Biochemical Journal

Research article

SUMO modification selectively regulates transcriptional activity of peroxisome-proliferator-activated receptor γ in C2C12 myotubes

Sung Soo Chung, Byung Yong Ahn, Min Kim, Jun Ho Kho, Hye Seung Jung, Kyong Soo Park

Abstract

PPAR (peroxisome-proliferator-activated receptor) γ, a nuclear receptor, can be conjugated with SUMO (small ubiquitin-like modifier), which results in the negative regulation of its transcriptional activity. In the present study, we tested whether de-SUMOylation of PPARγ affects the expression of PPARγ target genes in mouse muscle cells and investigated the mechanism by which de-SUMOylation increases PPARγ transcriptional activity. We found that the SUMO-specific protease SENP2 [SUMO1/sentrin/SMT3 (suppressor of mif two 3 homologue 1)-specific peptidase 2] effectively de-SUMOylates PPARγ–SUMO conjugates. Overexpression of SENP2 in C2C12 cells increased the expression of some PPARγ target genes, such as FABP3 (fatty-acid-binding protein 3) and CD36 (fatty acid translocase), both in the absence and presence of rosiglitazone. In contrast, overexpression of SENP2 did not affect the expression of another PPARγ target gene ADRP (adipose differentiation-related protein). De-SUMOylation of PPARγ increased ChIP (chromatin immunoprecipitation) of both a recombinant PPRE (PPAR-response element) and endogenous PPREs of the target genes CD36 and FABP3, but ChIP of the PPRE in the ADRP promoter was not affected by SENP2 overexpression. In conclusion, these results indicate that SENP2 de-SUMOylates PPARγ in myotubes, and de-SUMOylation of PPARγ selectively increases the expression of some PPARγ target genes.

  • myotube
  • peroxisome-proliferator-activated receptor γ (PPARγ)
  • peroxisome-proliferator-activated receptor-response element (PPRE)
  • small ubiquitin-like modifier (SUMO)
  • small ubiquitin-related modifier 1/sentrin/suppressor of mif two 3 homologue 1-specific peptidase 2 (SENP2)
  • transcriptional regulation

INTRODUCTION

PPARγ (peroxisome-proliferator-activated receptor γ) is a member of the nuclear hormone receptor superfamily. PPARγ forms a heterodimer with RXRα (retinoid X receptor α) and binds to PPREs (PPAR-response elements) of its target gene promoters [1]. PPARγ plays a critical role in lipogenesis and adipocyte differentiation [2,3]. TZDs (thiazolidinediones), such as troglitazone, rosiglitazone and pioglitazone, work as PPARγ ligands and increase insulin sensitivity in vivo and in vitro [4,5]. Some of these compounds have been used for the treatment of Type 2 diabetes and other conditions related to insulin resistance. Although PPARγ is mostly expressed in fat tissue, it is also expressed at a low level in other tissues, such as muscle and liver, and plays an important role in augmenting insulin sensitivity in these tissues [69]. In addition, PPARγ is also expressed in various vascular cells, including endothelial cells or vascular smooth muscle cells and monocytes, and affects vascular cell proliferation, migration and inflammatory responses [10].

Post-translational modification of PPARγ by SUMO (small ubiquitin-like modifier) has been reported. SUMOylation negatively regulates PPARγ transcriptional activity [11,12]. Lys107 of PPARγ is the major acceptor site for SUMO, and a mutation at this site (Lys→Arg) increases PPARγ transcriptional activity. Consistently, overexpression of the mutant form of PPARγ stimulates adipogenesis more efficiently than does wild-type [12]. We have reported that SUMOylation also occurs with lower affinity at Lys63 of PPARγ and that the mutation at Lys63 has little effect on the transcriptional activity of PPARγ [13]. In addition, it has been reported that SUMOylation at Lys395 involves transrepression of inflammatory responsive genes by PPARγ. Pascual et al. [14] have demonstrated that ligand-dependent SUMOylation at this site recruits co-repressor/HDAC3 (histone deaceylase 3) to inflammatory gene promoters. Although the mechanism by which SUMOylation at Lys395 affects transrepressive activity of PPARγ has been elucidated, how SUMOylation at Lys107 affects the transactivation of PPARγ remains unknown.

