Miano JM, Long X, Fujiwara K. Serum response factor: master regulator of the actin cytoskeleton and contractile apparatus.

Serum response factor (SRF) binds a 1216-fold degenerate cis element known as the CArG box. CArG boxes are found primarily in muscle- and growth-factor-associated genes although the full spectrum of functional CArG elements in the genome (the CArGome) has yet to be defined. Here we describe a genome-wide screen to further define the functional mammalian CArGome. A computational approach involving comparative genomic analyses of human and mouse orthologous genes uncovered >100 hypothetical SRF-dependent genes, including 10 previously identified SRF targets, harboring a conserved CArG element within 4000 bp of the annotated transcription start site (TSS). We PCR-cloned 89 hypothetical SRF targets and subjected each of them to at least two of several validations including luciferase reporter, gel shift, chromatin immunoprecipitation, and mRNA expression following RNAi knockdown of SRF; 60/89 (67%) of the targets were validated. Interestingly, 26 of the validated SRF target genes encode for cytoskeletal/contractile or adhesion proteins. RNAi knockdown of SRF diminishes expression of several SRF-dependent cytoskeletal genes and elicits an attending perturbation in the cytoarchitecture of both human and rodent cells. These data illustrate the power of integrating existing algorithms to interrogate the genome in a relatively unbiased fashion for cis-regulatory element discovery. In this manner, we have further expanded the mammalian CArGome with the discovery of an array of cyto-contractile genes that coordinate normal cytoskeletal homeostasis. We suggest one function of SRF is that of an ancient master regulator of the actin cytoskeleton.

Objective We previously showed that cholesterol loading in vitro converts mouse aortic vascular smooth muscle cells (VSMC) from a contractile state to one resembling macrophages. In human and mouse atherosclerotic plaques it has become appreciated that ~40% of cells classified as macrophages by histological markers may be of VSMC origin. We therefore sought to gain insight into the molecular regulation of this clinically relevant process. Approach and Results VSMC of mouse (or human) origin were incubated with cyclodextrin-cholesterol complexes for 72 hours, at which time the expression at the protein and mRNA levels of contractile-related proteins were reduced and of macrophage markers increased. Concurrent was down regulation of miR-143/145, which positively regulate the master VSMC-differentiation transcription factor myocardin (MYOCD). Mechanisms were further probed in mouse VSMC. Maintaining the expression of MYOCD or miR-143/145 prevented and reversed phenotypic changes caused by cholesterol loading. Reversal was also seen when cholesterol efflux was stimulated after loading. Notably, despite expression of macrophage markers, bioinformatic analyses showed that cholesterol-loaded cells remained closer to the VSMC state, consistent with impairment in classical macrophage functions of phagocytosis and efferocytosis. In apoE-deficient atherosclerotic plaques, cells positive for VSMC and macrophage markers were found lining the cholesterol-rich necrotic core. Conclusions Cholesterol loading of VSMC converts them to a macrophage–appearing state by downregulating the miR-143/145-myocardin axis. Though these cells would be classified by immunohistochemistry as macrophages in human and mouse plaques, their transcriptome and functional properties imply that their contributions to atherogenesis would not be those of classical macrophages.

Amyloid β-peptide (Aβ) deposition in cerebral vessels contributes to cerebral amyloid angiopathy (CAA) in Alzheimer’s disease (AD). Here, we report that in AD patients and two mouse models of AD, overexpression of serum response factor (SRF) and myocardin (MYOCD) in cerebral vascular smooth muscle cells (VSMCs) generates an Aβ non-clearing VSMC phenotype through transactivation of sterol regulatory element binding protein-2, which downregulates low density lipoprotein receptor-related protein-1, a key Aβ clearance receptor. Hypoxia stimulated SRF/MYOCD expression in human cerebral VSMCs and in animal models of AD. We suggest that SRF and MYOCD function as a transcriptional switch, controlling Aβ cerebrovascular clearance and progression of AD.

