Sall1 and Sall4 cooperatively interact with Myocd and SRF to promote cardiomyocyte proliferation by regulating CDK and cyclin genes.

Sall1 and Sall4 (Sall1/4), zinc-finger transcription factors, are expressed in the progenitors of the second heart field (SHF) and in cardiomyocytes during the early stages of mouse development. To understand the function of Sall1/4 in heart development, we generated heart-specific Sall1/4 functionally inhibited mice by forced expression of the truncated form of Sall4 (ΔSall4) in the heart. The ΔSall4-overexpression mice exhibited a hypoplastic right ventricle and outflow tract, both of which were derived from the SHF, and a thinner ventricular wall. We found that the numbers of proliferative SHF progenitors and cardiomyocytes were reduced in ΔSall4-overexpression mice. RNA-sequencing data showed that Sall1/4 act upstream of the cyclin-dependent kinase (CDK) and cyclin genes, and of key transcription factor genes for the development of compact cardiomyocytes, including myocardin (Myocd) and serum response factor (Srf). In addition, ChIP-sequencing and co-immunoprecipitation analyses revealed that Sall4 and Myocd form a transcriptional complex with SRF, and directly bind to the upstream regulatory regions of the CDK and cyclin genes (Cdk1 and Ccnb1). These results suggest that Sall1/4 are critical for the proliferation of cardiac cells via regulation of CDK and cyclin genes that interact with Myocd and SRF.

Notch signaling is a novel regulator of visceral smooth muscle cell differentiation in the murine ureter.

The contractile phenotype of smooth muscle cells (SMCs) is transcriptionally controlled by a complex of the DNA-binding protein SRF and the transcriptional co-activator MYOCD. The pathways that activate expression of Myocd and of SMC structural genes in mesenchymal progenitors are diverse, reflecting different intrinsic and extrinsic signaling inputs. Taking the ureter as a model, we analyzed whether Notch signaling, a pathway previously implicated in vascular SMC development, also affects visceral SMC differentiation. We show that mice with a conditional deletion of the unique Notch mediator RBPJ in the undifferentiated ureteric mesenchyme exhibit altered ureter peristalsis with a delayed onset, and decreased contraction frequency and intensity at fetal stages. They also develop hydroureter 2 weeks after birth. Notch signaling is required for precise temporal activation of Myocd expression and, independently, for expression of a group of late SMC structural genes. Based on additional expression analyses, we suggest that a mesenchymal JAG1-NOTCH2/NOTCH3 module regulates visceral SMC differentiation in the ureter in a biphasic and bimodal manner, and that its molecular function differs from that in the vascular system.

GATA6 is a crucial factor for Myocd expression in the visceral smooth muscle cell differentiation program of the murine ureter.

Smooth muscle cells (SMCs) are a crucial component of the mesenchymal wall of the ureter, as they account for the efficient removal of the urine from the renal pelvis to the bladder by means of their contractile activity. Here, we show that the zinc-finger transcription factor gene Gata6 is expressed in mesenchymal precursors of ureteric SMCs under the control of BMP4 signaling. Mice with a conditional loss of Gata6 in these precursors exhibit a delayed onset and reduced level of SMC differentiation and peristaltic activity, as well as dilatation of the ureter and renal pelvis (hydroureternephrosis) at birth and at postnatal stages. Molecular profiling revealed a delayed and reduced expression of the myogenic driver gene Myocd, but the activation of signaling pathways and transcription factors previously implicated in activation of the visceral SMC program in the ureter was unchanged. Additional gain-of-function experiments suggest that GATA6 cooperates with FOXF1 in Myocd activation and SMC differentiation, possibly as pioneer and lineage-determining factors, respectively.

The Function of RhoA/ROCK Pathway and MYOCD in Airway Remodeling in Asthma.

