Urokinase-induced smooth muscle cell responses require distinct signaling pathways: A role for the epidermal growth factor receptor

Objective: Urokinase plasminogen activator (uPA) a key serine protease during remodeling, is capable of inducing both smooth muscle cell migration and proliferation. However, the signals that produce these responses are poorly understood. Methods: Early passage rat aortic arterial smooth muscle cells were cultured in vitro and standard assays of DNA synthesis ([³H]thymidine incorporation), cell proliferation (manual cell counting), and migration (linear wound assay and Boyden chamber) were used to study the cells responses to uPA. Activation of the mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), p38^MAPK, Akt, MAP kinase/ERK kinase (MEK1/2), MAP kinase kinase (MKK)3/6, and epidermal growth factor receptor (EGFR) in response to uPA was assayed by Western blot analysis for the phosphorylated form of each kinase. These assays were repeated in the presence of the Gαi inhibitor pertussis toxin (PTx, 100 ng/mL), the Ras inhibitor manumycin A (MA, 10 μM), the phosphatidyl-inositol 3′ kinase (PI3K) inhibitor wortmannin (WN, 1 μM), the EGFR inhibitor AG1478 (AG, 10 nM), the MEK1 inhibitor PD98059 (PD, 10 μM), the p38^MAPK inhibitor SB203580 (SB, 10 μM), and the plasmin inhibitors aprotinin and ε-aminocaproic acid. Results: uPA induced a twofold increase in smooth muscle cell migration and increased smooth muscle cell DNA synthesis and proliferation. The ERK1/2 and p38^MAPK inhibitors PD98059 (PD) and SB203580 (SB) blocked cell proliferation, but only PD blocked cell migration. Although uPA-induced phosphorylation of both ERK1/2 and p38^MAPK was blocked by Gαi inhibition, inhibition of PI3K and Ras decreased the uPA-induced phosphorylation of ERK1/2 but not p38^MAPK. Activation of MEK1/2 was abrogated by inhibitors of Gαi and Ras, but not by PI3K inhibition. In contrast, activation of MKK3/6 was abrogated by inhibition of Gαi, but not by Ras or PI3K inhibition. uPA induced time-dependent phosphorylation of EGFR, which was dependent on plasmin activity. Inhibition of EGFR reduced both ERK1/2 and p38^MAPK activation. uPA activation of PI3K and MKK3/6 was EGFR-dependent and that of MEK1 was EGFR-independent. Conclusion: uPA induces smooth muscle cell proliferation through ERK1/2- and p38^MAPK-mediated pathways. Migration appears to be dependent on ERK1/2 activity alone. Activation of EGFR appears to be required. The differential activation of pathways for ERK1/2 and p38^MAPK by uPA allows for two distinct biologic responses that both require tyrosine kinase receptor transactivation. Clinical relevance. Elevated urokinase-like plasminogen activator (uPA) and decreased plasminogen activator inhibitor-1 (PAI-1) levels are predictors for restenosis. Matrix remodeling and smooth muscle cell responses are integrally linked. Changes in smooth muscle cell migration and proliferation are dependent on the extracellular matrix environment in which they are encased. Proteases such as uPA can effect smooth muscle cells and alter the matrix; their activity is controlled by a series of inhibitors (eg, PAI-1). The balance of activation and inhibition forms the basis of the proteolytic thermostat in the vessel wall. Understanding the biology of the proteolytic thermostat will allow for structured therapeutic interventions to control restenosis and thus improve patient care and avoid secondary interventions. Our study demonstrates that uPA is capable of inducing separate responses through more than one signaling pathway, in part, by transactivation of a nearby receptor for the unrelated ligand epidermal growth factor receptor (EGFR). Blockade of EGFR can inhibit both cell migration and proliferation induced by uPA. This is the first description of cross talk between uPA and EGFR in vascular smooth muscle cells. Targeting a pivotal receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases, is an attractive molecular target to control restenosis.