Plasminogen activator inhibitor type‐1 gene expression and induced migration in TGF‐β1‐stimulated smooth muscle cells is pp60c‐src/MEK‐dependent

Abstract

Transforming growth factor‐β1 (TGF‐β1) stimulates expression of plasminogen activator inhibitor type‐1 (PAI‐1), a serine protease inhibitor (SERPIN) important in the control of stromal barrier proteolysis and cell‐to‐matrix adhesion. Pharmacologic agents that target MEK (PD98059, U0126) or src family (PP1) kinases attenuated TGF‐β1‐dependent PAI‐1 transcription in R22 aortic smooth muscle cells. Pretreatment with PP1 at concentrations that inhibited TGF‐β1‐dependent PAI‐1 expression also blocked ERK1/2 activation/nuclear accumulation suggesting that the required src kinase activity is upstream of ERK1/2 in the TGF‐β1‐initiated signaling cascade. The IC₅₀ of the PP1‐sensitive kinase, furthermore, specifically implied involvement of pp60^c‐src in PAI‐1 induction. Indeed, addition of TGF‐β1 to quiescent R22 cells resulted in a 3‐fold increase in pp60^c‐src autophosphorylation and kinase activity. Transfection of a dominant‐negative pp60^c‐src construct, moreover, reduced TGF‐β1‐induced PAI‐1 expression levels to that of unstimulated controls or PP1‐pretreated cells. A ≥170 kDa protein that co‐immunoprecipitated with TGF‐β1‐activated pp60^c‐src was also phosphorylated transiently in response to TGF‐β1. TGF‐β1 is known to transactivate the 170 kDa EGF receptor (EGFR) by autocrine HB‐EGF or TGF‐α mechanisms suggesting involvement of EGFR activation in certain TGF‐β1‐initiated responses. Incubation of quiescent R22 cells with the EGFR‐specific inhibitor AG1478 prior to growth factor (EGF or TGF‐β1) addition effectively blocked EGFR activation as determined by direct visualization of receptor internalization. AG1478 suppressed (in a dose‐dependent fashion) EGF‐induced PAI‐1 protein levels and, at a final concentration of 2.5 μM, virtually eliminated EGF‐dependent PAI‐1 synthesis. More importantly, AG1478 similarly repressed inducible PAI‐1 levels in TGF‐β1‐stimulated R22 cultures. PP1, PD98059, and U0126 also inhibited TGF‐β1‐dependent cell motility at concentrations that significantly attenuated PAI‐1 expression. Consistent with the AG1478‐associated reductions in EGF‐ and TGF‐β1‐stimulated PAI‐1 expression, pretreatment of R22 cell cultures with AG1478 effectively suppressed growth factor‐stimulated cell motility. These data indicate that two major phenotypic characteristics of TGF‐β1‐exposure (i.e., transcription of specific target genes [e.g., PAI‐1], increased cell motility) are linked in the R22 vascular smooth muscle cell system, require pp60^c‐src kinase activity and MEK signaling and involve activation of an AG1478‐sensitive (likely EGFR‐dependent) pathway.