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

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.

BAG-1 Is a Novel Cytoplasmic Binding Partner of the Membrane Form of Heparin-binding EGF-like Growth Factor

“…NRK52E and COS7 cells were grown in Dulbecco's modified Eagle's medium, 10% fetal bovine serum. MC2 cells were cultured in T medium as described (13). All cells were maintained in a humidified atmosphere of 95% air, 5% CO 2 at 37°C.…”

“…The proHB-EGF-AP fusion construct (pRc/CMV-proHB-EGF-AP) and the tail-less form of this construct (pRc/CMV-proHB-EGF (⌬tail)-AP) have been described (13). A cDNA fragment encoding BAG-1 was excised from pJG45-mBAG-1 (clone B11) and subcloned into the EcoRI and XhoI sites of either pcDNA3.1/Myc-His (ϩ) or pcDNA6/His (Invitrogen, Carlsbad, CA) using standard protocols.…”

“…Each population was then transfected with either pcDNA6/His-BAG-1 or the control vector, pcDNA6/His-LacZ and stable transfectants were selected by blasticidin (Invitrogen). MC2-proHB-EGF-AP and MC2-proHB-EGF(⌬tail)-AP cloned cells have been described (13) and were double transfected by pcDNA6/His-BAG-1 or pcDNA6/His-LacZ as above. Cell populations were selected and maintained in medium supplemented with 300 g/ml G418 and/or 5 g/ml blasticidin.…”

“…HB-EGF Secretion Assay-Secretion of HB-EGF was measured by determining levels of alkaline phosphatase (AP) in the medium using cells expressing proHB-EGF-AP fusion proteins as described previously (13). Briefly, 40,000 cells/well were seeded in 24-well plates and 24 h later, cells were stimulated by etoposide at the different concentrations under serum-free conditions.…”

Detection of preneoplasia in histologically normal prostate biopsies

“…7 ± 10 Extracellular Ca 2 in¯ux also activates invasion and malignancyassociated enzyme metalloproteinases. 11 As an extension of studies on the role of P2X receptors in the urogenital system 12 ± 16 we examined their expression in clinical biopsies that had been previously diagnosed, using a unique suite of subtype-speci®c antibodies.…”