title: Insufficiency of pro-heparin-binding epidermal growth factor-like growth factor shedding enhances hypoxic cell death in H9c2 cardiomyoblasts via the activation of caspase-3 and c-Jun N-terminal kinase.
abstract: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cardiogenic and cardiohypertrophic growth factor. ProHB-EGF, a product of the Hb-egf gene and the precursor of HB-EGF, is anchored to the plasma membrane. Its ectodomain region is shed by a disintegrin and metalloproteases (ADAMs) when activated by various stimulations. It has been reported that an uncleavable mutant of Hb-egf, uc-Hb-egf, produces uc-proHB-EGF, which is not cleaved by ADAMs and causes dilation of the heart in knock-in mice. This suggests that the shedding of proHB-EGF is essential for the development and survival of cardiomyocytes: however, the molecular mechanism involved has remained unclear. In this study, we investigated the relationship between uc-proHB-EGF expression and cardiomyocyte survival. Human uc-proHB-EGF was adenovirally introduced into the rat cardiomyoblast cell line H9c2, and the cells were cultured under normoxic and hypoxic conditions. Uc-proHB-EGF-expressing H9c2 cells underwent apoptosis under normoxic conditions, which distinctly increased under hypoxic conditions. Furthermore, we observed an increased Caspase-3 activity, reactive oxygen species accumulation, and an increased c-Jun N-terminal kinase (JNK) activity in the uc-proHB-EGF-expressing H9c2 cells. Treatment of the uc-proHB-EGF transfectants with inhibitors of Caspase-3, reactive oxygen species, and JNK, namely, Z-VAD-fmk, N-acetylcysteine, and SP600125, respectively, significantly reduced hypoxic cell death. These data indicate that insufficiency of proHB-EGF shedding under hypoxic stress leads to cardiomyocyte apoptosis via Caspase-3- and JNK-dependent pathways.
authors.affiliation: Department of Biochemistry and Molecular Genetics, Center for Regenerative Medicine (CEREM), Ehime University, Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan.
authors.forename: Teruyoshi
authors.initials: T
authors.lastname: Uetani
authors.forename: Hironao
authors.initials: H
authors.lastname: Nakayama
authors.forename: Hideki
authors.initials: H
authors.lastname: Okayama
authors.forename: Takafumi
authors.initials: T
authors.lastname: Okura
authors.forename: Jitsuo
authors.initials: J
authors.lastname: Higaki
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Inoue
authors.forename: Shigeki
authors.initials: S
authors.lastname: Higashiyama
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D019483:Disintegrins / Q000235:genetics / Q000378:metabolism; D016166:Free Radical Scavengers / Q000494:pharmacology; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation; D032386:Myoblasts, Cardiac / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000873:Anthracenes; D015853:Cysteine Proteinase Inhibitors; D019483:Disintegrins; D016166:Free Radical Scavengers; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; C587885:Hbegf protein, rat; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D017382:Reactive Oxygen Species; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C432165:pyrazolanthrone; D048031:JNK Mitogen-Activated Protein Kinases; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; D053148:Caspase 3; D000111:Acetylcysteine