Molecular and Cellular Biology of Plasminogen Activation
Molecular and Cellular Biology of Plasminogen Activation
Molecular and Cellular Biology of Plasminogen Activation
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i016i<br />
TGF-b1-Induced <strong>Plasminogen</strong> Activator Inhibitor-<br />
1 Expression in Vascular Smooth Muscle Cells Requires<br />
Cooperative Rho/ROCK <strong>and</strong> EGFR Signaling<br />
Higgins PJ* <strong>and</strong> Samarakoon R<br />
Center for Cell <strong>Biology</strong> & Cancer Research, Albany Medical College, Albany, New York, USA<br />
Presenting author e-mail: higginp@mail.amc.edu<br />
TGF-b1 <strong>and</strong> its target gene encoding plasminogen activator inhibitor-1 (PAI-1) are major causative<br />
factors in the pathophysiology <strong>of</strong> arteriosclerosis <strong>and</strong> perivascular fibrosis. The definition <strong>of</strong> TGFb1-activated<br />
signaling networks that impact PAI-1 transcription in vascular smooth muscle cells<br />
(VSMC) may identify novel, therapeutically-relevant, opportunities to manage PAI-1-associated<br />
cardiovascular disease. TGF-b1-induced PAI-1 expression in VSMC required cooperative EGFR/<br />
MEK-ERK <strong>and</strong> Rho/ROCK signaling. Transient transfection <strong>of</strong> a dominant negative RhoA (DN-<br />
RhoA) expression construct or pretreatment <strong>of</strong> VSMC with C3 transferase (a Rho inhibitor) <strong>and</strong><br />
Y-27632 (an inhibitor <strong>of</strong> p160ROCK, a downstream effector <strong>of</strong> Rho) dramatically attenuated the<br />
TGF-b1-initiated PAI-1 inductive response. Genetic EGFR1 deficiency or pharmacologic inhibition<br />
<strong>of</strong> EGFR activity (with AG1478) virtually ablated TGF-b1-stimulated ERK1/2 activation as well<br />
as PAI-1 expression but not SMAD2 phosphorylation. Interference with Rho/ROCK signaling,<br />
in contrast, prevented SMAD2 activation <strong>and</strong> nuclear accumulation. Infection <strong>of</strong> VSMC with<br />
an adenovirus encoding a mutant Y845F-EGFR effectively decreased TGF-b1-induced PAI-1<br />
expression implicating the pp60c-src phosphorylation site (Y845) <strong>of</strong> the EGFR in the inductive<br />
response. This is consistent with previous findings that pp60c-src is activated by TGF-b1 in<br />
VSMC <strong>and</strong> that dominant negative pp60c-src (DN-Src) expression vectors significantly block<br />
TGF-b1 induced PAI-1 expression. SMAD2 activation, moreover, is not sufficient to induce PAI-<br />
1 expression by TGF-b1 in the absence <strong>of</strong> EGFR signaling both in VSMC <strong>and</strong> mouse embryonic<br />
fibroblasts. Thus, two distinct but cooperative pathways involving EGFR/MEK-ERK signaling<br />
<strong>and</strong> Rho-dependent SMAD2 activation are required for TGF-b1-induced PAI-1 expression in<br />
VSMC.<br />
28 X I t h I n t e r n a t i o n a l W o r k s h o p o n
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