Smooth Muscle-specific Gene Expression Research Articles

458-P: Proinflammatory Macrophage Phenotype Modulates Vascular Smooth Muscle Cell Differentiation via the Histone Demethylase JMJD3

Patients with diabetes are at increased risk of a multitude of vascular diseases, including atherosclerosis, which is driven by macrophage-mediated inflammation. While it is known that atherosclerosis is characterized by a proliferative, synthetic smooth muscle phenotype that underlies disease, the exact role of other cell types, namely myeloid cells and their interaction with smooth muscle cells is less understood. Furthermore, how diabetes modifies atherosclerotic severity is another important question. To address this, we performed an epigenetic superarray to identify key chromatin modifying enzymes in vascular smooth muscle cells (vSMC) from normal diet (ND) and diabetic-induced obese (DIO) aortas. We identified the histone demethylase, JMJD3, which was significantly decreased in DIO compared to ND vSMC (p<0.05). Furthermore, in our single cell RNA-seq data set from human atherosclerotic lesions, JMJD3 was expressed in smooth muscle cells. It is known that macrophages in atherosclerotic lesions exhibit a pro-inflammatory phenotype. To model this pro-inflammatory process, we stimulated bone marrow derived macrophages with LPS and found that supernatant from LPS-stimulated BMDMs significantly inhibited smooth muscle specific gene expression (SMA, SM22, CNN1, SM-MHC) as well as expression of JMJD3 compared to control media (p<0.05). Additionally, siRNA-mediated knockdown of JMJD3 in vSMC reduced SMC-specific gene expression, and treatment of mouse aortic vSMC with the JMJD3 selective inhibitor, GSK4J, decreased smooth muscle gene expression (p<0.05). Our data shows that pro-inflammatory macrophages drive reduced vascular smooth muscle gene expression and that JMJD3 in SMC is a regulator of this process. These results implicate JMJD3 in macrophage-smooth muscle cell crosstalk in the pro-inflammatory state and identify it as a potential therapeutic target in the management of atherosclerosis. Disclosure K.D.Mangum: None. A.Joshi: None. P.Mazumder: None. S.Wolf-fortune: None. E.Barrett: None. B.Moore: None. F.M.Davis: None. K.Gallagher: None. Funding National Institutes of Health (1F32DK131799-01); American College of Surgeons; Vascular & Endovascular Surgery Society

Silencing myostatin increases area fraction of smooth muscle in the corpus cavernosum of pigs

Myostatin (MSTN), an inhibitor of skeletal muscle growth, is also expressed in penile smooth muscle; however, it is unclear whether MSTN plays an inhibitory role in penile smooth muscle growth. We investigated the role of MSTN in the smooth muscle of the penile corpus cavernosum of pigs using MSTN homozygous mutant knockout (KO) and wild type (WT) pigs (n = 4 in each group). The mean of area fraction (%) of smooth muscle in the penile corpus cavernosum was 65.9 % ± 1.79 in the KO and approximately 41.7 % ± 5.39 in the WT (P < 0.001). KO pigs showed significantly increased expression of smooth muscle-specific genes, including smooth muscle protein 22 (TAGLN) (6.62-fold), smooth muscle myosin heavy chain (MYH11) (2.41-fold), myocardin (MYOCD) (3.05-fold), and serum response factor (SRF) (4.95-fold), and decreased expression of vimentin (VIM) (1.36-fold). Immunofluorescence staining and Western blotting showed smooth muscle-specific expression of α-smooth muscle actin (SMA) and calponin was higher in KO pigs (P < 0.05) than in WT pigs. KO pigs had less fat deposition inside the corpus cavernosum, and showed downregulation of adiponectin (ADIPOQ) and fatty acid synthase (FASN) (2.5-fold and 1.9-fold loss, respectively). In vitro experiments showed MSTN interference promoted corporal smooth muscle cell growth and expression of smooth muscle-specific markers, whereas it downregulated the expression of fat-specific genes, ADIPOQ and FASN. MSTN inhibition could promote smooth muscle growth and decrease fat deposition in the corpus cavernosum. MSTN, thus, could be a possible target for the treatment of smooth muscle dystrophy-related disorders such as erectile dysfunction.