SUMOylation is achieved by a serial enzymatic reaction conducted by the E1 SUMO-activating enzyme (SAE1/SAE2) and the E2-conjugating enzyme (ubc9) [15]. Unlike ubiquitination, in vitro SUMOylation does not require E3 ligase activity; however, SUMO E3 ligases such as PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] family members and RanBP2 (Ran-binding protein 2) enhances SUMOylation in vivo [16,17]. Modification of proteins by SUMO is reversible, and SUMO can be removed from its substrate by the SUMO-specific protease {SENP [SUMO1/sentrin/SMT3 (suppressor of mif two 3 homologue 1)-specific peptidase]} family members [18]. There are seven SENPs identified in the human genome, and they all have a conserved C-terminal catalytic domain and non-conserved N-terminal regions. They exhibit different cellular localization and different substrate specificity [1922].

As mentioned above, the mechanism by which SUMOylation of PPARγ regulates its transactivation ability has not been identified. Therefore we have tried to understand the mechanism involved in this process. First, we have demonstrated that SENP2 effectively removes SUMO from PPARγ–SUMO conjugates and increases the transcriptional activity of PPARγ. Next, we have demonstrated that overexpression of SENP2 in C2C12 myotubes increases the expression of some PPARγ target genes, such as FABP3 (fatty-acid-binding protein 3) and CD36 (fatty acid translocase), but has no effect on the expression of ADRP (adipose differentiation-related protein), another PPARγ target gene. Results from ChIP (chromatin immunoprecipitation) analysis showed the possibility that de-SUMOylation of PPARγ increases binding of PPARγ to the PPRE in a target gene-specific manner.

EXPERIMENTAL

Plasmids and adenovirus

The mouse PPARγ2 expression vector pSG-PPARγ2 was generously provided by Dr J.B. Kim (Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea). The HA (haemagglutinin)-tagged PPARγ expression vector (pcDNA-HA-PPARγ) was constructed by subcloning a PPARγ cDNA fragment downstream of the HA sequence that had been inserted into a pcDNA3 vector (Invitrogen). Lys107 was mutated to an arginine or asparagine residue using the QuikChange® Site-Directed Mutagenesis kit (Stratagene) to generate pcDNA-HA-PPARγK107R and pcDNA-HA-PPARγK107N. Myc-tagged SUMO1 and SENP1 expression vectors and FLAG-tagged SUMO1, SENP2 and SENP3 expression vectors were generously provided by Dr C. H. Chung (School of Biological Sciences, Seoul National University, Seoul, Korea). Two SENP2 mutants, C548S and RK576/577LM, were generated by site-directed mutagenesis (Stratagene). pPPRE-pk-Luc was generously provided by Dr S. H. Koo (Department of Molecular Cell Biology, Sungkyunkwan University, Gyeonggi-do, Korea). Adenoviruses harbouring the expression systems for PPARγ, PPARγ K107R, PPARγ DN (dominant negative) and SENP2 have been described previously [13].

Cell culture

COS-7 cells were maintained in DMEM (Dulbecco's modified Eagle's medium) (Invitrogen) supplemented with 10% (v/v) FBS (fetal bovine serum). C2C12 cells were maintained in DMEM (Invitrogen) supplemented with 10% (v/v) FBS, and differentiated into myotubes by incubation with 2% (v/v) horse serum for 5–7 days.

Immunoblotting

COS-7 cells were seeded on to 60-mm-diameter dishes and transfected with 1 μg of pcDNA-HA or pcDNA-HA-PPARγ, 1 μg of SENPs and 1 μg of SUMO1-expression vectors using Lipofectamine™ with Plus™ Reagent (Invitrogen). Cells were harvested 48 h after transfection and protein samples were subjected to SDS/PAGE and immunoblot analysis with anti-FLAG (Sigma), anti-Myc (Santa Cruz Biotechnology) and anti-PPARγ (Santa Cruz Biotechnology) antibodies.

