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Home > Vascular Annual Meeting > bakken_cell_migration

Cell Migration in Response to the Aminoterminal Fragment of Urokinase Requires Epidermal Growth Factor Receptor Transactivation Through an ADAM-mediated Mechanism

Andrew M. Bakken, Clinton D. Protack, Elisa Roztocil, Suzanne M. Nicholl, Mark G. Davies.
Methodist Debakey Heart Center, The Methodist Hospital, Houston, Texas.

OBJECTIVES: Cell migration is an integral component of intimal hyperplasia development and proteases are pivotal in the process. The study examines the role of aminoterminal fragment of urokinase (ATF) on a pivotal cross-talk receptor, Epidermal Growth Factor Receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor tyrosine kinases and is key to many of their responses. To determine the pathway of EGFR transactivation by ATF in human vascular smooth muscle cells (VSMC)

METHODS: VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to insulin in the presence and absence of the plasmin inhibitors (ε-aminocaproic acid and aprotinin) matrix metalloprotease (MMP) inhibitor GM6001, the ADAM inhibitors TAPI-0 and TAPI-1, Heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies and the EGFR inhibitor AG1478. siRNA against EGFR and ADAM (9,10,12) were also used.

RESULTS: ATF produced concentration dependent VSMC migration, which was inhibited by increasing concentrations of AG1478. ATF was shown to induce time-dependent EGFR phosphorylation, which peaked at 4-fold greater than control. This response was inhibited by AG1478 in a concentration-dependent manner. Incubation with siRNA against EGFR blocked the ATF mediated response. Application of the plasmin inhibitors did not block the response. EGFR phosphorylation by ATF was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPsm ADAMs, or HB-EGF. Direct blockade of the EGFR prevented activation by both ATF and EGF. Incubation with siRNA ADAM-9 and -10 significantly reduced EGFR activation in response to ATF. siRNA against ADAM-12 had no effect.

CONCLUSIONS: ATF can induce transactivation of EGFR by an ADAM-mediated, HB-EGF dependent process. This is the first description of crosstalk via ADAM between ATF and EGFR in VSMC. Targeting a pivotal cross-talk receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases is an attractive molecular target

AUTHOR DISCLOSURES: A.M. Bakken, None; C.D. Protack, None; E. Roztocil, None; S.M. Nicholl, None; M.G. Davies, NIH.