Smooth Muscle Cells

Vasoconstrictors such as arginine vasopressin (AVP) and angiotensin II (Ang II) have been shown to increase protein and mRNA levels of smooth muscle a-actin (SM-a-actin) in vascular smooth muscle cells. In the same cells, platelet-derived growth factor (PDGF) decreased SM-aactin protein and mRNA. The rat SM-a-actin promoter that has recently been isolated contains two E-boxes and three CC(A/T)6GG (CArG) elements. To examine regulation of the SM-a-actin promoter, a 765-bp region of the rat SM-a-actin gene was ligated into chloramphenicol acetyltransferase (CAT)-containing vectors and transfected into rat aortic vascular smooth muscle cells. Stimulation of cells with either AVP or Ang II increased CAT activity 5to 10-fold. PDGF was able to completely block the AVP-induced increase in CAT activity. To identify regions of the promoter responsible for both V urascular smooth muscle cells (VSMCs) undergo distinct patterns of growth in response to stimulation by polypeptide growth factors and vasoconstrictors. Stimulation by growth factors that signal through tyrosine kinase receptors, such as platelet-derived growth factor (PDGF) or epidermal growth factor, results in cell proliferation.1 Stimulation by vasoconstrictors such as angiotensin II (Ang II) or arginine vasopressin (AVP), in the absence of serum, results in hypertrophy of the cells with little, if any, increase in cell number.2A4 The molecular mechanisms underlying these distinct growth responses have not been established. In fact, a number of postreceptor signaling events are stimulated by both classes of agents, including activation of phospholipase C and mobilization of intracellular Ca2 ,5-7 activation of Na+-H+ exchange,8 and stimulation of mitogen-activated protein kinases.9-11 Recently, it has been shown that vasoconstrictors and PDGF have opposing effects on the levels of specific muscle proteins, including the smooth muscle isoform of a-actin (SM-a-actin). AVP and Ang II have been shown to increase both the protein and levels of steady-state mRNA for SM-a-actin.12 PDGF decreased both protein and mRNA levels.13 In the present study, Received June 24, 1994; accepted September 23, 1994. From the Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, Denver. Correspondence to Dr R.A. Nemenoff, Division of Renal Diseases and Hypertension, Box C-281, University of Colorado Health Sciences Center, 4200 East 9th Ave, Denver, CO 80262. C 1994 American Heart Association, Inc. the AVP stimulation and PDGF inhibition of promoter activity, a series of truncation mutants were prepared and transfected into vascular smooth muscle cells. Truncation of both E-boxes and the most distal CArG element did not qualitatively alter either AVP-induced stimulation of CAT activity or PDGF inhibition. However, removal of the middle CArG element resulted in a loss of AVP stimulation. These studies indicate that the AVP-induced elevation and PDGF-induced inhibition of SM-a-actin levels in vascular smooth muscle cells are mediated at least in part through regulation of the SM-a-actin promoter. The critical region of the promoter mediating this effect involves at a minimum one of the CArG elements. (Circ Res. 1994;75:1126-1130.)