title: TGF-beta1-induced plasminogen activator inhibitor-1 expression in vascular smooth muscle cells requires pp60(c-src)/EGFR(Y845) and Rho/ROCK signaling.
abstract: TGF-beta1 and its target gene encoding plasminogen activator inhibitor-1 (PAI-1) are major causative factors in the pathology of tissue fibrosis and vascular disease. The increasing complexity of TGF-beta1 action in the cardiovascular system requires analysis of specific TGF-beta1-initiated signaling events that impact PAI-1 transcriptional regulation in a physiologically-relevant cell system. TGF-beta1-induced PAI-1 expression in both primary cultures and in an established line (R22) of vascular smooth muscle cells (VSMC) was completely blocked by inhibition of epidermal growth factor receptor (EGFR) activity or adenoviral delivery of a kinase-dead EGFR(K721A) construct. TGF-beta1-stimulated PAI-1 expression, moreover, was preceded by EGFR phosphorylation on Y845 (a src kinase target residue) and required pp60(c-src) activity. Infection of VSMC with an adenovirus encoding the EGFR(Y845F) mutant or transfection with a dominant-negative pp60(c-src) (DN-Src) expression vector effectively decreased TGF-beta1-stimulated, but not PDGF-induced, PAI-1 expression implicating the pp60(c-src) phosphorylation site EGFR(Y845) in the inductive response. Consistent with these findings, TGF-beta1 failed to induce PAI-1 synthesis in src kinase-deficient (SYF(-/-/-)) fibroblasts and reexpression of a wild-type pp60(c-src) construct in SYF(-/-/-) cells rescued the PAI-1 response to TGF-beta1. TGF-beta1-induced EGFR activation, but not SMAD2 activation, moreover, was virtually undetectable in SYK(-/-/-) fibroblasts in comparison to wild type (SYK(+/+/+)) counterparts, confirming an upstream signaling role of src family kinases in EGFR(Y845) phosphorylation. Genetic EGFR deficiency or infection of VSMCs with EGFR(K721A) virtually ablated TGF-beta1-stimulated ERK1/2 activation as well as PAI-1 expression but not SMAD2 phosphorylation. Transient transfection of a dominant-negative RhoA (DN-RhoA) expression construct or pretreatment of VSMC with C3 transferase (a Rho inhibitor) or Y-27632 (an inhibitor of p160ROCK, a downstream effector of Rho) also dramatically attenuated the TGF-beta1-initiated PAI-1 inductive response. In contrast to EGFR pathway blockade, interference with Rho/ROCK signaling effectively inhibited TGF-betaR-mediated SMAD2 phosphorylation and nuclear accumulation. TGF-beta1-stimulated SMAD2 activation, moreover, was not sufficient to induce PAI-1 expression in the absence of EGFR signaling both in VSMC and mouse embryonic fibroblasts. Thus, two distinct pathways involving the EGFR/pp60(c-src)/MEK-ERK pathway and Rho/ROCK-dependent SMAD2 activation are required for TGF-beta1-induced PAI-1 expression in VSMC. The identification of such novel interactions between two TGF-beta1-activated signaling networks that specifically impact PAI-1 transcription in VSMC may provide therapeutically-relevant targets to manage the pathophysiology of PAI-1-associated cardiovascular/fibrotic diseases.
authors.affiliation: Albany Medical College, Center for Cell Biology and Cancer Research, MC-165, 47 New Scotland Avenue, Albany, NY 12208, USA.
authors.forename: Rohan
authors.initials: R
authors.lastname: Samarakoon
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Higgins
authors.forename: Craig E
authors.initials: CE
authors.lastname: Higgins
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Higgins
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011799:Quinazolines; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000378:metabolism; D051899:Smad2 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D020032:Tyrphostins / Q000494:pharmacology; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D011799:Quinazolines; D051785:Smad Proteins; D051899:Smad2 Protein; D053773:Transforming Growth Factor beta1; D020032:Tyrphostins; C101044:RTKI cpd; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src); D054460:rho-Associated Kinases; D020741:rho GTP-Binding Proteins