INTRODUCTION: Asthma is a common chronic respiratory disease characterized by chronic airway inflammation and abnormal airway remodeling. The RhoA/ROCK pathway and myocardin-related transcription factor A (MRTF-A) demonstrate significant associations with the proliferation of airway smooth muscle cells (ASCMs), which tightly correlates with the process of airway remodeling. MYOCD, which is homologous to MRTF-A but specifically expressed in smooth muscle cells, potentially regulates RhoA/ROCK activated cell proliferation and subsequent airway remodeling. METHODS: The RhoA/ROCK overexpression and silencing cell lines were constructed in vitro, as well as MYOCD overexpression/silencing. The cytoskeleton alterations induced by RhoA/ROCK pathway were identified by the measuring of globular actin and filamentous actin. RESULTS: The comparison between controls for overexpression/silencing and ROCK overexpression/silencing revealed that MYOCD presented consistent change trends with cytoskeleton and RhoA/ROCK pathway. The ROCK1 facilitates the proliferation and migration of ASCMs. The MYOCD enhanced the proliferation and migration of HASMCs. CONCLUSION: Our study indicates that Rho/ROCK/MYOCD is a key pathway involved in the migration and proliferation of airway smooth muscle cells. Inhibition of Rho/ROCK may be an effective approach to breaking the vicious cycle of asthmatic ASCMs proliferation, providing a novel strategy in treating asthma airway remodeling.

Myocd regulates airway smooth muscle cell remodeling in response to chronic asthmatic injury.

Abnormal growth of airway smooth muscle cells is one of the key features in asthmatic airway remodeling, which is associated with asthma severity. The mechanisms underlying inappropriate airway smooth muscle cell growth in asthma remain largely unknown. Myocd has been reported to act as a key transcriptional coactivator in promoting airway-specific smooth muscle development in fetal lungs. Whether Myocd controls airway smooth muscle remodeling in asthma has not been investigated. Mice with lung mesenchyme-specific deletion of Myocd after lung development were generated, and a chronic asthma model was established by sensitizing and challenging the mice with ovalbumin for a prolonged period. Comparison of the asthmatic pathology between the Myocd knockout mice and the wild-type controls revealed that abrogation of Myocd mitigated airway smooth muscle cell hypertrophy and hyperplasia, accompanied by reduced peri-airway inflammation, decreased fibrillar collagen deposition on airway walls, and attenuation of abnormal mucin production in airway epithelial cells. Our study indicates that Myocd is a key transcriptional coactivator involved in asthma airway remodeling. Inhibition of Myocd in asthmatic airways may be an effective approach to breaking the vicious cycle of asthmatic progression, providing a novel strategy in treating severe and persistent asthma. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Smooth Muscle Cell Phenotypic Switch Induced by Traditional Cigarette Smoke Condensate: A Holistic Overview.

Cigarette smoke (CS) is a risk factor for inflammatory diseases, such as atherosclerosis. CS condensate (CSC) contains lipophilic components that may represent a systemic cardiac risk factor. To better understand CSC effects, we incubated mouse and human aortic smooth muscle cells (SMCs) with CSC. We evaluated specific markers for contractile [i.e., actin, aortic smooth muscle (ACTA2), calponin-1 (CNN1), the Kruppel-like factor 4 (KLF4), and myocardin (MYOCD) genes] and inflammatory [i.e., IL-1ß, and IL-6, IL-8, and galectin-3 (LGALS-3) genes] phenotypes. CSC increased the expression of inflammatory markers and reduced the contractile ones in both cell types, with KLF4 modulating the SMC phenotypic switch. Next, we performed a mass spectrometry-based differential proteomic approach on human SMCs and could show 11 proteins were significantly affected by exposition to CSC (FC ≥ 2.7, p ≤ 0.05). These proteins are active in signaling pathways related to expression of pro-inflammatory cytokines and IFN, inflammasome assembly and activation, cytoskeleton regulation and SMC contraction, mitochondrial integrity and cellular response to oxidative stress, proteostasis control via ubiquitination, and cell proliferation and epithelial-to-mesenchymal transition. Through specific bioinformatics resources, we showed their tight functional correlation in a close interaction niche mainly orchestrated by the interferon-induced double-stranded RNA-activated protein kinase (alternative name: protein kinase RNA-activated; PKR) (EIF2AK2/PKR). Finally, by combining gene expression and protein abundance data we obtained a hybrid network showing reciprocal integration of the CSC-deregulated factors and indicating KLF4 and PKR as the most relevant factors.

Rho/ROCK-MYOCD in regulating airway smooth muscle growth and remodeling.

