Regulation of CD95 (Fas) Expression and Fas-Mediated Apoptotic Signaling in HLE B-3 Cells by 4-Hydroxynonenal ² Jie Li, ‡,§ Rajendra Sharma, ‡,§ Brad Patrick, ‡ Abha Sharma, ‡ Prince V. S. Jeyabal, ‡ Prasada M. R. V. Reddy, ‡ Manjit K. Saini, ‡ Seema Dwivedi, ‡ Shaheen Dhanani, ‡ Naseem H. Ansari, ‡ Piotr Zimniak, | Sanjay Awasthi, ⊥ and Yogesh C. Awasthi* ,‡ Department of Biochemistry and Molecular Biology, UniVersity of Texas Medical Branch, GalVeston, Texas 77555, Department of Pharmacology and Department of Biochemistry and Molecular Biology, UniVersity of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205, and Department of Chemistry and Biochemistry, UniVersity of Texas, Arlington, Texas 76019 ReceiVed April 21, 2006; ReVised Manuscript ReceiVed July 25, 2006 ABSTRACT: The Fas (apo/CD95) receptor which belongs to the TNF-R family is a transmembrane protein involved in the signaling for apoptosis through the extrinsic pathway. During this study, we have examined a correlation between intracellular levels of 4-HNE and expression of Fas in human lens epithelial (HLE B-3) cells. Our results show that in HLE B-3 cells, Fas is induced by 4-HNE in a concentration- and time-dependent manner, and it is accompanied by the activation of JNK, caspase 3, and the onset of apoptosis. Fas induction and activation of JNK are also observed in various tissues of mGsta4 null mice which have elevated levels of 4-HNE. Conversely, when 4-HNE is depleted in HLE B-3 cells by a transient transfection with hGSTA4, Fas expression is suppressed. However, upon the cessation of hGSTA4 expression in these transiently transfected cells, Fas and 4-HNE return to their basal levels. Fas-deficient transformed HLE B-3 cells stably transfected with hGSTA4 show remarkable resistance to apoptosis. Also, the wild- type HLE B-3 cells in which Fas is partially depleted by siRNA acquire resistance to 4-HNE-induced apoptosis, suggesting an at least partial role of Fas in 4-HNE-induced apoptosis in HLE B-3 cells. We also demonstrate that during 4-HNE-induced apoptosis of HLE B-3 cells, Daxx is induced and it binds to Fas. Together, these results show an important role of 4-HNE in regulation of the expression and functions of Fas. Fas antigen (apo-1/CD95) is a cell surface transmembrane glycoprotein that belongs to the tumor necrosis factor receptor (TNF-R) superfamily, which induces cell death by binding to its natural ligand (Fas L) (1, 2). The Fas-Fas L system is recognized as one of the major extrinsic pathways for induction of apoptosis in cells and is believed to have an essential role in maintaining homeostasis by regulating cell proliferation, differentiation, and apoptosis (3). Fas is expressed in many tissues; high expression levels are observed in the thymus, heart, liver, and kidney (4). Deficiency of Fas or Fas L in epithelial cells prevents apoptosis of these cells in ViVo as well as in Vitro (5). It has been suggested that resistance to apoptosis through the blockage of the Fas receptor might play an important role in tumorigenesis and tumor progression in several malignan- cies (6, 7). In addition to its important role in the immune system, it has been suggested that the Fas-Fas L system may play an important role in the pathogenesis of diseases which are characterized by the regulation of apoptosis (8). A multitude of studies during the past decade indicate a key role of 4-hydroxynonenal (4-HNE) 1 in various signaling pathways (9-26), including those for apoptosis. 4-HNE has been shown to modulate the expression and functions of certain key regulator membrane proteins such as tyrosine kinase receptors (21), PKC (27), and JNK (14). The role of 4-HNE in the regulation of Fas expression or its involvement in Fas-mediated apoptosis is not known and needs to be investigated. Our previous studies (28, 29) have shown that depletion of 4-HNE in human lens epithelial cells (HLE B-3) by transfection with the 4-HNE metabolizing glutathione S-transferase (GST) isozyme hGSTA4-4 leads to a dramatic phenotypic transformation of these cells. The transformed cells grow indefinitely in suspension as smaller, rounded cells as opposed to wild-type HLE B-3 cells which grow as attached cells with a limited life span. These transformed ² Support for this study was provided in part by NIH Grants EY 04396, ES021171 (Y.C.A.), CA77495 (S.A.), EY 013014 (N.H.A.), and ES 07804 (P.Z.). B.P. is supported by NIEHS Toxicology Training Grant ES 007254. Help from the core facilities of NIEHS Center Grant ES06676 is acknowledged. * To whom correspondence should be addressed: Department of Biochemistry and Molecular Biology, 551 Basic Science Bldg., Univers- ity of Texas Medical Branch, Galveston, TX 77555-0647. Telephone: (409) 772-2735. Fax: (409) 772-6603. E-mail: ycawasth@utmb.edu. ‡ University of Texas Medical Branch. § These authors contributed equally to this work. | University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System. ⊥ University of Texas. 1 Abbreviations: 4-HNE, 4-hydroxynonenal; PKC, protein kinase C; JNK, c-jun N-terminal terminal kinase; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; RIPA, radioimmunoprecipitation assay buffer; PMSF, phenylmethanesulfonyl fluoride; BSA, bovine serum albumin; ROS, reactive oxygen species; PARP, poly ADP-ribose polymerase; WT, wild-type; VT, vector-transfected; Tr, hGSTA4- transfected. 12253 Biochemistry 2006, 45, 12253-12264 10.1021/bi060780+ CCC: $33.50 © 2006 American Chemical Society Published on Web 09/16/2006