Transient transfection and reporter assay

NIH 3T3 cells were transfected with 0.3 μg of reporter plasmid (pPPRE-pk Luc), the indicated amount of expression vectors of PPARγ (pHA-PPARγ), SUMO1 (pFLAG-SUMO1), SENPs and β-galactosidase (0.1 μg) by Lipofectamine™ with Plus™ Reagent (Invitrogen). To transfect the same amount of DNA in each well, pcDNA was added. Cells were treated with rosiglitazone (10 μM) 24 h after transfection. Cells were harvested 2 days after transfection, and luciferase and β-galactosidase activity were determined according to the manufacturer's instructions (Promega).

RNA preparation and quantitative real-time PCR

Differentiated C2C12 cells were infected with the adenoviruses Ad-SENP2, Ad-PPARγ or Ad-GFP (green fluorescent protein) [50 MOI (multiplicity of infection)] for 24 h and then treated with rosiglitazone (10 μM) for an additional 24 h. For knockdown of PPARγ, PPARγ siRNAs (small interfering RNAs) (50 nM; Dharmacon) were transfected with Lipofectamine™ 2000 (Invitrogen) for 24 h before treatment with rosiglitazone. Total RNA was prepared using TRIzol® (Invitrogen) and was subjected to reverse transcription and real-time PCR. Real-time PCR was performed with TaqMan Master Mix reagents, TaqMan-specific primers (for CD36, FABP3 and ADRP) and a TaqMan ABI Prism 7000 sequence detector system (Applied Biosystems). 18S rRNA was used as an endogenous control, and experiments were performed in duplicate. For other experiments, PCR was performed with Prime Q-Mastermix (SYBR Green I) and ABI 7500 FAST real-time PCR systems (Applied Biosystems). Primers used for the real-time PCR are listed in Supplementary Table S1 (http://www.BiochemJ.org/bj/433/bj4330155add.htm).

ChIP

COS-7 cells were transfected with pPPRE-tk-Luc and the expression vectors for SUMO1, SENP2, HA–PPARγ or HA–PPARγ-K107R and treated with rosiglitazone for 24 h. After cross-linking and DNA fragmentation, nuclear extracts were immunoprecipitated with anti-PPARγ, anti-p300 (Santa Cruz Biotechnology), anti-NCoR1 (nuclear receptor co-repressor 1) (Santa Cruz Biotechnology) and anti-HA (Roche) antibodies or control IgG. Primers used for PCR were 5′-AAACGACGGCCAGTGCCAAGC-3′ and 5′-GGCGGGGCCGGATCCTCTAGAG-3′ for the PPRE-tk-Luc. For ChIP analysis of the PPREs on endogenous genes, C2C12 cells were treated with Ad-SENP2 for 48 h, and ChIP was performed with anti-PPARγ or control IgG antibodies. Primers for the PCR were: 5′-CGGCTCAAATCAGTTCCGTTG-3′ and 5′-GGTTGGTTGCCAAGGGAATTG-3′ for CD36-PPRE (−540/−339); 5′-CTTCGCGGAGTGAAGAACGAC-3′ and 5′-GGAGCCTGGACAGAGAGCATG-3′ for FABP3-PPRE (−1330/−1032); and 5′-CAAGAGGAAGTGACTCACTGGC-3′ and 5′-GGAAGGTTGAGAACCACTGCTC-3′ for ADRP-PPRE (−3412/−3246) [2325]. After PCR, band intensity was measured and normalized by total input using Labworks 4.0 (UVP).

Statistical analysis

SPSS version 10.0 was used for statistical analysis. Data are means±S.E.M., and statistical significance was calculated using the Mann–Whitney U test.