Issue Highlights

IUBMB LifeVolume 71, Issue 7 p. 770-770 Issue HighlightsFree Access Issue Highlights First published: 20 June 2019 https://doi.org/10.1002/iub.2106AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat 845 LncRNA MIAT Facilitates Osteosarcoma Progression by Regulating miR-128-3p/VEGFC Axis Chunyan Zhang, Linsen Xie, Huiling Liang and Yuanbo Cui In this study, the results display that lncRNA MIAT is highly expressed in osteosarcoma (OS) tissues and cell lines. High expression of MIAT is closely associated with large tumor size and distant metastasis in OS patients. Knockdown of MIAT inhibits cell proliferation and metastasis, and promotes apoptosis in OS cells in vitro. MIAT promotes OS progression by functioning as a ceRNA via sponging miR-128-3p to up-regulate VEGFC in vitro. These findings may provide new insight into the roles of MIAT in the tumorigenesis of OS. 908 MicroRNA-365 Promotes the Contractile Phenotype of Venous Smooth Muscle Cells and Inhibits Neointimal Formation in Rat Vein Grafts Bo-Jun Cao, Lei Zhu, Xiao-Wen Wang, Rong-Jiang Zou and Zhi-Qian Lu MicroRNA-365 (miR-365) levels are substantially reduced in rat vein grafts at 7, 14, and 28 days post-grafting. Overexpression of miR-365 promoted smooth muscle-specific gene expression and inhibited venous SMC proliferation and migration. Consistent with this, overexpression of miR-365 in a rat vein graft model significantly reduced graftinginduced neointimal formation and effectively improved the hemodynamics of the vein grafts, suggesting its potential therapeutic application for prevention of vein graft failure. REFERENCES 1Zhang C., Xie L., Liang H. and Cui Y. LncRNA MIAT Facilitates Osteosarcoma Progression by Regulating miR-128-3p/VEGFC Axis. IUBMB Life. 2019, 71, 845– 853. 2Cao B.-J., Zhu L., Wang X.-W., Zou R.-J. and Z.-Q. Lu. MicroRNA-365 Promotes the Contractile Phenotype of Venous Smooth Muscle Cells and Inhibits Neointimal Formation in Rat Vein Grafts. IUBMB Life. 2019, 71, 908– 916. Volume71, Issue7July 2019Pages 770-770 ReferencesRelatedInformation

MicroRNA‐365 promotes the contractile phenotype of venous smooth muscle cells and inhibits neointimal formation in rat vein grafts

The high rate of autologous vein graft failure caused by neointimal hyperplasia remains an unresolved issue in the field of cardiovascular surgery; therefore, it is important to explore new methods for protecting against neointimal hyperplasia. MicroRNA-365 has been reported to inhibit the proliferation of vascular smooth muscle cells (SMCs). This study aimed to test whether adenovirus-mediated miR-365 was able to attenuate neointimal formation in rat vein grafts. We found that miR-365 expression was substantially reduced in vein grafts following engraftment. In vitro, overexpression of miR-365 promoted smooth muscle-specific gene expression and inhibited venous SMC proliferation and migration. Consistent with this, overexpression of miR-365 in a rat vein graft model significantly reduced grafting-induced neointimal formation and effectively improved the hemodynamics of the vein grafts. Mechanistically, we identified that cyclin D1 as a potential downstream target of miR-365 in vein grafts. Specially, to increase the efficiency of miR-365 gene transfection, a 30% poloxamer F-127 gel containing 0.25% trypsin was mixed with adenovirus and spread around the vein grafts to increase the adenovirus contact time and penetration. We showed that adenovirus-mediated miR-365 attenuated venous SMC proliferation and migration in vitro and effectively inhibited neointimal formation in rat vein grafts. Restoring expression of miR-365 is a potential therapeutic approach for the treatment of vein graft failure. © 2019 IUBMB Life, 2019.

Molecular Regulation of Arterial Aneurysms: Role of Actin Dynamics and microRNAs in Vascular Smooth Muscle.

Aortic aneurysms are defined as an irreversible increase in arterial diameter by more than 50% relative to the normal vessel diameter. The incidence of aneurysm rupture is about 10 in 100,000 persons per year and ruptured arterial aneurysms inevitably results in serious complications, which are fatal in about 40% of cases. There is also a hereditary component of the disease and dilation of the ascending thoracic aorta is often associated with congenital heart disease such as bicuspid aortic valves (BAV). Furthermore, specific mutations that have been linked to aneurysm affect polymerization of actin filaments. Polymerization of actin is important to maintain a contractile phenotype of smooth muscle cells enabling these cells to resist mechanical stress on the vascular wall caused by the blood pressure according to the law of Laplace. Interestingly, polymerization of actin also promotes smooth muscle specific gene expression via the transcriptional co-activator MRTF, which is translocated to the nucleus when released from monomeric actin. In addition to genes encoding for proteins involved in the contractile machinery, recent studies have revealed that several non-coding microRNAs (miRNAs) are regulated by this mechanism. The importance of these miRNAs for aneurysm development is only beginning to be understood. This review will summarize our current understanding about the influence of smooth muscle miRNAs and actin polymerization for the development of arterial aneurysms.

