Depletion of Serum Response Factor by RNA Interference Mimics the Mitogenic Effects of Platelet Derived Growth Factor-BB in Vascular Smooth Muscle Cells

Abstract

Abstract-Promoters of many smooth muscle-specific genes (SM-genes) contain multiple CArG boxes, which represent a binding site for serum response factor (SRF). Transciptional control through these regions involves interactions with SRF and specific coactivators such as myocardin. We have previously reported that suppression of SM-gene expression by platelet derived growth factor (PDGF) is associated with redistribution of SRF, leading to lower intra-nuclear levels, and a reduction in SRF transactivation. To further assess the role of SRF depletion on VSMC phenotype, the current study used RNA interference (RNAi). Two SRF-specific sequences constructed as hairpins were stably expressed in rat VSMC. Clones expressing SRF RNAi had no detectable SRF expression by immunoblotting, and showed diminished levels of SM ␣-actin protein and promoter activity. Unexpectedly, depletion of VSMC resulted in increased rates of proliferation and migration. Several genes whose expression is increased by PDGF stimulation, including c-Jun, were similarly induced in cells lacking SRF. Key Words: vascular smooth muscle cells Ⅲ serum response factor Ⅲ RNA interference Ⅲ platelet derived growth factor P henotypic modulation of vascular smooth muscle cells (VSMC 1 ) is critical during development and in the onset of diseases such as atherosclerosis and hypertension. Atherosclerosis is a leading cause of heart disease and stroke and causes Ϸ50% of all deaths. 2,3 Therefore, understanding the physiology of large vessels will help to identify the cause of a primary cardiovascular disease. VSMC in mature animals express multiple contractile proteins to maintain vascular tone. These proteins are markers for the differentiated contractile phenotype. Unlike myocardial and skeletal muscle cells, VSMC are highly plastic and retain the ability to modulate their phenotype. 4 -6 Environmental factors can alter the expression levels of these contractile proteins, leading to a switch from a differentiated phenotype, characterized by high expression, to a proliferative, dedifferentiated phenotype characterized by low expression. This occurs in pathophysiologic states of large arteries such as atherosclerosis, where migration and proliferation of neointimal VSMC lead to vascular remodeling. 7,8 In adult cultured VSMC, vasoconstrictors, such as angiotensin II or arginine vasopressin (AVP), increase expression of smooth muscle (SM) markers such as SMA and SM22␣, whereas growth factors such as platelet derived growth factor (PDGF) decrease protein and mRNA levels of these proteins. 1,9 -11 We have shown that these effects are mediated through transcriptional regulation of SM specific promoter activity, and have defined specific signal transduction pathways that regulate SM specific gene expression by AVP or PDGF. AVP-induced increases in gene expression require c-jun amino terminal kinase and p38 MAP kinases, whereas PDGF-induced suppression is mediated by Ras, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. 1,[11][12][13] The promoters of these SM...

The Intermediate Conductance Calcium-activated Potassium Channel KCa3.1 Regulates Vascular Smooth Muscle Cell Proliferation via Controlling Calcium-dependent Signaling

“…HCSMCs treated with SKA-31 or NS309 also displayed similar morphological characteristics (ratio of cell length/width; PDGFϩSKA-31 25 Ϯ 2 and PDGFϩNS309 23 Ϯ 2, p Ͻ 0.05 versus PDGF alone 15 Ϯ 2, n ϭ 83 cells). Treatment of HCSMCs with PDGF increased the expression of l-caldesmon, a dedifferentiation marker, and decreased the expression of calponin-1, a differentiation marker, accompanied by a decreased expression of membranous PDGF ␤-receptors, a known negative feedback response (36) (Fig. 5B).…”

Serum response factor: master regulator of the actin cytoskeleton and contractile apparatus

“…Despite the evidence supporting SRF's role as a growth-associated transcription factor, there are some reports demonstrating an inverse relationship between SRF expression and cell growth. For example, SRF reverses the ras-transformed phenotype (54), and RNA interference (RNA i ) of SRF phenocopies growth factor-stimulated smooth muscle cell proliferation (49). Perhaps the most direct in vitro evidence blurring the concept of SRF being essential for growth are the studies done in embryonic stem cells null for SRF.…”

The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis

“…Experiments in cultured cells using dominant-negative SRF mutants or siRNA to down-regulate SRF expression have revealed essential roles for SRF in serum-dependent cell growth and skeletal muscle differentiation (Soulez et al 1996;Wei et al 1998;Kaplan-Albuquerque et al 2005). A dominant-negative SRF mutant also blocks differentiation of coronary smooth muscle cells (SMCs) in the proepicardial organ of chick embryos (Landerholm et al 1999) and disrupts skeletal and cardiac muscle differentiation in transgenic mice (Zhang et al 2001).…”