RESULTS

SENP2 de-SUMOylates PPARγ–SUMO conjugates

To identify which SENP family member can specifically remove SUMO from PPARγ–SUMO conjugates, PPARγ, SUMO and SENP (SENP1, SENP2 or SENP3) expression vectors were transfected into COS-7 cells. Immunoblot analysis clearly showed that SENP2 de-SUMOylated PPARγ (Figure 1A). SENP1, however, partially removed SUMO from the PPARγ–SUMO conjugates, and SENP3 had no effect. De-SUMOylation of PPARγ was not observed by overexpression of two inactive SENP2 mutants, C548S (C/S) and RK576/577LM (RK/LM) [26,27] (Figure 1B). These results demonstrate that SENP2 effectively de-SUMOylates PPARγ–SUMO conjugates. We also compared mRNA levels of seven SENPs (SENP1–SENP3 and SENP5–SENP8) in C2C12 myotubes. The mRNA levels of SENP2, SENP5 and SENP6 were higher than those of the other SENPs (Supplementary Figure S1 at http://www.BiochemJ.org/bj/433/bj4330155add.htm). SENP5 is localized primarily to the nucleoli and can translocate to the mitochondria [18,28]. SENP6 and SENP7 preferentially deconjugate SUMO2 and SUMO3 [18]. Therefore SENP5 and SENP6 may not play important roles in the regulation of PPARγ–SUMO modification.

Figure 1 SENP2 de-SUMOylates PPARγ–SUMO conjugates

(A) COS-7 cells were transfected with expression vectors of HA–PPARγ, SUMO1 and one of the SUMO proteases (SENP1, SENP2 or SENP3). Cell lysates were subjected to immunoblot analysis using an anti-PPARγ antibody. PPARγ–SUMO conjugates were detected after a long exposure. SENP1 was detected using an anti-Myc antibody, and SENP2 and SENP3 were detected using an anti-FLAG antibody. (B) Cells were transfected with expression vectors of HA-PPARγ, wild-type SENP2 (Wild) or the SENP2 mutants C548S (C/S) or RK576/577LM (RK/LM). Cell lysates were subjected to immunoblot analysis using an anti-PPARγ or anti-FLAG antibody. Con, control transfection.

SENP2 increases PPARγ transcriptional activity

To test whether SENP2 increases PPARγ transcriptional activity, cells were transfected with pcDNA-HA-PPARγ, pPPRE-pk-Luc and the expression vectors of SUMO and SENP2. Overexpression of SENP2 increased the transcriptional activity of PPARγ in a dose-dependent fashion both in the absence and presence of rosiglitazone, a PPARγ agonist (Figure 2A). In contrast, SENP2 did not affect the transcriptional activity of PPARγ K107N in which an asparagine residue was substituted for Lys107. These results indicate that SENP2 increased PPARγ activity through de-SUMOylation of PPARγ at Lys107, the major SUMOylation site. Two mutant forms of SENP2, C548S and RK576/577LM, did not increase PPARγ activity (Figure 2B). SENP1 slightly increased activity, but SENP3 had little effect on PPARγ transcriptional activity (Figure 2C), which is consistent with their de-SUMOylation abilities shown in Figure 1(B). These results reveal that SENP2 and SENP1 increase PPARγ activity by de-SUMOylation of PPARγ, and SENP2 has a greater effect on PPARγ activity than SENP1.

Figure 2 De-SUMOylation of PPARγ increases its transcriptional activity

(A) COS-7 cells were transfected with pPPRE-pk-Luc (0.3 μg), pCMV-β-gal (0.1 μg), pcDNA-HA-PPARγ wild-type (WT) or pcDNA-HA-PPARγ K107N mutant (0.1 μg), pFLAG-SUMO (0.1 μg) and pFLAG-SENP2 (0.05 and 0.1 μg). Cells were harvested 24 h after rosiglitazone (Rosi) treatment. Luciferase activity was normalized by β-galactosidase activity, and the value of the cells not transfected with either the PPARγ or SENP2 expression vectors was set to 1, and the other values are expressed as relative values. (B) Cells were transfected with expression vectors of wild-type SENP2 (WT) or one of its mutant forms, C548S (C/S) or RK576/577LM (RK/LM). The normalized luciferase activity of the cells not transfected with either the PPARγ or SENP2 expression vectors was set to 1, and the other values are expressed as relative values. (C) Cells were transfected with the SENP1, SENP2 or SENP3 expression vectors. *P< 0.05 compared with cells transfected with expression vectors of PPARγ WT and not treated with rosiglitazone; **P< 0.05 compared with cells transfected with expression vectors of PPARγ WT in the presence of rosiglitazone; †P< 0.05 compared with cells transfected with expression vectors of PPARγ WT and SENP2 (0.05 μg) in the absence of rosiglitazone; and ††P< 0.05 compared with cells transfected with expression vectors of PPARγ WT and SENP2 (0.05 μg) in the presence of rosiglitazone.