Abnormal airway remodeling is a common pathological change seen in chronic respiratory diseases. Altered proliferation and differentiation of airway smooth muscle cells (ASMCs) are the major components of airway remodeling, and the resultant structural abnormalities are difficult to restore. Understanding of airway smooth muscle regulation is urgently needed to identify potential intervention targets. MYOCD (or myocardin) and myocardin-related transcription factors (MRTFs) are key cotranscription factors in muscle growth, which have not been extensively investigated in airway smooth muscle cells. In addition, the RhoA/ROCK signaling pathway is known to play an important role in airway remodeling partly through regulating the proliferation and differentiation of ASMCs, which may be connected with MYOCD/MRTF cotranscription factors [Kumawat et al. (Am J Physiol Lung Cell Mol Physiol 311: L529-L537, 2016); Lagna et al. (J Biol Chem 282: 37244-37255, 2007)]. This review focuses on this newly recognized and potentially important RhoA/ROCK-MYOCD/MRTFs pathway in controlling airway smooth muscle growth and remodeling.

Requirement of miR-9-dependent regulation of Myocd in PASMCs phenotypic modulation and proliferation induced by hepatopulmonary syndrome rat serum.

Hepatopulmonary syndrome (HPS) is characterized by a triad of severe liver disease, intrapulmonary vascular dilation and hypoxaemia. Pulmonary vascular remodelling (PVR) is a key feature of HPS pathology. Our previous studies have established the role of the pulmonary artery smooth muscle cell (PASMC) phenotypic modulation and proliferation in HPS-associated PVR. Myocardin, a robust transcriptional coactivator of serum response factor, plays a critical role in the vascular smooth muscle cell phenotypic switch. However, the mechanism regulating myocardin upstream signalling remains unclear. In this study, treatment of rat PASMCs with serum drawn from common bile duct ligation rats, which model symptoms of HPS, resulted in a significant increase in miR-9 expression correlated with a decrease in expression of myocardin and the phenotypic markers SM-α-actin and smooth muscle-specific myosin heavy chain (SM-MHC). Furthermore, miRNA functional analysis and luciferase reporter assay demonstrated that miR-9 effectively regulated myocardin expression by directly binding to its 3'-untranslated region. Both the knockdown of miR-9 and overexpression of myocardin effectively attenuated the HPS rat serum-induced phenotype switch and proliferation of PASMCs. Taken together, the findings of our present study demonstrate that miR-9 is required in HPS rat serum-induced phenotypic modulation and proliferation of PASMCs for targeting of myocardin and that miR-9 may serve as a potential therapeutic target in HPS.

MYOCD and SRF-mediated MLCK transcription prevents polymorphonuclear neutrophils from ferroptosis in sepsis-related acute lung injury.

Persistent activation of polymorphonuclear neutrophils (PMNs) plays a crucial role in the development of sepsis-related acute lung injury (ALI). This study investigated key molecular mechanisms involved in the hyperactivation of PMNs during ALI. A mouse model of sepsis-related ALI was generated by lipopolysaccharide (LPS) injection. RNA sequencing identified myosin light chain kinase (MLCK) as the most significant differentially expressed gene (DEG) between PMNs isolated from model and control mice. Myocardin (MYOCD) and serum response factor (SRF) were two of the DEGs that could promote transcription of MLCK by binding to its promoter. Either knockdown of MLCK, MYOCD, or SRF ameliorated dysfunction and edema in the lungs of LPS-treated mice. Kyoto Encyclopedia of Genes and Genomes enrichment analysis suggested that the DEGs are enriched in a ferroptosis-related signaling pathway. The MLCK, MYOCD, or SRF knockdown increased contents of ROS, MDA, ferritin, and ferrous iron, and reduced levels of GSH and GPX4 in the PMNs. However, the MLCK overexpression restored ferroptosis resistance and activity of the PMNs, resulting in increased lung injury. Collectively, this study demonstrates that MYOCD and SRF-mediated MLCK upregulation is correlated with ferroptosis resistance and hyperactivation of PMNs in sepsis-related ALI.

Actin-related protein 5 suppresses the cooperative activation of cardiac gene transcription by myocardin and MEF2.

