Cyclin G2 regulates adipogenesis through PPAR gamma coactivation

Abstract

Cell cycle regulators such as cyclins, cyclin-dependent kinases, or retinoblastoma protein play important roles in the differentiation of adipocytes. In the present paper, we investigated the role of cyclin G2 as a positive regulator of adipogenesis. Cyclin G2 is an unconventional cyclin which expression is up-regulated during growth inhibition or apoptosis. Using the 3T3-F442A cell line, we observed an up-regulation of cyclin G2 expression at protein and mRNA levels throughout the process of cell differentiation, with a further induction of adipogenesis when the protein is transiently overexpressed. We show here that the positive regulatory effects of cyclin G2 in adipocyte differentiation are mediated by direct binding of cyclin G2 to peroxisome proliferator-activated receptor γ (PPARγ), the key regulator of adipocyte differentiation. The role of cyclin G2 as a novel PPARγ coactivator was further demonstrated by chromatin immunoprecipitation assays, which showed that the protein is present in the PPARγ-responsive element of the promoter of aP2, which is a PPARγ target gene. Luciferase reporter gene assays, showed that cyclin G2 positively regulates the transcriptional activity of PPARγ. The role of cyclin G2 in adipogenesis is further underscored by its increased expression in mice fed a high-fat diet. Taken together, our results demonstrate a novel role for cyclin G2 in the regulation of adipogenesis.

Figure 1. Cyclin G2 expression is increased during adipocyte differentiation

A. Quantification of mRNA expression levels by real time PCR of cyclin G2 at the indicated times of differentiation in mouse 3T3-F442A adipocytes or human primary adipocytes (C). Results were normalized by the expression levels of 18s mRNA. B. Protein expression of cyclin G2 during the indicated time points of differentiation. D. Comparative analysis of PPARγ and cyclin G2 expression by immunofluorescence in 3T3-F442A adipocytes during differentiation. Days of differentiation indicated are confluent (D0) early differentiation (D3) and terminally differentiated (D8). PPARγ expressing cells are labeled in red, cyclin G2 expressing cells in green and nuclei were visualized by Hoechst staining. E. Representative protein expression of cyclin G2 in mouse subcutaneous fat pads after 8 weeks normal diet (ND) or high fat diet (HFD). F. Densitometry analysis of cyclin G2 expression in subcutaneous fat pads of mice submited to ND (n=7) or HFD (n=7). Images were analysed by ImageJ software. G. Expression of cyclin G2 in stromal (SVF) and adipocyte (ADI) fraction of human WAT.

Figure 2. Cyclin G2 regulates adipogenesis

A. Representative micrographs of oil red O staining of 3T3-F442A cells during differentiation. Cells were either transfected with an expression vector of cyclin G2 (pCDNA3-cyclin G2) or empty vector (pCDNA3) five day after induction of differentiation. Oil red O staining was conducted at day 9 post differentiation. B. mRNA of adipocyte cells described in (A), at 7 days of differentiation was analyzed to asses the expression levels of cyclin G2 and the adipocyte markers PPARγ, lipoprotein lipase (LPL), adiponectin (Adipo), Glut 4 and aP2 by quantitative real time PCR. C. Representative micrographs of oil red O staining of 3T3-F442A cells at day 9 of differentiation. Cells were either transfected with control or mice cyclin G2 siRNAs at day 5 after induction of differentiation. D. mRNA of differentiating cells described in (C) at seven days of differentiation was analyzed to assess the expression levels of cyclin G2, PPARγ, LPL, Adipo, Glut4 and aP2 by quantitative real time PCR.

Figure 3. Cyclin G2 stimulates the PPARγ transcriptional activity

A. Activity of the PPRE-TK-Luc reporter carrying the PPARγ specific response elements measured in COS cells upon transfecting expression vectors for cyclin G2, PPARγ or both plasmids together. The experiments were performed in triplicate in the presence or absence of the PPARγ agonist rosiglitazone (10⁻⁴M) and were normalized for β-galactosidase activity. B. Activity of the UAS-TK-luc reporter measured in COS cells upon transfection of expression vectors for cyclin G2, Gal4-PPARγ–LBD or both plasmids together in the presence or absence of the PPARγ ligand rosiglitazone. C. Activity of the UAS-TK-luc reporter measured in COS cells upon transfection of expression vectors for cyclin G2, Gal4-PPARα-LBD or both plasmids together in the presence or absence of the PPARα ligand GW 3276. D. Activity of the UAS-TK-luc reporter measured in COS cells upon transfection of expression vectors for cyclin G2, Gal4-PPARδ-LBD in the presence or absence of the PPARδ ligand GW 61072. E. ChIP assay demonstrating binding of cyclin G2 to the aP2 promoter. Cross-linked chromatin from either confluent 3T3-F442A preadipocytes (lower panel) or 3T3-F442A adipocytes differentiated during 6 days (upper panels) was incubated with antibodies against PPARγ, cyclin G2 or with purified rabbit IgGs as control. Immunoprecipitates were analyzed by PCR using primers specific for the promoter region containing a PPRE of aP2, LPL and Tmeme143 genes. The input included in the PCR was conducted with 20% of the total chromatin. A region of the aP2 promoter outside the PPRE was amplified as negative control. F. Chip and Rechip assay. Cross-linked chromatin from 3T3-F442A adipocytes differentiated during 6 days was incubated with antibodies against cyclin G2. The immunoprecipitated chromatin was incubated with antibodies against PPARγ, or with purified rabbit IgGs as control. Immunoprecipitates were analyzed by PCR using primers specific for the aP2 promoter region containing a PPRE.

Figure 4. Cyclin G2 interacts with PPARγ during adipogenesis

A. Coimmunoprecipitation of PPARγ and cyclin G2 from differentiated 3T3-F442A. Extracts were imunoprecipitated with a PPARγ, cyclin G2 or rabbit IgGs and revealed with an anti-PPARγ antibody. One twentieth of the total extract is shown as control input. B. Schematic representation of the deletion GST-PPARγ constructs used in the subsequent experiments (upper panel). GST pull-down assay showing the interaction of in vitro translated cyclin G2 with the GST-DEF domain of PPARγ (lower panel). C. GST pull-down assay showing the interaction of GST-cycling G2 with in vitro translated PPARγ in the presence or absence or the PPARγ drug co-activator pioglitazone used at 100 nM. D. Schematic representation of the deletion GST-cyclin G2 constructs used in the subsequent experiments. Mutations in the LXXLL motifs are indicated. E–F. GST pull-down assay showing the interaction of in vitro translated cyclin G2 constructs as represented in D, with the GST-DEF domain of PPARγ.