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E-box/basic helix-loop-helix (bHLH)-dependent regulation of promoters for skeletal muscle-specific genes is well established, but similar regulation of smooth muscle-selective promoters has not been reported. Using transient transfection assays of smooth muscle alpha-actin (SMalphaA) promoter-chloramphenicol acetyltransferase (CAT) reporter constructs in rat vascular smooth muscle cells (SMCs) and L6 skeletal myotubes, we identified two activator elements, smE1 and smE2, with sequences corresponding to E-box (5'-CAnnTG-3') motifs. In L6 myotubes, 4-bp mutations of smE1 or smE2 E-box motif alone completely abolished promoter activity. In contrast, mutation of smE1 and smE2 was required to reduce promoter activity in SMCs. Supershift analyses identified a myogenin-containing complex as the predominant smE1 and smE2 binding activity in skeletal muscle, and myogenin overexpression transactivated the promoter. Supershift analyses with SMC extracts demonstrated that the bHLH protein upstream stimulatory factor (USF) bound smE1, and USF overexpression transactivated the promoter in an smE1-dependent manner. In summary, our results provide novel evidence implicating E-box elements in directing expression of the SMalphaA promoter through distinct bHLH factor complexes in skeletal vs. smooth muscle.

Transcriptional activity of the smooth muscle (SM) alpha-actin gene is differentially regulated in SM vs. non-SM cells. Contained within the rat SM alpha-actin promoter are two MCAT motifs, binding sites for transcription enhancer factor 1 (TEF-1) transcriptional factors implicated in the regulation of many muscle-specific genes. Transfections of SM alpha-actin promoter-CAT constructs containing wild-type or mutagenized MCAT elements were performed to evaluate their functional significance. Mutation of the MCAT elements resulted in increased transcriptional activity in SM cells, whereas these mutations either had no effect or decreased activity in L6 myotubes or endothelial cells. High-resolution gel shift assays resolved several complexes of different mobilities that were formed between MCAT oligonucleotides and nuclear extracts from the different cell types, although no single band was unique to SM. Western blot analysis of nuclear extracts with polyclonal antibodies to conserved domains of the TEF-1 gene family revealed multiple reactive bands, some that were similar and others that differed between SM and non-SM. Supershift assays with a polyclonal antibody to the TEF-related protein family demonstrated that TEF-1 or TEF-1-related proteins were contained in the shifted complexes. Results suggest that the MCAT elements may contribute to cell type-specific regulation of the SM alpha-actin gene. However, it remains to be determined whether the differential transcriptional activity of MCAT elements in SM vs. non-SM is due to differences in expression of TEF-1 or TEF-1-related proteins or to unique (cell type specific) combinatorial interactions of the MCAT elements with other cis-elements and trans-factors.

Smooth muscle cells (SMC) within atherosclerotic lesions proliferate and exhibit phenotypic modulation, but the contribution of vascular endothelium to this process is poorly understood. Our aim was to examine the effects of endothelial cell-conditioned medium (ECCM) on vascular SMC growth and differentiation. Rat aortic ECCM stimulated a ninefold increase in [3H]thymidine incorporation and downregulated smooth muscle-specific myosin heavy chain and alpha-actin synthesis in rat aortic SMC. These effects were not inhibited by antibodies to platelet-derived growth factor (PDGF)-BB or PDGF-AB or with a PDGF beta-receptor subunit. Treatment with PDGF-BB (at a concentration found in ECCM), PDGF-AA, basic fibroblast growth factor, endothelin-1, or transforming growth factor-beta did not reproduce these effects. The ECCM activities were sensitive to heat and trypsinization, were >30 kDa in molecular mass, and bound weakly to heparin-Sepharose. Our data indicate that cultured endothelial cells produce a factor(s) that downregulates contractile protein expression in SMC, which may contribute to SMC dedifferentiation and proliferation.

Thrombin has been implicated in vascular smooth muscle cell (VSMC) proliferation after vessel injury. Its proliferative effects, which are mediated via proteolytic activation of a receptor similar or identical to the cloned thrombin receptor (TR), have markedly delayed kinetics. The present study demonstrates that, despite rapid thrombin receptor activation and similar time to S phase entry compared with classic polypeptide growth factors, prolonged thrombin exposure is required to promote maximal VSMC mitogenesis. Flow cytometric analysis of thrombin-stimulated cells revealed that thrombin induced a progressive increase in the growth fraction over 3 days in culture, an effect that was blocked by hirudin even late after thrombin addition. Northern blot hybridization after thrombin stimulation demonstrated that thrombin upregulates TR mRNA expression within 6 h. These findings indicate that VSMC proliferate in response to prolonged thrombin exposure and suggest that the mitogenic delay may involve not only the thrombin-dependent synthesis and activation of newly made TR but also the progressive thrombin-dependent recruitment of cells into the growth fraction.