Similar regulatory mechanisms of caveolins and cavins by myocardin family coactivators in arterial and bladder smooth muscle.

Caveolae are membrane invaginations present at high densities in muscle and fat. Recent work has demonstrated that myocardin family coactivators (MYOCD, MKL1), which are important for contractile differentiation and cell motility, increase caveolin (CAV1, CAV2, CAV3) and cavin (CAVIN1, CAVIN2, CAVIN3) transcription, but several aspects of this control mechanism remain to be investigated. Here, using promoter reporter assays we found that both MKL1/MRTF-A and MKL2/MRTF-B control caveolins and cavins via their proximal promoter sequences. Silencing of MKL1 and MKL2 in smooth muscle cells moreover reduced CAV1 and CAVIN1 mRNA levels by well over 50%, as did treatment with second generation inhibitors of MKL activity. GATA6, which modulates expression of smooth muscle-specific genes, reduced CAV1 and CAV2, whereas the cavins were unaffected or increased. Viral overexpression of MKL1 and myocardin induced caveolin and cavin expression in bladder smooth muscle cells from rats and humans and MYOCD correlated tightly with CAV1 and CAVIN1 in human bladder specimens. A recently described activator of MKL-driven transcription (ISX) failed to induce CAV1/CAVIN1 which may be due to an unusual transactivation mechanism. In all, these findings further support the view that myocardin family coactivators are important transcriptional drivers of caveolins and cavins in smooth muscle.

Cooperative signaling by TGF-β1 and WNT-11 drives sm-α-actin expression in smooth muscle via Rho kinase-actin-MRTF-A signaling.

Airway smooth muscle (ASM) remodeling is a key feature in asthma and includes changes in smooth muscle-specific gene and protein expression. Despite this being a major contributor to asthma pathobiology, our understanding of the mechanisms governing ASM remodeling remains poor. Here, we studied the functional interaction between WNT-11 and TGF-β1 in ASM cells. We demonstrate that WNT-11 is preferentially expressed in contractile myocytes and is strongly upregulated following TGF-β1-induced myocyte maturation. Knock-down of WNT-11 attenuated TGF-β1-induced smooth muscle (sm)-α-actin expression in ASM cells. We demonstrate that TGF-β1-induced sm-α-actin expression is mediated by WNT-11 via RhoA activation and subsequent actin cytoskeletal remodeling, as pharmacological inhibition of either Rho kinase by Y27632 or actin remodeling by latrunculin A attenuated sm-α-actin induction. Moreover, we show that TGF-β1 regulates the nuclear expression of myocardin-related transcription factor-A (MRTF-A) in a Rho kinase-dependent fashion, which in turn mediates sm-α-actin expression. Finally, we demonstrate that TGF-β1-induced MRTF-A nuclear translocation is dependent on endogenous WNT-11. The present study thus demonstrates a WNT-11-dependent Rho kinase-actin-MRTF-A signaling axis that regulates the expression of sm-α-actin in ASM cells.

Increased IGF-IEc expression and mechano-growth factor production in intestinal muscle of fibrostenotic Crohn's disease and smooth muscle hypertrophy.

