Serum response factor (SRF) is a highly conserved and widely expressed, single copy transcription factor that theoretically binds up to 1,216 permutations of a 10-base pair cis element known as the CArG box. SRF-binding sites were defined initially in growth-related genes. Gene inactivation or knockdown studies in species ranging from unicellular eukaryotes to mice have consistently shown loss of SRF to be incompatible with life. However, rather than being critical for proliferation and growth, these genetic studies point to a crucial role for SRF in cellular migration and normal actin cytoskeleton and contractile biology. In fact, recent genomic studies reveal nearly half of the >200 SRF target genes encoding proteins with functions related to actin dynamics, lamellipodial/filopodial formation, integrin-cytoskeletal coupling, myofibrillogenesis, and muscle contraction. SRF has therefore emerged as a dispensable transcription factor for cellular growth but an absolutely essential orchestrator of actin cytoskeleton and contractile homeostasis. This review summarizes the recent genomic and genetic analyses of CArG-SRF that support its role as an ancient, master regulator of the actin cytoskeleton and contractile machinery.

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. Therefore, we 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 was reduced and of macrophage markers increased. Concurrent was downregulation of miR-143/145, which positively regulate the master VSMC differentiation transcription factor myocardin. Mechanisms were further probed in mouse VSMC. Maintaining the expression of myocardin 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. Although 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.

Serum response factor (SRF) binds a 1216-fold degenerateciselement 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 forcis-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—: Long noncoding 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 (RNA-seq) of human coronary artery smooth muscle cells revealed 31 unannotated lncRNAs, including a vascular cell–enriched lncRNA (Smooth muscle and Endothelial cell–enriched migration/differentiation-associated long NonCoding RNA [ SENCR ]). Strand-specific reverse transcription polymerase chain reaction (PCR) and rapid amplification of cDNA ends indicate that SENCR is transcribed antisense from the 5′ end of the FLI1 gene and exists as 2 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-seq experiments in smooth muscle cells after SENCR knockdown disclose decreased expression of Myocardin and numerous smooth muscle contractile genes, whereas several promigratory genes are increased. Reverse transcription PCR and Western blotting experiments validate several differentially expressed genes after SENCR knockdown. Loss-of-function studies in scratch wound and Boyden chamber assays support SENCR as an inhibitor of smooth muscle cell migration. Conclusions—: SENCR is a new vascular cell–enriched, cytoplasmic lncRNA that seems 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-β1, a known stimulus for SMC differentiation. Array analysis revealed a number of TGF-β1-induced miRs, including miR143/145. Validation studies showed that TGF-β1 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-β1-induced miR143/145 expression. siRNA studies demonstrated that the effect of TGF-β1 on miR143/145 was dependent upon the myocardin and serum response factor transcriptional switch as well as SMAD4. TGF-β1 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-β1-dependent activation of the enhancer. Chromatin immunoprecipitation assays documented TGF-β1-mediated enrichment of SMAD3 and SMAD4 binding over the enhancer region containing the SMAD-binding element. Pre-miR145 strongly promoted SMC differentiation, whereas an anti-miR145 partially blocked TGF-β1-induced SMC differentiation. These results demonstrate a dual pathway for TGF-β1-induced transcription of miR143/145, thus revealing a novel mechanism underlying TGF-β1-induced human vascular SMC differentiation.