Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C

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Abstract
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 surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB‐EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB‐EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB‐EGF, in NbMC‐2 prostate epithelial cells. The proHB‐EGF‐AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB‐EGF‐AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage‐secretion responses of proHB‐EGF to extracellular 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 extracellular Ca2+ ions in the secretion mechanism. Ionomycin‐induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA‐mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca2+ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin‐ and TPA‐induced HB‐EGF‐AP secretion was not dependent on the presence of the proHB‐EGF cytoplasmic domain and 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 membrane‐associated metalloproteinase to process proHB‐EGF by a pathway that does not require PKC. J. Cell. Biochem. 69:143–153, 1998.

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