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Heparin-Binding Epidermal Growth Factor Cleavage Mediates Zinc-Induced Epidermal Growth Factor Receptor Phosphorylation

Center for Environmental Medicine, Asthma and Lung Biology, and Department of Pharmacology, University of North Carolina at Chapel Hill; Human Studies Division, National Health Effects and Environmental Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina; and Department of Medicine, University of California San Francisco, San Francisco, California

Address correspondence to: Weidong Wu, Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. E-mail: Weidong_Wu{at}med.unc.edu

We have previously shown that exposure to zinc ions can activate epidermal growth factor (EGF) receptor (EGFR) signaling in murine fibroblasts and A431 cells through a mechanism involving Src kinase. While studying the effects of zinc ions in normal human bronchial epithelial cell, we uncovered evidence for an additional mechanism of Zn²⁺-induced EGFR activation. Exposure to Zn²⁺ induced phosphorylation of EGFR at tyrosine 1068, a major autophosphorylation site, in a dose- and time-dependent fashion. This effect of Zn²⁺ on EGFR was significantly blocked with an antibody against the ligand-binding domain of the receptor. Neutralizing antibodies against EGFR ligands revealed the involvement of heparin-binding EGF (HB-EGF) in Zn²⁺-induced EGFR phosphorylation. This observation was further supported by immunoblots showing elevated levels of HB-EGF released by Zn²⁺-exposed cells. Zymography showed the existence of matrix metalloproteinase-3 in Zn²⁺-challenged cells. Incubation with a specific matrix metalloproteinase-3 inhibitor suppressed Zn²⁺-induced EGFR phosphorylation as well as HB-EGF release. Therefore, these data support an autocrine or paracrine mechanism whereby Zn²⁺ induces EGFR phosphorylation through the extracellular release of EGFR ligands, which may be mediated by metalloproteinases.

Abbreviations: bronchial epithelial cell basal medium, BEBM • cyclooxygenase-2, COX-2 • epidermal growth factor, EGF • epidermal growth factor receptor, EGFR • heparin-binding EGF, HB-EGF • mitogen-activated protein kinases, MAPK • MAPK/ERK kinase, MEK • matrix metalloproteinase, MMP • normal human bronchial epithelial cells, NHBE • N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid, NNGH • polyacrylamide gel electrophoresis, PAGE • phosphate-buffered saline, PBS • sodium dodecyl sulfate, SDS • transforming growth factor-, TGF- • N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, TPEN