MYOCD is a transcription factor important for cardiac and smooth muscle development. We previously identified that actin-related protein 5 (ARP5) binds to the N-terminus of MYOCD. Here, we demonstrate that ARP5 inhibits the cooperative action of the cardiac-specific isoform of MYOCD with MEF2. ARP5 overexpression in murine hearts induced cardiac hypertrophy and fibrosis, whereas ARP5 knockdown in P19CL6 cells significantly increased cardiac gene expression. ARP5 was found to bind to a MEF2-binding motif of cardiac MYOCD and inhibit MEF2-mediated transactivation by MYOCD. RNA-seq analysis revealed 849 genes that are upregulated by MYOCD-MEF2 and 650 genes that are repressed by ARP5. ARP5 expression increased with cardiomyopathy and was negatively correlated with the expression of Tnnt2 and Ttn, which were regulated by cardiac MYOCD-MEF2. Overall, our data suggest that ARP5 is a potential suppressor of cardiac MYOCD during physiological and pathological processes.

MYOCD is Required for Cardiomyocyte-like Cells Induction from Human Urine Cells and Fibroblasts Through Remodeling Chromatin.

Despite direct reprogramming of human cardiac fibroblasts into induced cardiomyocytes (iCM) holds great potential for heart regeneration, the mechanisms are poorly understood. Whether other human somatic cells could be reprogrammed into cardiomyocytes is also unknown. Here, we report human urine cells (hUCs) could be converted into CM-like cells from different donors and the related chromatin accessibility dynamics (CAD) by assay for transposase accessible chromatin(ATAC)-seq. hUCs transduced by MEF2C, TBX5, MESP1 and MYOCD but without GATA4 expressed multiple cardiac specific genes, exhibited Ca2+ oscillation potential and sarcomeric structures, and contracted synchronously in coculture with mouse CM. Additionally, we found that MYOCD is required for both closing and opening critical loci, mainly by hindering the opening of loci enriched with motifs for the TEAD and AP1 family and promoting the closing of loci enriched with ETS motifs. These changes differ partially from CAD observed during iCM induction from human fibroblasts. Collectively, our study offers one practical platform for iCM generation and insights into mechanisms for iCM fate determination.

Role of smooth muscle YAP and TAZ in protection against phenotypic modulation, inflammation, and aneurysm development.

A ruptured arterial aneurysm, especially in the aorta, represents one of the most acute and mortal conditions encountered in clinical medicine. Population-based screening in elderly men, treatment of risk factors, such as hypertension, and endovascular or open repair of rupture-prone lesions, represent cornerstones in management. Surgical repair has a sizeable effect on life-expectancy, but medical therapy that retards aneurysm growth still represents a considerable and unmet clinical need. In the current review we survey recent findings implicating the mechano-responsive transcriptional co-activators YAP and TAZ in protection from aneurysm development. Arteries from mouse mutants that lack YAP and TAZ in vascular smooth muscle respond inadequately to mechanical stimulation, and they develop aneurysms characterized by elastin fragmentation, proteoglycan infiltration, and severe inflammation at breathtaking speed. This seems to be due, at least in part, to unscheduled activation of STING (stimulator of interferon genes), an arm of innate immunity that responds to double-stranded DNA in the cytoplasm. YAP and TAZ protect from STING activation by securing nuclear integrity. These novel insights suggest unanticipated medical therapies for sporadic and genetic aneurysms alike, involving inhibition of kinases in the Hippo pathway using small molecules, or inhibition of STING signaling itself. Translation of these novel findings into clinical therapies now represents an important priority.

Fusion of the Paired Box 3 (PAX3) and Myocardin (MYOCD) Genes in Pediatric Rhabdomyosarcoma.

BACKGROUND/AIM: Fusions of the paired box 3 gene (PAX3 in 2q36) with different partners have been reported in rhabdomyosarcomas and biphenotypic sinonasal sarcomas. We herein report the myocardin (MYOCD on 17p12) gene as a novel PAX3-fusion partner in a pediatric tumor with adverse clinical outcome. MATERIALS AND METHODS: A rhabdomyo-sarcoma found in a 10-year-old girl was studied using a range of genetic methodologies. RESULTS: The karyotype of the tumor cells was 48,XX,add(2)(q11),+del(2)(q35),add(3)(q?25),-7, del(8)(p 21),-15, add(17)(p 11), + 20, +der(?) t(?; 15) (?;q15),+mar[8]/46,XX[2]. Fluorescence in situ hybridization detected PAX3 rearrangement whereas array comparative genomic hybridization revealed genomic imbalances affecting hundreds of genes, including MYCN, MYC, FOXO3, and the tumor suppressor gene TP53. A PAX3-MYOCD fusion transcript was found by RNA sequencing and confirmed by Sanger sequencing. CONCLUSION: The investigated rhabdomyosarcoma carried a novel PAX3-MYOCD fusion gene and extensive additional aberrations affecting the allelic balance of many genes, among them TP53 and members of MYC and FOXO families of transcription factors.

