EXTRACELLULAR CALCIUM INFLUX STIMULATES METALLOPROTEINASE CLEAVAGE AND SECRETION OF HEPARIN-BINDING EGF-LIKE GROWTH-FACTOR INDEPENDENTLY OFPROTEIN-KINASE-C

The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro-form of heparin -binding epidermal growth factor-like growth factor (HB-EGF) at cell s urfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expre ssed a chimeric protein, consisting of proHB-EGF fused to placental al kaline phosphatase (AP) near the amino terminus of processed HB-EGF, i n NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Se creted HB-EGF-AP bound to heparin and exhibited potent growth factor a ctivity. The presence of the AP moiety allowed highly quantitative mea surements of cleavage-secretion responses of proHB-EGF to extracellula r stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time-and dose-dependent manner by TPA. However, this was also observed with the Ca2+ ionophore, ionomycin, suggesting the involvement of ext racellular Ca2+ ions in the secretion mechanism. Ionomycin-induced sec retion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-m ediated secretion effect was inhibited by staurosporine, GF109203X, an d by pretreatment with TPA, but not by calcium chelation. A small secr etion response was induced by thapsigargin, which releases Ca2+ from i ntracellular stores, but this was completely eliminated by extracellul ar calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion w as not dependent on the presence of the proHB-EGF cytoplasmic domain a nd was specifically inhibited by the metalloproteinase inhibitors 1,10 -phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca2+ influx activates a memb rane-associated metalloproteinase to process proHB-EGF by a pathway th at does not require PKC. (C) 1998 Wiley-Liss, Inc.