Metalloprotease inhibitor blocks angiotensin II-induced migration through inhibition of epidermal growth factor receptor transactivation

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Abstract

In vascular smooth muscle cells (VSMCs), angiotensin II (AngII) induces transactivation of the EGF receptor (EGFR) which involves a metalloprotease that stimulates processing of heparin-binding EGF from its precursor. However, the identity and pharmacological sensitivity of the metalloprotease remain unclear. Here, we screened the effects of several metalloprotease inhibitors on AngII-induced EGFR transactivation in VSMCs. We found that an N-phenylsulfonyl-hydroxamic acid derivative {2R-[(4-biphenylsulfonyl)amino]-N-hydroxy-3-phenylpropinamide} (BiPS), previously known as matrix metalloprotease (MMP)-2/9 inhibitor, markedly inhibited AngII-induced EGFR transactivation, whereas the MMP-2 or -9 inhibition by other MMP inhibitors failed to block the transactivation. BiPS markedly inhibited AngII-induced ERK activation and protein synthesis without affecting AngII-induced intracellular Ca2+ elevation. VSMC migration induced by AngII was also inhibited not only by an EGFR inhibitor but also by BiPS. Thus, BiPS is a specific candidate to block AngII-induced EGFR transactivation and subsequent growth and migration of VSMCs, suggesting its potency to prevent vascular remodeling.

Section snippets

Materials and methods

Reagents and antibodies. AngII and H2O2 was obtained from Sigma. All MMP inhibitors, tissue inhibitor of metalloproteases (TIMP)-1, A23187, and AG1478 were purchased from Calbiochem. Phospho-specific anti-EGFR antibody and phospho-specific anti-JAK2 antibody were purchased from Bio-Source International. Anti-EGFR antibody and phospho-specific anti-ERK antibody were purchased from Santa Cruz Biotechnology.

Cell culture. VSMCs were prepared from thoracic aorta of 12-week-old male Sprague–Dawley

Screening of various MMP inhibitors on AngII-induced EGFR transactivation

To determine whether AngII-induced EGFR transactivation involves MMP activation, the effects of various MMP inhibitors on EGFR phosphorylation at Tyr1068, a Grb2-binding site, were examined (Fig. 1A). An N-phenylsulfonyl-hydroxamic acid derivative, BiPS, previously known as MMP-2/9 inhibitor-II [25] and CGS27023, also known as MMP-3 inhibitor-II [28], markedly inhibited AngII-induced EGFR transactivation. However, thiadiazol urea matrix metalloprotease inhibitors, also known as MMP-3

Discussion

The major findings of the present study are (i) a N-phenylsulfonyl-hydroxamic acid-derived metalloprotease inhibitor, BiPS, selectively inhibited AngII-induced EGFR transactivation without affecting intracellular Ca2+ elevation or JAK2 activation in VSMCs, (ii) Ca2+ ionophore- and H2O2-induced EGFR transactivation and AngII-induced ERK activation were inhibited by BiPS, and (iii) AngII-induced VSMC growth and migration were also blocked by BiPS through inhibition of EGFR transactivation. These

Acknowledgements

We thank Trinita Fitzgerald and Kunie Eguchi for their excellent technical assistance. This work was supported in part by the research Grants HL58205 (T. Inagami), HL03320 (E.D. Motley), and DK20593 (T. Inagami) from the National Institute of Health, Merck Postdoctoral Science Fellowship (G.D. Frank), an American Heart Association Scientist Development Grant (S. Eguchi), and Vanderbilt University Diabetes Center Pilot and Feasibility Proposal (S. Eguchi).

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