Myocardin suppression increases lipid retention and atherosclerosis via downregulation of ABCA1 in vascular smooth muscle cells.

Myocardin (MYOCD) plays an important role in cardiovascular disease. However, its underlying impact on atherosclerosis remains to be elucidated. ATP binding cassette transporter A1 (ABCA1), a key membrane-associated lipid transporter which maintains intracellular lipid homeostasis, has a protective function in atherosclerosis progress. The purpose of this study was to investigate whether and how the effect of MYOCD on atherosclerosis is associated with ABCA1 in vascular smooth muscle cells (VSMCs). We found both MYOCD and ABCA1 expression were dramatically decreased in atherosclerotic patient aortas compared to control. MYOCD knockdown inhibited ABCA1 expression in human aortic vascular smooth muscle cells (HAVSMCs), leading to reduced cholesterol efflux and increased intracellular cholesterol contents. MYOCD overexpression exerted the opposite effect. Mechanistically, MYOCD regulates ABCA1 expression in an SRF-dependent manner. Consistently, apolipoprotein E-deficient mice treated with MYOCD shRNA developed more plaques in the aortic sinus, which is associated with reduced ABCA1 expression, increased cholesterol retention in the aorta, and decreased high-density lipoprotein cholesterol levels in the plasma. Our data suggest that MYOCD deficiency exacerbates atherosclerosis by downregulating ABCA1 dependent cholesterol efflux from VSMCs, thereby providing a novel strategy for the therapeutic treatment of atherosclerotic cardiovascular disease.

Sall4 and Myocd Empower Direct Cardiac Reprogramming From Adult Cardiac Fibroblasts After Injury.

Direct conversion of fibroblasts into induced cardiomyocytes (iCMs) holds promising potential to generate functional cardiomyocytes for drug development and clinical applications, especially for direct in situ heart regeneration by delivery of reprogramming genes into adult cardiac fibroblasts in injured hearts. For a decade, many cocktails of transcription factors have been developed to generate iCMs from fibroblasts of different tissues in vitro and some were applied in vivo. Here, we aimed to develop genetic cocktails that induce cardiac reprogramming directly in cultured cardiac fibroblasts isolated from adult mice with myocardial infarction (MICFs), which could be more relevant to heart diseases. We found that the widely used genetic cocktail, Gata4, Mef2c, and Tbx5 (GMT) were inefficient in reprogramming cardiomyocytes from MICFs. In a whole well of a 12-well plate, less than 10 mCherry+ cells (<0.1%) were observed after 2 weeks of GMT infection with Myh6-reporter transgenic MICFs. By screening 22 candidate transcription factors predicted through analyzing the gene regulatory network of cardiac development, we found that five factors, GMTMS (GMT plus Myocd and Sall4), induced more iCMs expressing the cardiac structural proteins cTnT and cTnI at a frequency of about 22.5 ± 2.7% of the transduced MICFs at day 21 post infection. What is more, GMTMS induced abundant beating cardiomyocytes at day 28 post infection. Specifically, Myocd contributed mainly to inducing the expression of cardiac proteins, while Sall4 accounted for the induction of functional properties, such as contractility. RNA-seq analysis of the iCMs at day 28 post infection revealed that they were reprogrammed to adopt a cardiomyocyte-like gene expression profile. Overall, we show here that Sall4 and Myocd play important roles in cardiac reprogramming from MICFs, providing a cocktail of genetic factors that have potential for further applications in in vivo cardiac reprogramming.

Smooth Muscle Differentiation Is Essential for Airway Size, Tracheal Cartilage Segmentation, but Dispensable for Epithelial Branching.

Airway smooth muscle is best known for its role as an airway constrictor in diseases such as asthma. However, its function in lung development is debated. A prevalent model, supported by in vitro data, posits that airway smooth muscle promotes lung branching through peristalsis and pushing intraluminal fluid to branching tips. Here, we test this model in vivo by inactivating Myocardin, which prevented airway smooth muscle differentiation. We found that Myocardin mutants show normal branching, despite the absence of peristalsis. In contrast, tracheal cartilage, vasculature, and neural innervation patterns were all disrupted. Furthermore, airway diameter is reduced in the mutant, counter to the expectation that the absence of smooth muscle constriction would lead to a more relaxed and thereby wider airway. These findings together demonstrate that during development, while airway smooth muscle is dispensable for epithelial branching, it is integral for building the tracheal architecture and promoting airway growth.