Objective Long non-coding RNAs (lncRNAs) represent a rapidly growing class of RNA genes with functions related primarily to transcriptional and post-transcriptional control of gene expression. There is a paucity of information about lncRNA expression and function in human vascular cells. Thus, we set out to identify novel lncRNA genes in human vascular smooth muscle cells and to gain insight into their role in the control of smooth muscle cell phenotypes. Approach and Results RNA-sequencing of human coronary artery smooth muscle cells revealed 31 unannotated lncRNAs, including a vascular cell-enriched lncRNA we call SENCR (Smooth muscle and Endothelial cell enriched migration/differentiation-associated long Non-Coding RNA). Strand-specific RT-PCR and rapid amplification of cDNA ends indicate that SENCR is transcribed antisense from the 5’ end of the FLI1 gene and exists as two splice variants. RNA fluorescence in situ hybridization and biochemical fractionation studies demonstrate SENCR is a cytoplasmic lncRNA. Consistent with this observation, knockdown studies reveal little to no cis-acting effect of SENCR on FLI1 or neighboring gene expression. RNA-sequencing experiments in smooth muscle cells following SENCR knockdown disclose decreased expression of Myocardin and numerous smooth muscle contractile genes, while a number of pro-migratory genes are increased. RT-PCR and Western blotting experiments validate several differentially expressed genes following SENCR knockdown. Loss-of-function studies in scratch wound and Boyden chamber assays support SENCR as an inhibitor of smooth muscle cell migration. Conclusion SENCR is a new vascular cell-enriched, cytoplasmic lncRNA that appears to stabilize the smooth muscle cell contractile phenotype.

MicroRNA 143/145 (miR143/145) is restricted to adult smooth muscle cell (SMC) lineages and mediates, in part, the expression of several SMC contractile genes. Although the function of miR143/145 has begun to be elucidated, its transcriptional regulation in response to various signaling inputs is poorly understood. In an effort to define a miR signature for SMC differentiation, we screened human coronary artery SMCs for miRs modulated by TGF-␤1, a known stimulus for SMC differentiation. Array analysis revealed a number of TGF-␤1-induced miRs, including miR143/145. Validation studies showed that TGF-␤1 stimulated miR143/145 expression in a dose-and time-dependent manner. We utilized several chemical inhibitors and found that SB203580, a specific inhibitor of p38MAPK, significantly decreased TGF-␤1-induced miR143/145 expression. siRNA studies demonstrated that the effect of TGF-␤1 on miR143/145 was dependent upon the myocardin and serum response factor transcriptional switch as well as SMAD4. TGF-␤1 stimulated a 580-bp human miR143/145 enhancer, and mutagenesis studies revealed a critical role for both a known CArG box and an adjacent SMAD-binding element for full TGF-␤1-dependent activation of the enhancer. Chromatin immunoprecipitation assays documented TGF-␤1-mediated enrichment of SMAD3 and SMAD4 binding over the enhancer region containing the SMAD-binding element. Pre-miR145 strongly promoted SMC differentiation, whereas an antimiR145 partially blocked TGF-␤1-induced SMC differentiation. These results demonstrate a dual pathway for TGF-␤1-induced transcription of miR143/145, thus revealing a novel mechanism underlying TGF-␤1-induced human vascular SMC differentiation. SMCs3 display remarkable phenotypic adaptation in response to physical, chemical, and biological perturbations. Such altered SMC phenotypes play a major role in the pathogenesis of many human diseases, including asthma, atherosclerosis, restenosis, hypertension, transplant arteriopathy, and Alzheimer angiopathy (1-3). Accumulating evidence has shown SMC differentiation to be tightly regulated by an interacting network of environmental stimuli, signaling pathways, and various transcription factors, most notably SRF and MYOCD (2, 4 -8). TGF-␤1 is among the most potent soluble growth factors that activate SMC contractile gene expression in both specified SMC and non-SMC types (9 -15). Members of the TGF-␤1 superfamily transmit signals through both SMADdependent and SMAD-independent pathways (16). The classic pathway is through transmembrane serine-threonine kinase receptors, which mediate the phosphorylation of receptor-specific SMAD2 and SMAD3. The phosphorylated SMAD2-SMAD3 complex then interacts with the common SMAD4 to form a heteromeric complex, which translocates to the nucleus and binds to Smad-binding elements (SBE) located in the regulatory region of a number of target genes (16). SMAD-independent pathways, such as MAPK and PI3K, can also be triggered by TGF-␤ to initiate signal transduction and gene regulation (17,18). Both SMAD-dependent ...

