Plasminogen activator inhibitor type-1 gene expression and induced migration in TGF-beta1-stimulated smooth muscle cells is pp60(c-src)/MEK-dependent.
Transforming growth factor-beta1 (TGF-beta1) 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-beta1-dependent PAI-1 transcription in R22 aortic smooth muscle cells. Pretreatment with PP1 at concentrations that inhibited TGF-beta1-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-beta1-initiated signaling cascade. The IC(50) of the PP1-sensitive kinase, furthermore, specifically implied involvement of pp60(c-src) in PAI-1 induction. Indeed, addition of TGF-beta1 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-beta1-induced PAI-1 expression levels to that of unstimulated controls or PP1-pretreated cells. A >/=170 kDa protein that co-immunoprecipitated with TGF-beta1-activated pp60(c-src) was also phosphorylated transiently in response to TGF-beta1. TGF-beta1 is known to transactivate the 170 kDa EGF receptor (EGFR) by autocrine HB-EGF or TGF-alpha mechanisms suggesting involvement of EGFR activation in certain TGF-beta1-initiated responses. Incubation of quiescent R22 cells with the EGFR-specific inhibitor AG1478 prior to growth factor (EGF or TGF-beta1) 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 muM, virtually eliminated EGF-dependent PAI-1 synthesis. More importantly, AG1478 similarly repressed inducible PAI-1 levels in TGF-beta1-stimulated R22 cultures. PP1, PD98059, and U0126 also inhibited TGF-beta1-dependent cell motility at concentrations that significantly attenuated PAI-1 expression. Consistent with the AG1478-associated reductions in EGF- and TGF-beta1-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-beta1-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.
Samarakoon
Rohan
R
Center for Cell Biology & Cancer Research, Albany Medical College, Albany, New York, USA.
Higgins
Craig E
CE
Higgins
Stephen P
SP
Kutz
Stacie M
SM
Higgins
Paul J
PJ
D000818:Animals; D001011:Aorta / Q000166:cytology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D020935:MAP Kinase Signaling System / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000502:physiology*; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011799:Quinazolines; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D020032:Tyrphostins / Q000494:pharmacology
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D004791:Enzyme Inhibitors; D017395:Plasminogen Activator Inhibitor 1; D011720:Pyrazoles; D011743:Pyrimidines; D011799:Quinazolines; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D020032:Tyrphostins; C101044:RTKI cpd; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src); D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3