovascular diseases.

Deletion of yes-associated protein (YAP) specifically in cardiac and vascular smooth muscle cells reveals a crucial role for YAP in mouse cardiovascular development.

Rationale: Our previous study has shown that yes-associated protein (YAP) plays a crucial role in the phenotypic modulation of vascular smooth muscle cells (SMCs) in response to arterial injury. However, the role of YAP in vascular SMC development is unknown. Objective: The goal of this study was to investigate the functional role of YAP in cardiovascular development in mice and determine the mechanisms underlying YAP’s actions. Methods and Results: YAP was deleted in cardiomyocytes and vascular SMCs by crossing YAP flox mice with SM22&agr;-Cre transgenic mice. Cardiac/SMC-specific deletion of YAP directed by SM22&agr;-Cre resulted in perinatal lethality in mice because of profound cardiac defects including hypoplastic myocardium, membranous ventricular septal defect, and double outlet right ventricle. The cardiac/SMC-specific YAP knockout mice also displayed severe vascular abnormalities including hypoplastic arterial wall, short/absent brachiocephalic artery, and retroesophageal right subclavian artery. Deletion of YAP in mouse vascular SMCs induced expression of a subset of cell cycle arrest genes including G-protein–coupled receptor 132 (Gpr132). Silencing Gpr132 promoted SMC proliferation, whereas overexpression of Gpr132 attenuated SMC growth by arresting cell cycle in G0/G1 phase, suggesting that ablation of YAP-induced impairment of SMC proliferation was mediated, at least in part, by induction of Gpr132 expression. Mechanistically, YAP recruited the epigenetic repressor histone deacetylase-4 to suppress Gpr132 gene expression via a muscle CAT element in the Gpr132 gene. Conclusions: YAP plays a critical role in cardiac/SMC proliferation during cardiovascular development by epigenetically regulating expression of a set of cell cycle suppressors.

The Induction of Yes-Associated Protein Expression After Arterial Injury Is Crucial for Smooth Muscle Phenotypic Modulation and Neointima Formation

Objective—Abnormal proliferation and migration of vascular smooth muscle cells (SMCs) are the key events in the progression of neointima formation in response to vascular injury. The goal of this study is to investigate the functional role of a potent oncogene yes-associated protein (YAP) in SM phenotypic modulation in vitro and in vivo. Methods and Results—In vitro cell culture and in vivo in both mouse and rat arterial injury models YAP expression is significantly induced and correlated with the vascular SMC synthetic phenotype. Overexpression of YAP promotes SMC migration and proliferation while attenuating SM contractile gene expression. Conversely, knocking down endogenous YAP in SMCs upregulates SM gene expression but attenuates SMC proliferation and migration. Consistent with this, knocking down YAP expression in a rat carotid balloon injury model and genetic deletion of YAP, specifically, in vascular SMCs in mouse after carotid artery ligation injury attenuates injury-induced SM phenotypic switch and neointima formation. Conclusion—YAP plays a novel integrative role in SM phenotypic modulation by inhibiting SM-specific gene expression while promoting SM proliferation and migration in vitro and in vivo. Blocking the induction of YAP would be a potential therapeutic approach for ameliorating vascular occlusive diseases.

Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.

MicroRNA-15b/16 Attenuates Vascular Neointima Formation by Promoting the Contractile Phenotype of Vascular Smooth Muscle Through Targeting YAP

Objective—To investigate the functional role of the microRNA (miR)-15b/16 in vascular smooth muscle (SM) phenotypic modulation. Approach and Results—We found that miR-15b/16 is one of the most abundant mRs expressed in contractile vascular smooth muscle cells (VSMCs). However, when contractile VSMCs get converted to a synthetic phenotype, miR-15b/16 expression is significantly reduced. Knocking down endogenous miR-15b/16 in VSMCs attenuates SM-specific gene expression but promotes VSMC proliferation and migration. Conversely, overexpression of miR-15b/16 promotes SM contractile gene expression while attenuating VSMC migration and proliferation. Consistent with this, overexpression of miR-15b/16 in a rat carotid balloon injury model markedly attenuates injury-induced SM dedifferentiation and neointima formation. Mechanistically, we identified the potent oncoprotein yes-associated protein (YAP) as a downstream target of miR-15b/16 in VSMCs. Reporter assays validated that miR-15b/16 targets YAP’s 3′ untranslated region. Moreover, overexpression of miR-15b/16 significantly represses YAP expression, whereas conversely, depletion of endogenous miR-15b/16 results in upregulation of YAP expression. Conclusions—These results indicate that miR-15b/16 plays a critical role in SM phenotypic modulation at least partly through targeting YAP. Restoring expression of miR-15b/16 would be a potential therapeutic approach for treatment of proliferative vascular diseases.