PPAR-Dependent Alteration of GRP94 Expression in Mouse Hepatocytes Neil Macdonald,* Katie Barrow,* Robert Tonge,† Matthew Davison,† Ruth A. Roberts,* ,1 and Stephan Chevalier* ,2 *Cancer Biology Group, Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield SK10 4TJ, Cheshire, United Kingdom; and †Proteomics Group EST-Biology, AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield SK10 4TJ, United Kingdom Received September 28, 2000 The adverse effects of the peroxisome proliferators (PPs), a class of rodent nongenotoxic hepatocarcino- gens, include suppression of apoptosis, induction of hepatocyte proliferation, and liver enlargement which eventually leads to tumours. The response to PPs is mediated by the peroxisome proliferator activated re- ceptor  (PPAR). We carried out proteomic analyses of PP-treated hepatocytes from wild-type and PPAR- null mice to identify the molecular pathways underly- ing the adverse effects of PPs. We have identified eigh- teen protein spots exhibiting differential expression in PP-treated wild-type mouse hepatocytes. Several proteins involved in lipid metabolism pathways, but also ATP synthase  subunit, which are regulated by PPs were identified. In addition, both 2D silver- stained gels and Western blotting analysis indicated that the anti-apoptotic glucose-regulated protein 94 (GRP94) is consistently overexpressed upon stimula- tion with PPs, providing us with novel insights into the anti-apoptotic mechanism activated by PPs. © 2000 Academic Press Key Words: GRP94; peroxisome proliferator; pro- teome; apoptosis; PPAR; hepatocyte. The peroxisome proliferator-activated receptor  (PPAR) is a ligand activated transcription factor that belongs to the nuclear receptor superfamily (1). PPAR is activated by a diverse group of ligands including fatty acids as well as chemicals belonging to the per- oxisome proliferator (PP) class of rodent nongenotoxic hepatocarcinogens, which includes chemicals with many applications in healthcare and industry. Activa- tion of PPAR is responsible for the pleiotropic effects of PPs in rodents such as enzyme induction, hepatocyte DNA synthesis, liver enlargement, and tumours (2– 4). However, the available evidence indicates that PPs do not induce tumours in humans (4 – 6). PPs such as nafenopin are thought to cause tumours in rodents by stimulating cell proliferation and suppressing apopto- sis, but the proteins or molecular mechanisms involved are unknown (7, 8). The liver response to PPs involves changes in the expression of liver proteins such as the peroxisomal -oxidation enzymes and the fatty acid transport proteins (9). While only a few of the proteins regulated by PPs have been identified at least some are nonperoxisomal, suggesting that the response to PPs involves many diverse metabolic and regulatory changes in the livers of PP-susceptible species (9). Con- ditions and factors other than PPs such as diabetes, changes in thyroid hormone levels, endotoxins, and high-fat diet can also cause peroxisome proliferation (10). The role of PPAR in many of these effects is not yet known (9) but PP-induced proteome changes in rodent liver are consistent over a range of structurally varied PPs acting through a single PPAR-mediated mechanism (11). In order to identify proteins that control suppression of apoptosis and stimulation of DNA replication in response to PPs, we have used two-dimensional poly- acrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) to identify changes in protein ex- pression after 24 h of nafenopin treatment of wild-type versus PPAR null mice primary hepatocyte cultures. Under these conditions, we identified several proteins involved in lipid metabolism pathways which are reg- ulated by PPs, such as hydroxymethylglutaryl-CoA synthase and  3,5- 2,4 dienoyl-CoA isomerase (12, 13). We have also identified nonperoxisomal protein targets such as ATP synthase  subunit and glucose- regulated protein 94 (GRP94). We show by Western blotting that expression of GRP94, which possess anti- apoptotic properties (14), is stimulated by PPs, but not 1 To whom correspondence should be addressed. Fax: +44 (0) 1625582897. E-mail: ruth.roberts@ctl.zeneca.com. 2 Present address: Pfizer, Discovery Support Laboratory, ZI de Poce-sur-Cisse, BP 159, 37401 Amboise Cedex, France. Biochemical and Biophysical Research Communications 277, 699 –704 (2000) doi:10.1006/bbrc.2000.3741, available online at http://www.idealibrary.com on 699 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.