Effect of SENP2 overexpression on the transcription levels of PPARγ target genes in C2C12 myotubes

We also tested whether overexpression of SENP2 increases the expression of endogenous PPARγ target genes in C2C12 myotubes. C2C12 cells were differentiated into myotubes and infected with adenovirus harbouring the SENP2 expression system (Ad-hSENP2). SENP2 overexpression by infection of Ad-hSENP2 was confirmed at its protein and mRNA levels (Figure 3). SENP2 overexpression increased the transcription levels of two PPARγ target genes CD36 and FABP3 in the absence of rosiglitazone; this effect was similar to the effect of rosiglitazone alone (Figure 3B). In the presence of rosiglitazone, expression of these genes were increased further by SENP2 overexpression. In contrast, expression of another PPARγ target gene ADRP was not affected by SENP2. Taken together, these results suggest that overexpression of SENP2 selectively increases the expression of some PPARγ target genes in myotubes.

Figure 3 Overexpression of SENP2 increases the expression of the PPARγ target genes CD36 and FABP3 in C2C12 myotubes

(A) C2C12 cells were differentiated to myotubes and infected with various concentrations of Ad-hSENP2 (20, 50 and 100 MOI). Expression of hSENP2 (human SENP2) was detected by immunoblot with an anti-SENP2 antibody. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) C2C12 myotubes were infected with Ad-hSENP2 (50 MOI) for 24 h and then treated with rosiglitazone (10 μM) for another 24 h. Total RNA was isolated and subjected to reverse transcription and real-time PCR with specific probes. Normalized mRNA levels of mouse CD36, FABP3 or ADRP (mCD36, mFABP3 or mADRP) in the absence of either hSENP2 overexpression or rosiglitazone treatment was set to 1, and the other values are expressed as relative values (n=3). Expression of hSENP2 was detected only after the infection of Ad-hSENP2. *P<0.05 compared with cells not treated with either Ad-hSENP2 or rosiglitazone; and **P<0.05 compared with cells treated with Ad-hSENP2 but not treated with rosiglitazone.

Effect of SENP2 overexpression on target genes is mediated by PPARγ

To confirm that the effect of SENP2 on the expression of CD36 and FABP3 was mediated by PPARγ activation, PPARγ DN was overexpressed by infection of Ad-PPARγ DN. The PPARγ DN mutant L466A is transcriptionally inactive, but binds to PPREs and inhibits the activity of wild-type PPARγ [29]. The effect of SENP2 on the expression of CD36 and FABP3 was abolished when PPARγ activity was inhibited by overexpression of PPARγ DN (Figure 4A). In addition, the effect of SENP2 on the transcriptional levels of CD36 and FABP3 was determined after knocking down PPARγ by siRNA. The PPARγ transcript was dramatically reduced by transfection of the specific siRNA against PPARγ (siPPARγ) (Figure 4B). In the presence of siPPARγ, overexpression of SENP2 barely increased the transcription level of CD36 or FABP3. These results indicate that SENP2 increases the expression of CD36 and FABP3 by enhancing PPARγ activity, probably through de-SUMOylation of PPARγ. To confirm whether PPARγ K107R had a greater effect on the transcription of CD36 and FABP3 than wild-type PPARγ in C2C12 myotubes, Ad-PPARγ wild-type or Ad-PPARγ K107R was infected into the cells at two different doses. The mRNA levels of CD36 and FABP3 increased with the expression of PPARγ (Figure 4C). Consistent with the results obtained from SENP2 overexpression, PPARγ K107R increased the expression of CD36 and FABP3 more efficiently than wild-type PPARγ, but the expression level of ADRP was not affected by the SUMOylation status of PPARγ.