Efficient differentiation of vascular smooth muscle cells from Wharton's Jelly mesenchymal stromal cells using human platelet lysate: A potential cell source for small blood vessel engineering.

BACKGROUND: The development of fully functional small diameter vascular grafts requires both a properly defined vessel conduit and tissue-specific cellular populations. Mesenchymal stromal cells (MSCs) derived from the Wharton's Jelly (WJ) tissue can be used as a source for obtaining vascular smooth muscle cells (VSMCs), while the human umbilical arteries (hUAs) can serve as a scaffold for blood vessel engineering. AIM: To develop VSMCs from WJ-MSCs utilizing umbilical cord blood platelet lysate. METHODS: WJ-MSCs were isolated and expanded until passage (P) 4. WJ-MSCs were properly defined according to the criteria of the International Society for Cell and Gene Therapy. Then, these cells were differentiated into VSMCs with the use of platelet lysate from umbilical cord blood in combination with ascorbic acid, followed by evaluation at the gene and protein levels. Specifically, gene expression profile analysis of VSMCs for ACTA2, MYH11, TGLN, MYOCD, SOX9, NANOG homeobox, OCT4 and GAPDH, was performed. In addition, immunofluorescence against ACTA2 and MYH11 in combination with DAPI staining was also performed in VSMCs. HUAs were decellularized and served as scaffolds for possible repopulation by VSMCs. Histological and biochemical analyses were performed in repopulated hUAs. RESULTS: WJ-MSCs exhibited fibroblastic morphology, successfully differentiating into "osteocytes", "adipocytes" and "chondrocytes", and were characterized by positive expression (> 90%) of CD90, CD73 and CD105. In addition, WJ-MSCs were successfully differentiated into VSMCs with the proposed differentiation protocol. VSMCs successfully expressed ACTA2, MYH11, MYOCD, TGLN and SOX9. Immunofluorescence results indicated the expression of ACTA2 and MYH11 in VSMCs. In order to determine the functionality of VSMCs, hUAs were isolated and decellularized. Based on histological analysis, decellularized hUAs were free of any cellular or nuclear materials, while their extracellular matrix retained intact. Then, repopulation of decellularized hUAs with VSMCs was performed for 3 wk. Decellularized hUAs were repopulated efficiently by the VSMCs. Biochemical analysis revealed the increase of total hydroyproline and sGAG contents in repopulated hUAs with VSMCs. Specifically, total hydroxyproline and sGAG content after the 1st, 2nd and 3rd wk was 71 ± 10, 74 ± 9 and 86 ± 8 µg hydroxyproline/mg of dry tissue weight and 2 ± 1, 3 ± 1 and 3 ± 1 µg sGAG/mg of dry tissue weight, respectively. Statistically significant differences were observed between all study groups (P < 0.05). CONCLUSION: VSMCs were successfully obtained from WJ-MSCs with the proposed differentiation protocol. Furthermore, hUAs were efficiently repopulated by VSMCs. Differentiated VSMCs from WJ-MSCs could provide an alternative source of cells for vascular tissue engineering.

Early growth response 2 (Egr2) plays opposing roles in committing C3H10T1/2 stem cells to adipocytes and smooth muscle-like cells.

Early growth response 2 (Egr2) is a zinc-finger transcription factor that acts as an important modulator of a variety of physiological processes, such as cell differentiation, proliferation and apoptosis. Here we showed that Egr2 was downregulated by bone morphogenetic protein (BMP) signaling pathways during the commitment of C3H10T1/2 stem cells to adipocyte lineage. Overexpression of Egr2 completely prevented BMP4-induced adipocyte lineage commitment of C3H10T1/2 stem cells, while simultaneously stimulating early smooth muscle-like differentiation. We also demonstrated that Egr2-induced early smooth muscle-like differentiation is transforming growth factor ß1-independent. Egr2 can activate the transcription of early smooth muscle cell specific genes smooth muscle protein 22α and calponin 1. Together, the results indicated a novel role for Egr2 in repressing adipocyte lineage commitment and promoting early smooth muscle-like cell differentiation.