The igf1 gene is alternatively spliced as IGF-IEa and IGF-IEc variants in humans. In fibrostenotic Crohn's disease, the fibrogenic cytokine TGF-β1 induces IGF-IEa expression and IGF-I production in intestinal smooth muscle and results in muscle hyperplasia and collagen I production that contribute to stricture formation. Mechano-growth factor (MGF) derived from IGF-IEc induces skeletal and cardiac muscle hypertrophy following stress. We hypothesized that increased IGF-IEc expression and MGF production mediated smooth muscle hypertrophy also characteristic of fibrostenotic Crohn's disease. IGF-IEc transcripts and MGF protein were increased in muscle cells isolated from fibrostenotic intestine under regulation by endogenous TGF-β1. Erk5 and MEF2C were phosphorylated in vivo in fibrostenotic muscle; both were phosphorylated and colocalized to nucleus in response to synthetic MGF in vitro. Smooth muscle-specific protein expression of α-smooth muscle actin, γ-smooth muscle actin, and smoothelin was increased in affected intestine. Erk5 inhibition or MEF2C siRNA blocked smooth muscle-specific gene expression and hypertrophy induced by synthetic MGF. Conditioned media of cultured fibrostenotic muscle induced muscle hypertrophy that was inhibited by immunoneutralization of endogenous MGF or pro-IGF-IEc. The results indicate that TGF-β1-dependent IGF-IEc expression and MGF production in patients with fibrostenotic Crohn's disease regulates smooth muscle cell hypertrophy a critical factor that contributes to intestinal stricture formation.

The Transcription Factor TEAD1 Represses Smooth Muscle-specific Gene Expression by Abolishing Myocardin Function*♦

The TEAD (transcriptional enhancer activator domain) proteins share an evolutionarily conserved DNA-binding TEA domain, which binds to the MCAT cis-acting regulatory element. Previous studies have shown that TEAD proteins are involved in regulating the expression of smooth muscle α-actin. However, it remains undetermined whether TEAD proteins play a broader role in regulating expression of other genes in vascular smooth muscle cells. In this study, we show that the expression of TEAD1 is significantly induced during smooth muscle cell phenotypic modulation and negatively correlates with smooth muscle-specific gene expression. We further demonstrate that TEAD1 plays a novel role in suppressing expression of smooth muscle-specific genes, including smooth muscle α-actin, by abolishing the promyogenic function of myocardin, a key mediator of smooth muscle differentiation. Mechanistically, we found that TEAD1 competes with myocardin for binding to serum response factor (SRF), resulting in disruption of myocardin and SRF interactions and thereby attenuating expression of smooth muscle-specific genes. This study provides the first evidence demonstrating that TEAD1 is a novel general repressor of smooth muscle-specific gene expression through interfering with myocardin binding to SRF.

Abstract 9922: The Induction of YAP Expression Following Arterial Injury is Crucial for Smooth Muscle Phenotypic Modulation and Neointima Formation

Rationale: Abnormal proliferation and migration of vascular smooth muscle cells (SMCs) are the key events in the progression of neointimal formation in response to vascular injury. Previous studies have shown that YAP is a potent oncogene promoting cell migration and proliferation, while the role of YAP in vascular diseases remain unknown. Objective: The goal of this study is to investigate the functional role of YAP in smooth muscle phenotypic modulation in vitro and in vivo. Methods and Results: In vitro in 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. YAP is also highly expressed in human carotid atherosclerotic plaque and localized in the majority of SMCs. Over-expression of YAP promotes SMC migration and proliferation while attenuating smooth muscle contractile gene expression. Conversely, knocking-down endogenous YAP in SMCs up-regulates smooth muscle gene expression but attenuates SMC proliferation and migration. Consistent with this, knocking-down YAP expression in a rat carotid balloon injury model attenuates injury-induced smooth muscle dedifferentiation and neointima formation. Furthermore, carotid artery ligation in mice with genetic deletion of YAP specifically in SMCs leads to increase smooth muscle-specific gene expression and reduction of neointima formation. Conclusion: YAP plays a novel integrative role in smooth muscle phenotypic modulation by inhibiting smooth muscle-specific gene expression while promoting smooth muscle proliferation and migration in vitro and in vivo. Blocking the induction of YAP would be a potential therapeutic approach for ameliorating vascular occlusive diseases.

Effect of sinomenine on vascular smooth muscle cell dedifferentiation and neointima formation after vascular injury in mice