Figure 4 Effect of SENP2 overexpression is mediated by PPARγ

(A) C2C12 myotubes were infected with Ad-GFP (−), Ad-hSENP2 (50 MOI) and/or Ad-hPPARγ DN (50 MOI) for 48 h. Total RNA was isolated and subjected to reverse transcription and real-time PCR. The value from cells treated with Ad-GFP was set to 1, and the others are expressed as relative values. *P< 0.05 compared with cells treated with Ad-GFP (n=3). (B) C2C12 myotubes were transfected with siRNA against PPARγ (siPPARγ) for 24 h and then infected with Ad-GFP (−) (50 MOI) or Ad-hSENP2 (50 MOI) for an additional 48 h. Total RNA was isolated and then subjected to reverse transcription and real-time PCR (n=3). The value from cells not treated with Ad-SENP2 or siPPARγ was set to 1, and the others were expressed as relative values. *P< 0.05 compared with cells treated with Ad-GFP. h, human; m, mouse. (C) C2C12 myotubes were infected with Ad-GFP (10 or 20 MOI), Ad-PPARγ wild-type (Wild) or Ad-PPARγ K107R for 48 h. Total RNA was isolated and subjected to reverse transcription and real-time PCR. The value from cells treated with Ad-GFP (10 MOI) was set to 1, and the others are expressed as relative values (n=3). *P< 0.05 compared with cells treated with Ad-PPARγ wild-type (10 MOI); **P< 0.05 compared with cells treated with Ad-PPARγ wild-type (20 MOI); and †P< 0.05 compared with cells treated with Ad-PPARγ K107R (10 MOI).

Effect of SUMOylation of PPARγ on its binding to PPREs

We have also investigated the mechanism by which SUMOylation of PPARγ regulates its transcriptional activity. Because SUMOylation of PPARγ did not affect cellular localization of PPARγ (results not shown), we tested whether SUMOylation of PPARγ modulates the interaction between co-activator/co-repressors and PPARγ. COS-7 cells were transfected with pPPRE-tk-Luc, HA–PPARγ, SENP2 and SUMO-1 expression vectors, and ChIP was performed with anti-PPARγ, anti-p300 or anti-NCoR1 antibodies. PCR was then performed to amplify the DNA fragment that contains the PPRE sequence of the reporter construct. Binding of PPARγ to the PPRE was slightly increased by rosiglitazone and, interestingly, it was also increased by SENP2 overexpression (Figure 5A). Although rosiglitazone increased the binding of the p300 co-activator and decreased the binding of the NCoR1 co-repressor, any dramatic change in the binding of p300 or NCoR1 was not observed by SENP2 overexpression. ChIP using the anti-p300 antibody was increased by SENP2 overexpression; however, it is not certain whether it was caused by the increase in PPARγ binding to the PPRE region or by an increase in PPARγ affinity to the cofactor. For further investigation, the wild-type PPARγ or PPARγ K107R expression vectors were transfected into COS-7 cells, and ChIP was performed as described above. Consistent with the results in Figure 5(A), the binding of PPARγ K107R to the PPRE was much greater than that of wild-type PPARγ (immunoprecipitation with the anti-HA antibody) (Figure 5B). Western blot analysis of cell lysates showed that the expression level of wild-type PPARγ was similar to that of PPARγ K107R. These results suggest that SUMOylation of PPARγ may affect the binding of PPARγ to the PPRE. We also performed ChIP analysis with the endogenous PPREs of the CD36, FABP3 or ADRP promoters (Figure 5C). ChIP of the PPREs in the CD36 and FABP3 promoters was increased by the overexpression of SENP2. In contrast, ChIP of the PPRE in the ADRP promoter was not affected by overexpression of SENP2. This result is correlated well with the effect of SENP2 on the expression of these genes, indicating the possibility that the transcriptional increase in CD36 and FABP3 by SENP2 overexpression is caused by enhanced binding of PPARγ to the promoters.

