title: ZnT-1 protects HL-1 cells from simulated ischemia-reperfusion through activation of Ras-ERK signaling.
abstract: Activation of ERK signaling may promote cardioprotection from ischemia-reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells overexpressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.
authors.affiliation: Department of Physiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
authors.forename: Ofer
authors.initials: O
authors.lastname: Beharier
authors.forename: Shani
authors.initials: S
authors.lastname: Dror
authors.forename: Shiri
authors.initials: S
authors.lastname: Levy
authors.forename: Joy
authors.initials: J
authors.lastname: Kahn
authors.forename: Merav
authors.initials: M
authors.lastname: Mor
authors.forename: Sharon
authors.initials: S
authors.lastname: Etzion
authors.forename: Daniel
authors.initials: D
authors.lastname: Gitler
authors.forename: Amos
authors.initials: A
authors.lastname: Katz
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Muslin
authors.forename: Arie
authors.initials: A
authors.lastname: Moran
authors.forename: Yoram
authors.initials: Y
authors.lastname: Etzion
mesh_terms: D000818:Animals; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death; D002460:Cell Line; D002470:Cell Survival; D004789:Enzyme Activation / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D051381:Rats; D015427:Reperfusion Injury / Q000139:chemically induced* / Q000378:metabolism*; D015032:Zinc / Q000633:toxicity
chemical_list: D027682:Cation Transport Proteins; D005419:Flavonoids; C485803:Slc30a1 protein, mouse; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras); D015032:Zinc; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one