Background-Myocardin (Myocd) is a strong coactivator that binds the serum response factor (SRF) transcription factor over CArG elements embedded within smooth muscle cell (SMC) and cardiac muscle cyto-contractile genes. Here, we sought to ascertain whether Myocd-mediated gene expression confers a structural and physiological cardiac or SMC phenotype. Methods and Results-Adenoviral-mediated expression of Myocd in the BC 3 H1 cell line induces cardiac and SMC genes while suppressing both skeletal muscle markers and cell growth. Key Words: smooth muscle Ⅲ serum response factor Ⅲ myocardin Ⅲ contraction Ⅲ knockdown D ifferentiated vascular smooth muscle cells (SMCs) have 2 important phenotypic characteristics. First, they replicate infrequently within the normal vessel wall. 1 Second, they express a unique cyto-contractile gene program encoding a subproteome necessary for the principal function of these cells, namely contraction. After physical or chemical injury to the vessel wall however, vascular SMCs increase their replication rate and reduce the expression of many cyto-contractile genes including the smooth muscle isoforms of alpha actin, myosin heavy chain, and calponin. 2 Such changes in SMC-restricted genes are thought to contribute to the pathogenesis of atherosclerosis, transplant arteriopathy, hypertension, bypass-graft failure, and the malignant phenotype. 2,3 Although the majority of vascular diseases correlate with reductions in SMC contractile proteins, at least one example exists in which increases in SMC contractile proteins are associated with a disorder. 4 Elucidating the intrinsic and extrinsic cues that specify one vascular SMC phenotype over another has therefore been the subject of intense study, with myriad proteins and signal transduction pathways identified. 2 See accompanying article on page 1416The differentiated phenotype of a muscle cell is largely determined by the expression of both ubiquitous and cell-specific transcription factors (TFs). The latter are exemplified by members of the MyoD family of basic helix-loop-helix TF which can convert a variety of cells, including cultured SMCs, into skeletal muscle cells. 5 Widely expressed TFs such as serum response factor (SRF) are critical for normal skeletal muscle, cardiomyocyte, and SMC differentiated phenotypes. 6 Ubiquitous TFs such as SRF orchestrate specific programs of gene expression through combinatorial associations with coregulators, some of which display cell-specific patterns of expression. 7 Among SRF coregulators, myocardin (Myocd) has emerged as one of central importance for the establishment of SMC identity. First cloned in a bioinformatic screen for unknown cardiac-specific genes, Myocd was shown initially to stimulate a battery of SRF target genes associated with cardiac muscle differentiation. 8 17 Here, we show that despite the activation of both cardiac and SMC genes, Myocd confers an ultrastructural and contractile phenotype that most closely resembles that seen in SMCs; no evidence for structural or physiolo...

deacetylase ͉ myogenic ͉ SRF ͉ MEF2 ͉ promoter S keletal muscle identity is controlled primarily by four skeletal muscle-specific myogenic regulatory factors (MRFs), MyoD, myogenin (Myog), Myf5, and MRF4, which cooperate with the myocyte enhancer factor-2 (MEF2) transcription factor to activate skeletal muscle gene expression (1). Although the MRFs act in a dominant manner and can convert a variety of cell types, including smooth muscle, into skeletal muscle (2), there are settings in which skeletal muscle can be induced to transdifferentiate into other cell types, suggesting that the MRFs may be subordinate to other cell-specific transcription factors (3, 4).Much of the work related to transcriptional regulation of smooth muscle cell (SMC) differentiation has focused on serum response factor (SRF), a widely expressed transcription factor that binds the CArG box found in the regulatory regions of many SMC-specific genes (5). Genetic inactivation of SRF (6) and CArG mutagenesis studies in transgenic mice (7) have confirmed the necessity of CArG-SRF in controlling SMC differentiation. However, SRF is only a weak transcriptional activator and requires interacting cofactors that recruit proteins to promote a permissive state for gene transcription. One such cofactor is myocardin (Myocd), which is expressed primarily in cardiac and SMCs and displays high transcriptional activity (8). Myocd can activate SMC-specific genes (9), and genetic deletion of Myocd in mice leads to defective vascular SMC differentiation (10). Thus, Myocd displays features of a master regulator of the SMC phenotype.In an effort to define the cells of the cardiovascular system derived from Myocd-dependent lineages, we performed lineage tracing in mouse embryos by introducing Cre recombinase into the Myocd locus and monitoring the expression of a Credependent lacZ from the ROSA26 reporter (R26R) mouse line. Consistent with previous expression data, cardiac and vascular SMCs are derived from Myocd-dependent lineages. Surprisingly, skeletal muscle in these mice also expressed lacZ, indicating its derivation from a Myocd-dependent lineage. However, rather than functioning as an activator of skeletal muscle gene expression, Myocd represses MyoD-mediated stimulation of the Myog promoter and blocks skeletal muscle differentiation in vitro. At the same time, Myocd transactivates SMC contractile protein genes, thereby converting skeletal myoblasts to an SMC phenotype. These results suggest that Myocd acts as a bifunctional switch for muscle differentiation by concurrently opposing the gene program for skeletal muscle differentiation and specifying a SMC fate. Results Myocd Is Expressed in Progenitors of Skeletal Muscle.Myocd is expressed throughout the atrial and ventricular myocardium and in a subset of vascular and visceral SMCs (8). To trace the embryonic origins of Myocd-expressing lineages, we performed lineage tracing by creating a mouse in which the first exon of Myocd was replaced with a Cre-recombinase cassette [supporting information (SI) Fig. ...