Sinomenine, a pure alkaloid extract from Sinomenium acutum, has anti-inflammatory and immunoregulatory functions. This study investigated the efficiency and the signalling pathways involved in the effect of sinomenine on vascular smooth muscle cell (VSMC) dedifferentiation in response to platelet-derived growth factor (PDGF)-BB stimulation and vascular injury. VSMCs were isolated from rat aorta and preincubated with sinomenine before being stimulated with PDGF-BB. WST and BrdU incorporation assays were used to evaluate VSMC proliferation. Flow cytometric analysis was performed for testing the cell cycle progression. The cell migration of VSMCs were analysed using a Transwell system. The expression of VSMC specific genes and signalling proteins were tested by Western blot. For the animal study, C57/BL6 mice were fed either normal rodent chow diets or sinomenine chow diets that supplemented with 0.09 % sinomenine (w/w) in the normal chows for 14 days before carotid artery wire injury. PDGF-BB activated the dedifferentiation of VSMCs characterised by decreased expression of SMA, Smoothelin and SM22α. However, sinomenine treatment preserved the dedifferentiation in response to PDGF-BB. The activations of mitogen-activated protein kinase extracellular signal-regulated kinases, Akt, GSK3β and STAT3 induced by PDGF-BB were also inhibited in sinomenine-treated VSMCs. In vivo evidence with wire-injured mice exhibited a reduction in neointimal area and an increase in smooth muscle-specific gene expression in the sinomenine-treated group. In this study, we found that sinomenine-suppressed VSMC phenotype switching induced by PDGF-BB in vitro and neointimal formation in vivo. Therefore, sinomenine is a potential candidate to be used in the treatment of vascular proliferative disease.

Gastrodin inhibits cell proliferation in vascular smooth muscle cells and attenuates neointima formation in vivo

Vascular smooth muscle cell (VSMC) proliferation plays a critical role in the development of vascular diseases. In the present study, we tested the efficacy and the mechanisms of action of gastrodin, a bioactive component of the Chinese herb Gastrodia elata Bl, in relation to platelet-derived growth factor-BB (PDGF-BB)-dependent cell proliferation and neointima formation after acute vascular injury. Cell experiments were performed with VSMCs isolated from rat aortas. WST and BrdU incorporation assays were used to evaluate VSMC proliferation. Eight-week-old C57BL/6 mice were used for the animal experiments. Gastrodin (150 mg/kg/day) was administered in the animal chow for 14 days, and the mice were subjected to wire injury of the left carotid artery. Our data demonstrated that gastrodin attenuated the VSMC proliferation induced by PDGF-BB, as assessed by WST assay and BrdU incorporation. Gastrodin influenced the S-phase entry of VSMCs and stabilised p27Kip1 expression. In addition, pre-incubation with sinomenine prior to PDGF-BB stimulation led to increased smooth muscle-specific gene expression, thereby inhibiting VSMC dedifferentiation. Gastrodin treatment also reduced the intimal area and the number of PCNA-positive cells. Furthermore, PDGF-BB-induced phosphorylation of ERK1/2, p38 MAPK, Akt and GSK3β was suppressed by gastrodin. Our results suggest that gastrodin can inhibit VSMC proliferation and attenuate neointimal hyperplasia in response to vascular injury. Furthermore, the ERK1/2, p38 MAPK and Akt/GSK3β signalling pathways were found to be involved in the effects of gastrodin.

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

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.

Interferon-γ-mediated Inhibition of Serum Response Factor-dependent Smooth Muscle-specific Gene Expression

IFNγ exerts multiple biological effects on effector cells by regulating many downstream genes, including smooth muscle-specific genes. However, the molecular mechanisms underlying IFNγ-induced inhibition of smooth muscle-specific gene expression remain unclear. In this study, we have shown that serum response factor (SRF), a common transcriptional factor important in cell proliferation, migration, and differentiation, is targeted by IFNγ in a STAT1-dependent manner. We show that the molecular mechanism by which IFNγ regulates SRF is via activation of the 2-5A-RNase L system, which triggers SRF mRNA decay and reduced SRF expression. As a result, decreased SRF expression reduces expression of SRF target genes such as smooth muscle α-actin and smooth muscle myosin heavy chain. Additionally, IFNγ reduced p300 and acetylated histone-3 binding in both smooth muscle α-actin and SRF promoters, epigenetically decreasing smooth muscle α-actin and SRF transcriptional activation. Our data reveal that SRF is a novel IFNγ-regulated gene and further elucidate the molecular pathway between IFNγ, IFNγ-regulated genes, and SRF and its target genes.

