title: Multiple cyclin kinase inhibitors promote bile acid-induced apoptosis and autophagy in primary hepatocytes via p53-CD95-dependent signaling.
abstract: Previously, using primary hepatocytes residing in early G1 phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21Cip-1/WAF1/mda6 (p21) enhanced the toxicity of deoxycholic acid (DCA) + MEK1/2 inhibitor. This study examined the mechanisms regulating this apoptotic process. Overexpression of p21 or p27(Kip-1) (p27) enhanced DCA + MEK1/2 inhibitor toxicity in primary hepatocytes that was dependent on expression of acidic sphingomyelinase and CD95. Overexpression of p21 suppressed MDM2, elevated p53 levels, and enhanced CD95, BAX, NOXA, and PUMA expression; knockdown of BAX/NOXA/PUMA reduced CDK inhibitor-stimulated cell killing. Parallel to cell death processes, overexpression of p21 or p27 profoundly enhanced DCA + MEK1/2 inhibitor-induced expression of ATG5 and GRP78/BiP and phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2alpha, and it increased the numbers of vesicles containing a transfected LC3-GFP construct. Incubation of cells with 3-methyladenine or knockdown of ATG5 suppressed DCA + MEK1/2 inhibitor-induced LC3-GFP vesicularization and enhanced DCA + MEK1/2 inhibitor-induced toxicity. Expression of dominant negative PERK blocked DCA + MEK1/2 inhibitor-induced expression of ATG5, GRP78/BiP, and eIF2alpha phosphorylation and prevented LC3-GFP vesicularization. Knock-out or knockdown of p53 or CD95 abolished DCA + MEK1/2 inhibitor-induced PERK phosphorylation and prevented LC3-GFP vesicularization. Thus, CDK inhibitors suppress MDM2 levels and enhance p53 expression that facilitates bile acid-induced, ceramide-dependent CD95 activation to induce both apoptosis and autophagy in primary hepatocytes.
authors.affiliation: Department of Biochemistry, Virginia Commonwealth University, Richmond, Virginia 23298-0035, USA.
authors.forename: Guo
authors.initials: G
authors.lastname: Zhang
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Park
authors.forename: Clint
authors.initials: C
authors.lastname: Mitchell
authors.forename: Teneille
authors.initials: T
authors.lastname: Walker
authors.forename: Hossein
authors.initials: H
authors.lastname: Hamed
authors.forename: Elaine
authors.initials: E
authors.lastname: Studer
authors.forename: Martin
authors.initials: M
authors.lastname: Graf
authors.forename: Mohamed
authors.initials: M
authors.lastname: Rahmani
authors.forename: Seema
authors.initials: S
authors.lastname: Gupta
authors.forename: Philip B
authors.initials: PB
authors.lastname: Hylemon
authors.forename: Paul B
authors.initials: PB
authors.lastname: Fisher
authors.forename: Steven
authors.initials: S
authors.lastname: Grant
authors.forename: Paul
authors.initials: P
authors.lastname: Dent
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D000071187:Autophagy-Related Protein 5; D001646:Bile / Q000378:metabolism; D002478:Cells, Cultured; D002756:Cholagogues and Choleretics / Q000494:pharmacology*; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003840:Deoxycholic Acid / Q000378:metabolism / Q000494:pharmacology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D015870:Gene Expression; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
chemical_list: D000225:Adenine; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; D002756:Cholagogues and Choleretics; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D003840:Deoxycholic Acid; D000091342:Endoplasmic Reticulum Chaperone BiP; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D008869:Microtubule-Associated Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051736:Proto-Oncogene Proteins c-mdm2; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D051028:bcl-2-Associated X Protein; D019892:eIF-2 Kinase; D019014:fas Receptor; C495773:Atg5 protein, mouse; C494827:Bax protein, mouse; C494828:Bax protein, rat; C000717248:Hspa5 protein, mouse; C488588:PUMA protein, mouse; C408009:Pmaip1 protein, mouse; C025946:3-methyladenine; C497207:Mdm2 protein, mouse; C497208:Mdm2 protein, rat; C482127:Map2k1 protein, mouse; C482129:Map2k2 protein, mouse