Use of a PPAR gamma-specific monoclonal antibody to demonstrate thiazolidinediones induce PPAR gamma receptor expression in vitro.
Troglitazone and rosiglitazone (BRL49653), members of the thiazolidinedione (TZD) class of antidiabetic drugs, are peroxisome proliferator-activated receptor gamma (PPARgamma) ligands that induce adipocyte differentiation and increase the expression of PPARgamma protein. Here, we report the characterization of a PPARgamma specific monoclonal antibody (MAb), PgammaA53.25, and its use to monitor PPARgamma expression in the noncommitted pluripotent murine mesenchymal stem cell line, C3H10T1/2, treated with TZDs. MAb PgammaA53.25 was raised against a region in the N-terminal domain of human PPARgamma shared by splice variants PPARgamma1 and PPARgamma2. It recognizes immunizing antigen in enzyme-linked immunoadsorbent assay (ELISA), and does not cross-react with the N-terminal domains of PPARalpha or PPARdelta. In Western blotting, PgammaA53.25 reacts with the immunizing antigen as well as distinct protein bands corresponding to the molecular weight of full length PPARgamma from C3H10T1/2 cells and rat tissue lysates. In fluorescent microscopy, PgammaA53.25 immunostains nuclei of C3H10T1/2 cells treated with PPARgamma ligands. The fluorescence intensity of the treated cells is TZD dose-dependent, and correlates with lipid accumulation consistent with adipogenesis. Based on these results, we propose that MAb PgammaA53.25 will be a useful tool for elucidating the role of PPARgamma in fatty acid metabolism and adipocyte differentiation.
Su
J L
JL
Department of Molecular Sciences, Glaxo Wellcome Research and Development, Research Triangle Park, NC 27709, USA.
Winegar
D A
DA
Wisely
G B
GB
Sigel
C S
CS
Hull-Ryde
E A
EA
D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002839:Chromans / Q000494:pharmacology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007004:Hypoglycemic Agents / Q000494:pharmacology; D066298:In Vitro Techniques; D051379:Mice; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D000077154:Rosiglitazone; D013045:Species Specificity; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology*; D045162:Thiazolidinediones; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D000077288:Troglitazone
D000911:Antibodies, Monoclonal; D002839:Chromans; D007004:Hypoglycemic Agents; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D013844:Thiazoles; D045162:Thiazolidinediones; D014157:Transcription Factors; D000077154:Rosiglitazone; D000077288:Troglitazone