Mechanisms of divergent effects of activated peroxisome proliferator-activated receptor-γ on mitochondrial citrate carrier expression in 3T3-L1 fibroblasts and mature adipocytes

• Published in: Biochimica et biophysica acta 2013-06, Vol.1831 (6), p.1027-1036
• Main Authors: Bonofiglio, Daniela, Santoro, Antonella, Martello, Emanuela, Vizza, Donatella, Rovito, Daniela, Cappello, Anna Rita, Barone, Ines, Giordano, Cinzia, Panza, Salvatore, Catalano, Stefania, Iacobazzi, Vito, Dolce, Vincenza, Andò, Sebastiano
• Format: Article
• Language: English
• Subjects: 3T3-L1 Cells; acetyl coenzyme A; Adipocyte; adipocytes; Adipocytes - cytology; Adipocytes - drug effects; Adipocytes - metabolism; adipogenesis; Adipogenesis - physiology; Animals; antagonists; Apoptosis - drug effects; Blotting, Western; Cell Differentiation - drug effects; Cell Proliferation - drug effects; Cells, Cultured; cholesterol; Chromatin Immunoprecipitation; Citrate carrier; citrates; cytosol; Electrophoretic Mobility Shift Assay; fatty acids; fibroblasts; Fibroblasts - cytology; Fibroblasts - drug effects; Fibroblasts - metabolism; gene expression regulation; Hypoglycemic Agents - pharmacology; Luciferases - metabolism; messenger RNA; Mice; Mitochondria - drug effects; Mitochondria - metabolism; mitochondrial membrane; Mitochondrion; mutagenesis; Nuclear Receptor Co-Repressor 2 - antagonists & inhibitors; Nuclear Receptor Co-Repressor 2 - genetics; Nuclear Receptor Co-Repressor 2 - metabolism; PPAR gamma - genetics; PPAR gamma - metabolism; PPARgamma; Promoter Regions, Genetic - genetics; rats; Real-Time Polymerase Chain Reaction; reporter genes; Repressor Proteins - genetics; Repressor Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; RNA, Small Interfering - genetics; Sp1; Sp1 Transcription Factor - genetics; Sp1 Transcription Factor - metabolism; Thiazolidinediones - pharmacology; Transcriptional Activation; Up-Regulation
• ISSN: 1388-1981; 0006-3002; 1879-2618
• DOI: 10.1016/j.bbalip.2013.01.014

The citrate carrier (CIC), a nuclear-encoded protein located in the mitochondrial inner membrane, plays an important metabolic role in the transport of acetyl-CoA from the mitochondrion to the cytosol in the form of citrate for fatty acid and cholesterol synthesis. Citrate has been reported to be essential for fibroblast differentiation into fat cells. Because peroxisome proliferator-activated receptor-gamma (PPARγ) is known to be one of the master regulators of adipogenesis, we aimed to study the regulation of CIC by the PPARγ ligand rosiglitazone (BRL) in 3T3-L1 fibroblasts and in adipocytes. We demonstrated that BRL up-regulated CIC mRNA and protein levels in fibroblasts, while it did not elicit any effects in mature adipocytes. The enhancement of CIC levels upon BRL treatment was reversed using the PPARγ antagonist GW9662, addressing how this effect was mediated by PPARγ. Functional experiments using a reporter gene containing rat CIC promoter showed that BRL enhanced CIC promoter activity. Mutagenesis studies, electrophoretic-mobility-shift assay and chromatin-immunoprecipitation analysis revealed that upon BRL treatment, PPARγ and Sp1 are recruited on the Sp1-containing region within the CIC promoter, leading to an increase in CIC expression. In addition, mithramycin, a specific inhibitor for Sp1-DNA binding activity, abolished the PPARγ-mediated up-regulation of CIC in fibroblasts. The stimulatory effects of BRL disappeared in mature adipocytes in which PPARγ/Sp1 complex recruited SMRT corepressor to the Sp1 site of the CIC promoter. Taken together, our results contribute to clarify the molecular mechanisms by which PPARγ regulates CIC expression during the differentiation stages of fibroblasts into mature adipocytes. [Display omitted] ► PPARγ regulates CIC expression during fibroblast differentiation into fat cells. ► Activated PPARγ up-regulates CIC expression in fibroblasts, but not in adipocytes. ► In fibroblasts PPARγ ligand enhances the recruitment of PPARγ/Sp1/PGC1α、ARA70 coactivators to CIC promoter. ► In adipocytes PPARγ/Sp1/SMRT corepressor are recruited on CIC promoter. ► Divergent effects of PPARγ on CIC expression during adipogenesis are proposed.