Repression of Smooth Muscle Differentiation by a Novel High Mobility Group Box-containing Protein, HMG2L1

The molecular mechanisms regulating smooth muscle-specific gene expression during smooth muscle development are poorly understood. Myocardin is an extraordinarily powerful cofactor of serum response factor (SRF) that stimulates expression of smooth muscle-specific genes. In an effort to search for proteins that regulate myocardin function, we identified a novel HMG box-containing protein HMG2L1 (high mobility group 2 like 1). We found that HMG2L1 expression is correlated with the smooth muscle cell (SMC) synthetic phenotype. Overexpression of HMG2L1 in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous HMG2L1 in SMCs increases smooth muscle-specific gene expression. Furthermore, we found HMG2L1 specifically abrogates myocardin-induced activation of smooth muscle-specific genes. By GST pulldown assays, the interaction domains between HMG2L1 and myocardin were mapped to the N termini of each of the proteins. Finally, we demonstrated that HMG2L1 abrogates myocardin function through disrupting its binding to SRF and abolishing SRF-myocardin complex binding to the promoters of smooth muscle-specific genes. This study provides the first evidence of this novel HMG2L1 molecule playing an important role in attenuating smooth muscle differentiation.

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.

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.

Abstract 5431: Activation of Myocardin Function by an Ubiquitin E3 Ligase, EDD

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of contractile proteins. The expression of these proteins is markedly attenuated during the de-differentiation of smooth muscle that occurs under many pathological conditions. SRF is a transcription factor that plays a central role in the expression of these smooth muscle-specific genes through physical association with various cell-restricted or signal dependent accessory factors. Of the SRF-associated proteins identified, myocardin is the most potent for stimulating expression of smooth muscle-specific genes. Although several proteins have been identified that can modify myocardin function, the mechanism of how myocardin regulation is still poorly understood. To identify additional myocardin associated proteins that are required for myocardin-dependent activation smooth muscle-specific genes we performed a yeast 2-hybrid screen of mouse embryo cDNA library using myocardin as bait. From this screen we identified a HECT domain containing protein EDD 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. EDD has however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that the amino-terminal domain of myocardin bound to the HECT domain of EDD. We also show that EDD specifically enhanced trans-activation of smooth muscle-specific promoters by myocardin. Furthermore, EDD significantly augmented myocardin’s ability to induce expression of endogenous SMC marker genes, and these effects of EDD were independent on its E3 ligase function. Conversely, depletion of endogenous EDD in fibroblast cells by RNAi attenuated myocardin-induced smooth muscle-specific gene expression, and EDD knockdown in smooth muscle cells resulted in down-regulation of smooth muscle-specific genes. Ongoing experiments are investigating the mechanism by which EDD activates myocardin function. This study reveals an unexpected role for the ubiquitin E3 ligase EDD as an activator of smooth muscle differentiation through its ability to potentiate myocardin function. This research has received full or partial funding support from the American Heart Association, National Center.

PKA-Dependent Phosphorylation of Serum Response Factor Inhibits Smooth Muscle–Specific Gene Expression

Our goal was to identify phosphorylation sites that regulate serum response factor (SRF) activity to gain a better understanding of the signaling mechanisms that regulate SRF's involvement in smooth muscle cell (SMC)-specific and early response gene expression. By screening phosphorylation-deficient and mimetic mutations in SRF(-/-) embryonic stem cells, we identified T159 as a phosphorylation site that significantly inhibits SMC-specific gene expression in an embryonic stem cell model of SMC differentiation. This residue conforms to a highly conserved consensus cAMP-dependent protein kinase (PKA) site, and in vitro and in vivo labeling studies demonstrated that it was phosphorylated by PKA. Results from gel shift and chromatin immunoprecipitation assays demonstrated that T159 phosphorylation inhibited SRF binding to SMC-specific CArG elements. Interestingly, the myocardin factors could at least partially rescue the effects of the T159D mutation under some conditions, but this response was promoter specific. Finally, PKA signaling had much less of an effect on c-fos promoter activity and SRF binding to the c-fos CArG. Our results indicate that phosphorylation of SRF by PKA inhibits SMC-specific transcription suggesting a novel signaling mechanism for the control of SMC phenotype.

Regulation of smooth muscle-specific gene expression by cGMP-dependent protein kinase

Background Vascular smooth muscle cells (VSMCs) in normal adult blood vessels exhibit a differentiated, phenotype, but in atherosclerotic disease and after acute vascular injury, they de-differentiate into a proliferative, synthetic phenotype; the latter is characterized by decreased smooth muscle (SM)-specific gene expression and decreased cGMP-dependent protein kinase (PKG) activity [1,2]. Prolonged in vitro culture of primary aortic VSMCs, or exposure of pulmonary VSMCs to hypoxia also leads to de-differentiation associated with loss of PKG, and restoring PKG activity increases SM-specific gene expression and re-differentiates cells into the contractile phenotype, but the mechanism(s) whereby PKG regulates SM genes are poorly understood [2,3].