Platelet-derived growth factor-BB and Ets-1 transcription factor negatively regulate transcription of multiple smooth muscle cell differentiation marker genes

Abstract

Dandré, Frédéric, and Gary K. Owens. Platelet-derived growth factor-BB and Ets-1 transcription factor negatively regulate transcription of multiple smooth muscle cell differentiation marker genes. Am J Physiol Heart Circ Physiol 286: H2042-H2051, 2004. First published January 29, 2004 10.1152/ajpheart.00625.2003.-Platelet-derived growth factor (PDGF)-BB, a potent mitogen for mesenchymal cells, also downregulates expression of multiple smooth muscle (SM) cell (SMC)-specific markers. However, there is conflicting evidence whether PDGF-BB represses SMC marker expression at a transcriptional or posttranscriptional level, and little is known regarding the mechanisms responsible for these effects. Results of the present studies provide clear evidence that PDGF-BB treatment strongly repressed SM ␣-actin, SM myosin heavy chain (MHC), and SM22␣ promoters in SMCs. Of major significance for resolving previous controversies in the field, we found PDGF-BB-induced repression of SMC marker gene promoters in subconfluent, but not postconfluent, cultures. Treatment of postconfluent SMCs with a tyrosine phosphatase inhibitor restored PDGF-BB-induced repression, whereas treatment of subconfluent SMCs with a tyrosine kinase blocker abolished PDGF-BB-induced repression, suggesting that a tyrosine phosphorylation event mediates cell density-dependent effects. On the basis of previous observations that Ets-1 transcription factor is upregulated within phenotypically modulated neointimal SMCs, we tested whether Ets-1 would repress SMC marker expression. Consistent with this hypothesis, results of cotransfection experiments indicated that Ets-1 overexpression reduced transcriptional activity of SMC marker promoter constructs in SMCs, whereas it increased activity of SM ␣-actin promoter in endothelial cells. PDGF-BB treatment increased expression of Ets-1 in cultured SMCs, and SM ␣-actin mRNA expression was reduced in multiple independent clones of SMCs stably transfected with an Ets-1-overexpressing construct. Taken together, results of these experiments provide novel insights regarding possible mechanisms whereby PDGF-BB and Ets-1 may contribute to SMC phenotypic switching associated with vascular injury. vascular injury; promoter; cell density; signaling SMOOTH MUSCLE (SM) cells (SMCs) are highly specialized cells expressing a unique repertoire of contractile proteins, ion channels, and signaling molecules necessary for their contractile function (38). The differentiation state of SMCs is controlled by a complex combination of local environmental cues such as cell-cell and cell-matrix interactions, neuronal influences, and hemodynamic and mechanical forces (38), as well as intrinsic factors such as clonal origins (6). In contrast to cardiac and skeletal muscle cells, which are terminally differentiated, SMCs can undergo major changes in phenotype, depending on physiological or pathological conditions. For example, in tissue culture or in vascular neointimal lesions, SMCs display diminished expression of a number of proteins that are c...

PDGF-DD, a novel mediator of smooth muscle cell phenotypic modulation, is upregulated in endothelial cells exposed to atherosclerosis-prone flow patterns

“…Cultured rat aortic SMCs were grown to subconfluence, transiently transfected in insulin-free serum-free medium (IFSFM), and stimulated with PDGF-DD or the vehicle as previously described for PDGF-BB (8). These cells and the associated conditions are used routinely by our laboratory and consistently express all the SMC differentiation markers that have been identified.…”

“…Cleaved PDGF-DD decreased SMC gene promoter activity in a dose-dependent manner. Growth-arrested subconfluent rat aortic SMCs were transfected with the Ϫ2555 to ϩ2813 SM ␣-actin enhancer, the Ϫ4200 to ϩ11600 SM MHC promoter-enhancer, or the Ϫ447 to ϩ47 SM22␣ promoter-enhancer luciferase constructs as previously described (8). In addition, c-fos (Ϫ356/ϩ190 bp), aortic carboxypeptidase-like protein (ACLP; Ϫ2502/ϩ176), and myocardin promoters were also used to transfect SMCs.…”

“…In support of this notion, recent evidence suggests that unstable atherosclerotic lesions prone to rupture are characterized by cells expressing lower levels of SM ␣-actin, indicating a lack of SMCs and/or the preponderance of phenotypically modulated SMCs within these types of lesions (10,22,39). Thus an understanding of the factors and mechanisms that control the phenotypic state of SMCs within atherosclerotic lesions is of critical importance.Platelet-derived growth factor (PDGF)-BB is a secreted molecule known to decrease SMC gene expression (5,8,19,42). PDGF-BB is a homodimer of two B subunits, which can bind to the PDGF ␣␣-, ␣␤-, or ␤␤-receptor (37).…”

“…Platelet-derived growth factor (PDGF)-BB is a secreted molecule known to decrease SMC gene expression (5,8,19,42). PDGF-BB is a homodimer of two B subunits, which can bind to the PDGF ␣␣-, ␣␤-, or ␤␤-receptor (37).…”

Searching for transcriptional regulators of Ang II-induced vascular pathology

“…Ang II activates the Ras-MAPK pathway, which in turn induces both the expression and phosphorylation of Ets-1, leading to expression of a number of genes involved in vascular responses. Besides the important role of Ets-1 as an effector of Ang II responses, several other mechanisms of transcriptional control involving nuclear factor of activated T cells, PI3K, AKT, RhoA, or myocardin, acting in concert with or independently of Ets-1, have begun to be characterized (6)(7)(8). For example, Ang II induces the binding of serum response factor (SRF) to the specific smooth and cardiac muscle coactivator myocardin; furthermore, the homeodomain protein Prx1 strongly promotes SRF binding to its specific response element after Ang II stimulation.…”