Transforming Growth Factor-β1-induced Expression of Smooth Muscle Marker Genes Involves Activation of PKN and p38 MAPK

Abstract

Differentiated vascular smooth muscle cells (SMCs) exhibit a work phenotype characterized by expression of several well documented contractile apparatus-associated proteins. However, SMCs retain the ability to de-differentiate into a proliferative phenotype, which is involved in the progression of vascular diseases such as atherosclerosis and restenosis. Understanding the mechanisms involved in maintaining SMC differentiation is critical for preventing proliferation associated with vascular disease. In this study, the molecular mechanisms through which transforming growth factor-␤1 (TGF-␤1) induces differentiation of SMCs were examined. TGF-␤1 stimulated actin re-organization, inhibited cell proliferation, and up-regulated SMC marker gene expression in PAC-1 SMCs. These effects were blocked by pretreatment of cells with either HA1077 or Y-27632, which inhibit the kinases downstream of RhoA. Moreover, TGF-␤1 activated RhoA and its downstream target PKN. Overexpression of active PKN alone was sufficient to increase the transcriptional activity of the promoters that control expression of smooth muscle (SM) ␣-actin, SM-myosin heavy chain, and SM22␣. In addition, PKN increased the activities of serum-response factor (SRF), GATA, and MEF2-dependent enhancer-reporters. RNA interference-mediated inhibition of PKN abolished TGF-␤1-induced activation of SMC marker gene promoters. Finally, examination of MAPK signaling demonstrated that TGF-␤1 increased the activity of p38 MAPK, which was required for activation of the SMC marker gene promoters. Co-expression of dominant negative p38 MAPK was sufficient to block PKN-mediated activation of the SMC marker gene promoters as well as the serumresponse factor, GATA, and MEF2 enhancers. Taken together, these results identify components of an important intracellular signaling pathway through which TGF-␤1 activates PKN to promote differentiation of SMCs.

Transforming Growth Factor-β1 (TGF-β1) Utilizes Distinct Pathways for the Transcriptional Activation of MicroRNA 143/145 in Human Coronary Artery Smooth Muscle Cells

“…Finally, luciferase assays reveal that SB203580 suppresses TGF-␤1-induced miR143/145 promoter activity, and mutation of a previously defined conserved CArG box (35,37) causes a loss in response to TGF-␤1. Our results are in agreement with previous studies showing an involvement of p38MAPK in regulating SMC differentiation (14,57). Importantly, we offer evidence that p38MAPK directly regulates the expression of SRF and MYOCD, thus providing new insight into the mechanism of TGF-␤1-p38MAPK in modulating SMC differentiation.…”

“…SMAD-independent pathways, such as MAPK and PI3K, can also be triggered by TGF-␤ to initiate signal transduction and gene regulation (17,18). Both SMAD-dependent and SMAD-independent pathways have been demonstrated to contribute to SMC differentiation in a context-dependent manner (11,14).…”

“…SMAD-independent pathways, such as MAPK and PI3K, can also be triggered by TGF-␤ to initiate signal transduction and gene regulation (17,18). Both SMAD-dependent and SMAD-independent pathways have been demonstrated to contribute to SMC differentiation in a context-dependent manner (11,14).MicroRNAs (miRs) represent a class of conserved, small non-coding RNAs that bind to and target mRNA for degradation or a block in translation (19,20). Aberrant expression of miRs frequently leads to abnormalities in development and the pathogenesis of disease (21-25), and a growing number of miRs have been discovered as powerful modulators in cardiovascular biology.…”

A Novel RhoA/ROCK-CPI-17-MEF2C Signaling Pathway Regulates Vascular Smooth Muscle Cell Gene Expression

“…Interestingly, in VSMCs PP1␣ serves as the catalytic subunit of the myosin light chain phosphatase complex (MLCP) and is regulated by RhoA signaling to control calcium sensitivity during contraction (14). In addition, signals emanating from RhoA in VSMCs have been previously shown to activate p38 MAP kinase (MAPK) signaling (15), a known activator of MEF2 transcriptional activity in multiple cell types (16 -18). In this report we document for the first time, a novel signaling pathway in VSMCs that links RhoA-mediated regulation of calcium sensitivity to MEF2-dependent expression of myocardin.…”

Primary and immortalized mouse epicardial cells undergo differentiation in response to TGFβ

“…These activated RSmads complex with Smad 4 and translocate into the nucleus to alter gene transcription. TGF␤ can activate additional downstream effectors including RhoA (Bhowmick et al, 2001a(Bhowmick et al, , 2003Edlund et al, 2002;Masszi et al, 2003;Deaton et al, 2005), Ras (Ward et al, 2002), mitogen activated protein (MAP) kinases (Bhowmick et al, 2001b;Bakin et al, 2002;Xie et al, 2004;Deaton et al, 2005), and PI3K/AKt (Bakin et al, 2000), although the mechanisms by which TGF␤ regulates these effectors is less well described. A second class of TGF␤ binding proteins contains two transmembrane proteins termed the type III TGF␤ receptor (TGF␤R3), or betaglycan, and endoglin.…”