title: Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity.
abstract: Vero cell heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a 20- to 30-kDa membrane-anchored HB-EGF precursor (proHB-EGF). Localization and processing of proHB-EGF, both constitutive and 12-O-tetradecanoylphorbol 13-acetate (TPA)-inducible, was examined in Vero cells overexpressing recombinant HB-EGF (Vero H cells). Flow cytometry and fluorescence immunostaining demonstrated that Vero cell proHB-EGF is cell surface-associated and localized at the interface of cell to cell contact. Cell surface biotinylation and immunoprecipitation detected a 20- to 30-kDa heterogeneous proHB-EGF species. Vero H cell surface proHB-EGF turned over constitutively with a half-life of 1.5 h. Some of the 20- to 30-kDa cell surface-associated proHB-EGF was processed and a 14-kDa species of bioactive HB-EGF was released slowly, but most of the proHB-EGF was internalized, displaying a diffuse immunofluorescent staining pattern and accumulation of proHB-EGF in endosomes. Addition of TPA induced a rapid processing of proHB-EGF at a Pro148-Val149 site with a half-life of 7min. The TPA effect was abrogated by the protein kinase C inhibitors, staurosporine and H7. Kinetic analysis showed that loss of cell surface proHB-EGF is maximal at 30 min after addition of TPA and that proHB-EGF is resynthesized and the initial cell surface levels are regained within 12-24 h. Loss of cell surface proHB-EGF was concomitant with appearance of 14- and 19-kDa soluble HB-EGF species in conditioned medium. Vero H cell-associated proHB-EGF is a juxtacrine growth factor for EP170.7 cells in coculture. Processing of proHB-EGF resulted in loss of juxtacrine activity and a simultaneous increase in soluble HB-EGF paracrine mitogenic activity. It was concluded that processing regulates HB-EGF bioactivity by converting it from a cell-surface juxtacrine growth factor to a processed, released soluble paracrine growth factor.
authors.affiliation: Department of Biochemistry, Osaka University Medical School, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Goishi
authors.forename: S
authors.initials: S
authors.lastname: Higashiyama
authors.forename: M
authors.initials: M
authors.lastname: Klagsbrun
authors.forename: N
authors.initials: N
authors.lastname: Nakano
authors.forename: T
authors.initials: T
authors.lastname: Umata
authors.forename: M
authors.initials: M
authors.lastname: Ishikawa
authors.forename: E
authors.initials: E
authors.lastname: Mekada
authors.forename: N
authors.initials: N
authors.lastname: Taniguchi
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives*; D000470:Alkaloids / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry; D002522:Chlorocebus aethiops; D004247:DNA / Q000096:biosynthesis; D004167:Diphtheria Toxin / Q000378:metabolism; D011992:Endosomes / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D018942:Macrolides; D008970:Molecular Weight; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011498:Protein Precursors / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014709:Vero Cells
chemical_list: D000470:Alkaloids; D000596:Amino Acids; D000900:Anti-Bacterial Agents; D004167:Diphtheria Toxin; D004791:Enzyme Inhibitors; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007546:Isoquinolines; D018942:Macrolides; D010879:Piperazines; D011498:Protein Precursors; D004815:Epidermal Growth Factor; C080501:1-(5-isoquinolinylsulfonyl)-3-methylpiperazine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C040929:bafilomycin A1; D004247:DNA; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate