title: Protein kinase Cδ protects against bile acid apoptosis by suppressing proapoptotic JNK and BIM pathways in human and rat hepatocytes.
abstract: Retained bile acids, which are capable of inducing cell death, activate protein kinase Cδ (PKC-δ) in hepatocytes. In nonhepatic cells, both pro- and antiapoptotic effects of PKC-δ are described. The aim of this study was to determine the role of PKC-δ in glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes and human HUH7-Na-taurocholate-cotransporting polypeptide (Ntcp) cells. Apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for caspase 3 cleavage. The role of PKC-δ was evaluated with a PKC activator (phorbol myristate acetate, PMA) and PKC inhibitors (chelerythrine, H-7, or calphostin), PKC-δ knockdown, and wild-type (WT) or constitutively active (CA) PKC-δ. PKC-δ activation was monitored by immunoblotting for PKC-δ Thr505 and Tyr311 phosphorylation or by membrane translocation. JNK and Akt phosphorylation and the amount of total bisindolylmaleimide (BIM) were determined by immunoblotting. GCDC induced the translocation of PKC-δ to the mitochondria and/or plasma membrane in rat hepatocytes and HUH7-Ntcp cells and increased PKC-δ phosphorylation on Thr505, but not on Tyr311, in HUH7-Ntcp cells. GCDC-induced apoptosis was attenuated by PMA and augmented by PKC inhibition in rat hepatocytes. In HUH-Ntcp cells, transfection with CA or WT PKC-δ attenuated GCDC-induced apoptosis, whereas knockdown of PKC-δ increased GCDC-induced apoptosis. PKC-δ silencing increased GCDC-induced JNK phosphorylation, decreased GCDC-induced Akt phosphorylation, and increased expression of BIM. GCDC translocated BIM to the mitochondria in rat hepatocytes, and knockdown of BIM in HUH7-Ntcp cells decreased GCDC-induced apoptosis. Collectively, these results suggest that PKC-δ does not mediate GCDC-induced apoptosis in hepatocytes. Instead PKC-δ activation by GCDC stimulates a cytoprotective pathway that involves JNK inhibition, Akt activation, and downregulation of BIM.
authors.affiliation: Department of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University, Grafton, Massachusetts; cynthia.leveille-webster@tufts.edu.
authors.forename: Cynthia R L
authors.initials: CR
authors.lastname: Webster
authors.affiliation: Department of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University, Grafton, Massachusetts;
authors.forename: Andrea N
authors.initials: AN
authors.lastname: Johnston
authors.affiliation: Department of Biomedical Sciences, Cummings School of Veterinary Medicine at Tufts University, Grafton, Massachusetts.
authors.forename: M Sawkat
authors.initials: MS
authors.lastname: Anwer
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005999:Glycochenodeoxycholic Acid / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology
chemical_list: D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C000606778:Bcl2l11 protein, rat; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D005999:Glycochenodeoxycholic Acid; D051745:Protein Kinase C-delta; D048031:JNK Mitogen-Activated Protein Kinases
keywords: BCl2 proteins; HUH7; cell death; exchange protein activated by cAMP; glycochenodeoxycholate