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CELLULAR AND MOLECULAR

Collagenase-2 and -3 Mediate Epidermal Growth Factor Receptor Transactivation by Bradykinin B₂ Receptor in Kidney Cells

The Medical and Research Services of the Ralph H. Johnson Veterans Affairs Medical Center and Department of Medicine (Nephrology Division) of the Medical University of South Carolina, Charleston, South Carolina

We have previously shown that stimulation of extracellular signal-regulated protein kinase (ERK) by bradykinin (BK) in murine inner medullary collecting duct (mIMCD)-3 cells is mediated by epidermal growth factor receptor (EGFR) transactivation. The mechanism of EGFR transactivation seemed to be novel, because it does not require phospholipase C, Ca²⁺, calmodulin, protein kinase C, G_i subunits, or EGFR-B₂ receptor heterodimerization. In this study, we demonstrated the involvement of matrix metalloproteinases (MMPs) in B₂ receptor-induced EGFR transactivation using their broad-spectrum inhibitors batimastat and N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (Galardin) (GM-6001). Selective inhibitors for collagenase-2 and -3 (MMP-8 and MMP-13, respectively) blocked BK-induced EGFR phosphorylation and ERK activation, whereas inhibitors for MMP-1, -2, -3, -7, or -9 were without effect. Transfection of mIMCD-3 cells with MMP-8 small interfering RNA (siRNA) resulted in 50% decrease of BK-induced ERK activation. A neutralizing antibody against MMP-13 as well as transfection with MMP-13 siRNA produced a similar effect. Inhibition of both collagenases resulted in 65% decrease of BK-induced ERK activation, supporting roles for both enzymes. Stimulation of mIMCD-3 cells with 10 nM BK increased the activity of collagenases in concentrated culture media within 10 min. Moreover, recombinant MMP-13 and MMP-8, when applied to mIMCD-3 cells for 10 min without BK, stimulated tyrosine phosphorylation of EGFR and caused 250% increase over basal ERK phosphorylation comparable with BK-induced ERK activation. Collagenases-induced ERK activation was inhibited by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478) and thus dependent on EGFR tyrosine kinase activity. This study demonstrates a novel role for collagenase-2 and -3 in signaling of the G_q-coupled BK B₂ receptor in mIMCD-3 cells.

Address correspondence to: Dr. Maria N. Garnovskaya, Medical University of South Carolina, 96 Jonathan Lucas St., Room 829 CSB, P.O. Box 250623, Charleston, SC 29425-2227. E-mail: garnovsk{at}musc.edu