Identification of the regulators of smooth muscle cell (SMC) gene expression is critical to understanding SMC differentiation and alterations in SMC phenotype in vascular disease. Previous studies revealed positive transcriptional activity within the chicken smooth muscle (SM) alpha-actin promoter region from -209 to -257. In the present study, transient transfections of wild-type and mutant chicken SM alpha-actin promoter/reporter gene constructs into rat aortic SMC demonstrated that the positive transcriptional activity of this region was abolished with a two base pair mutation in a conserved sequence motif at -225 to -233 (TGTTTATC to TACTTATC). Electrophoretic mobility shift assays revealed that nuclear factors bound promoter fragments containing this sequence and that specific mutations in the TGTTTATC motif abolished nuclear factor binding. Studies thus provide evidence for binding of a nuclear factor to a positive cis-acting element within the SM alpha-actin promoter. Further characterization of this factor may contribute to a better understanding of the molecular mechanisms that regulate differentiation of SMC in vascular disease.

Previous studies have shown that alpha-thrombin (Thr) is a mitogen for cultured smooth muscle cells (SMC), but controversy exists concerning mechanisms of growth stimulation. The purpose of these studies was to determine whether autocrine production of platelet-derived growth factor-AA (PDGF-AA) mediated Thr-induced SMC mitogenesis. SMC derived from aortae of Sprague-Dawley rats were grown to confluence, growth arrested in serum-free medium, and treated. Conditioned medium (CM) was harvested by adding bovine serum albumin as a carrier and hirudin to neutralize residual Thr. Results demonstrated that CM from Thr-treated SMC (Thr CM) had increased mitogenic activity compared with control CM (Cnt CM) and that PDGF-AA levels were increased 12-fold in Thr CM compared with Cnt CM. The excess mitogenic activity in Thr CM was markedly inhibited by PDGF-AA-neutralizing antibodies. Cotreatment with Thr and PDGF-AA-neutralizing antibodies resulted in a 30% decrease in Thr-induced [3H]thymidine incorporation. These results demonstrate that autocrine production of PDGF-AA partially mediated Thr-induced SMC mitogenesis.

Our objectives were to 1) determine how growth state and cell density affect the expression of the smooth muscle (SM) and nonmuscle (NM) isoforms of the 20-kDa myosin regulatory light chains (MLC20) in cultured rat aortic smooth muscle cells (SMC) and 2) to determine whether angiotensin II stimulates differential phosphorylation of SM and NM MLC20 isoforms in an effort to assess whether the SM and NM isoforms may subserve different cellular functions. The results demonstrated that changes in the SM MLC20 isoform content were inversely correlated with cell growth but independent of cell density. MLC20 phosphorylation levels were 20.8 +/- 2.9 and 19.4 +/- 3.7% for SM and NM isoforms, respectively, in unstimulated, substrate-attached SMC. Angiotensin II transiently elevated phosphorylation levels of both the SM and NM MLC20 isoforms to 60-70%. No differences in either the magnitude or the kinetics of phosphorylation were observed for the SM vs. NM isoforms. Forskolin, 3-isobutyl-1-methylxanthine, or isoproterenol treatment led to parallel dephosphorylation of the SM- and NM-specific isoforms followed by depolymerization of stress fibers and cell arborization. The studies provide evidence that growth arrest of cultured SMC enhances expression of cell-specific/-selective proteins characteristic of differentiated SM. However, there was no evidence for differential phosphorylation changes of SM and NM MLC20 isoforms in response to activating or relaxing agents as expected if these isoforms subserve different cellular functions.

Previous studies have demonstrated that angiotensin II (ANG II) and arginine vasopressin (AVP) stimulate increased protein synthesis and cellular hypertrophy in cultured rat aortic smooth muscle cells (SMC). The aim of this study was to explore the hypothesis that ANG II- and/or AVP-induced increases in protein synthesis are mediated by autocrine secretion of platelet-derived growth factor (PDGF)-AA. Results demonstrated that ANG II or AVP increased expression of PDGF-A, but not -B, chain mRNA. Additionally, conditioned media from ANG II- and AVP-treated SMC had increased mitogenic activity for Swiss 3T3 cells, which could be inhibited with a neutralizing antibody to PDGF-AA. However, PDGF-AA-neutralizing antibodies did not inhibit ANG II- or AVP-induced increases in protein synthesis, and exogenous PDGF-AA did not stimulate increased protein synthesis. Furthermore, no PDGF-alpha receptors were evident based on 125I-labeled PDGF-AA binding studies. In summary, results indicate that ANG II- or AVP-induced increases in protein synthesis were not dependent on autocrine secretion of PDGF-AA.

A long-term objective of my laboratory has been to understand the mechanisms that regulate both normal and developmental growth of vascular smooth muscle as well as the accelerated growth of smooth muscle that occurs in atherosclerotic lesions or arteries of hypertensive patients and animals. Previous studies in this and other laboratories have demonstrated that smooth muscle cells are capable of two distinct growth responses in vivo, depending on the nature of the growth stimulus. Smooth muscle cell growth in large vessels of chronically hypertensive animals appears to occur primarily by enlargement of preexisting cells (i.e., cellular hypertrophy) with little or no cell proliferation (hyperplasia) and is accompanied by development of polyploidy in a large fraction of the cells. In contrast, in experimental injury models of atherogenesis, or after induction of severe acute hypertension, aortic smooth muscle cells undergo a classic proliferative response. This lecture focuses on possible control mechanisms for hypertrophy of vascular smooth muscle, with particular emphasis on examination of the possible role of contractile agonists as hypertrophic agents, exploration of how this process differs from cellular hyperplasia, discussion of the possible mechanisms for formation of polyploid cells, and examination of the role of mechanical factors in growth regulation of vascular smooth muscle cells.