Transforming growth factor-beta1-induced expression of smooth muscle marker genes involves activation of PKN and p38 MAPK.
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-beta1 (TGF-beta1) induces differentiation of SMCs were examined. TGF-beta1 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-beta1 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) alpha-actin, SM-myosin heavy chain, and SM22alpha. 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-beta1-induced activation of SMC marker gene promoters. Finally, examination of MAPK signaling demonstrated that TGF-beta1 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 serum-response factor, GATA, and MEF2 enhancers. Taken together, these results identify components of an important intracellular signaling pathway through which TGF-beta1 activates PKN to promote differentiation of SMCs.
Cardiovascular Research Institute, Department of Integrative Physiology, University of North Texas Health Science Center, Fort Worth, Texas 76107-2699, USA.
Rebecca A
RA
Deaton
Chang
C
Su
Thomas G
TG
Valencia
Stephen R
SR
Grant
D000199:Actins / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005819:Genetic Markers; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D009130:Muscle, Smooth / Q000166:cytology / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000199:Actins; D005819:Genetic Markers; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C061570:protein kinase N; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein