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J. Biol. Chem., Vol. 283, Issue 36, 24343-24358, September 5, 2008

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Multiple Cyclin Kinase Inhibitors Promote Bile Acid-induced Apoptosis and Autophagy in Primary Hepatocytes via p53-CD95-dependent Signaling^*

Guo Zhang, Margaret A. Park, Clint Mitchell, Teneille Walker, Hossein Hamed, Elaine Studer, Martin Graf^¶, Mohamed Rahmani, Seema Gupta, Philip B. Hylemon, Paul B. Fisher^||**, Steven Grant^**, and Paul Dent^**1

From the Departments of Biochemistry and Molecular Biology, Hematology/Oncology, Microbiology and Immunology, ^¶Neurosurgery, ^||Human and Molecular Genetics, and ^**Institute for Molecular Medicine, Virginia Commonwealth University, Richmond, Virginia 23298-0035

Previously, using primary hepatocytes residing in early G₁ phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21^{Cip-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 eIF2, 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 eIF2 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.

Received for publication, May 6, 2008 , and in revised form, June 11, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants R01-DK52825, P01-CA104177, and R01-CA108520 (to P. D.) and R01-CA63753 and R01-CA77141 (to S. G.) from the USPHS, Department of Defense Award DAMD17-03-1-0262, The V Foundation, and a Leukemia Society of America Grant 6405-97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical College of Virginia, Virginia Commonwealth University, 401 College St., Richmond, VA 23298-0035. Tel.: 804-628-0861; Fax: 804-827-1309; E-mail: pdent{at}vcu.edu.

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