Figure 5 Effect of SUMOylation of PPARγ on its binding to PPREs

(A) COS-7 cells were transfected with pPPRE-tk-Luc, pFLAG-SUMO-1 and pFLAG-SENP2, and then treated with rosiglitazone (Rosi) for 24 h. ChIP was performed with anti-PPARγ, anti-p300 or anti-NCoR1 antibodies. PCR was performed with the specific primers flanking the PPRE of pPPRE-tk-Luc. For the input, 1:10 vol. of samples for immunoprecipitation was used for PCR. Values were expressed as a percentage of the total input and are means±S.E.M. of four independent experiments. *P<0.05 compared with cells not treated with SENP2 or rosiglitazone. (B) COS-7 cells were transfected with pPPRE-tk-Luc, pcDNA-HA-PPARγ wild-type or pcDNA-HA-PPARγ K107R and pFLAG-SUMO-1, and then treated with rosiglitazone for 24 h. ChIP was performed with anti-HA, anti-p300 or anti-NCoR1 antibodies (n=4). Cell lysates were also analysed by Western blot (WB) with an anti-PPARγ antibody. *P<0.05 compared with cells transfected with HA–PPARγ without rosiglitazone; **P<0.05 compared with cells transfected with HA–PPARγ with rosiglitazone. (C) C2C12 myotubes were infected with Ad-GFP or Ad-SENP2 for 48 h and then subjected to ChIP with an anti-PPARγ antibody. PCR was performed with specific primers on the flanking regions of the PPREs of CD36, FABP3 or ADRP genes (n=4). *P<0.05 compared with cells infected with Ad-GFP.

DISCUSSION

As with other nuclear receptors, PPARγ transcriptional activity is regulated by binding to several co-activators and co-repressors [30,31]. Upon binding of a ligand to PPARγ, co-repressors such as NCoRI and SMRT (silencing mediator of retinoid and thyroid receptors) dissociate from PPARγ, and co-activators including p300 and SRC1 (steroid receptor co-activator 1) are recruited to PPARγ. However, it has been reported that the PPRE of the aP2 (adipocyte fatty-acid-binding protein 2) gene is constitutively associated with co-activators and that the PPRE of the glycerol kinase gene is associated with co-repressors in the absence of ligands in adipocytes. These data suggest that co-activators/co-repressors selectively control PPARγ target genes [32]. Related to SUMOylation of PPARγ, SUMOylation of PPARγ at Lys396 has been reported to recruit NCoR1 and repress inflammatory response genes in macrophages [14]. Therefore it seems that diverse mechanisms are involved in the transcriptional regulation mediated by PPARγ, co-repressors and co-activators. In our present study, overexpression of SENP2 increased the transcription of CD36 and FABP3 by modulating PPARγ activity but did not affect the expression of ADRP, indicating that only some PPARγ target genes are selectively regulated by de-SUMOylation/SUMOylation of PPARγ at Lys107. Considering the other reports described above, the selective effect of SENP2 overexpression on PPARγ target genes is not a unique phenomenon.

In addition to ChIP, we also performed EMSAs (electrophoretic mobility-shift assays) to determine whether SUMOylation of PPARγ affected the DNA-binding activity of PPARγ. The consensus PPRE, CD36 PPRE, FABP3 PPRE or ADRP PPRE was used as a probe (approx. 35–40 bp), and nuclear extracts were prepared from C2C12 myotubes infected with Ad-GFP or Ad-SENP2. Unexpectedly, binding activity of PPARγ to the PPRE probes was not affected by SENP2 overexpression (Supplementary Figure S2 at http://www.BiochemJ.org/bj/433/bj4330155add.htm). Whereas the DNA-binding affinity of PPARγ was not affected by SUMOylation in EMSAs, results from the ChIP in Figure 5 consistently showed the effect of SUMOylation status on the binding of PPARγ to the PPREs. We cannot rule out the possibility that the affinities of the antibodies used in the ChIP were affected by SUMOylation of PPARγ and the results were misinterpreted. However, similar results were obtained using both anti-PPARγ and anti-HA antibodies (Figures 5A and 5B) and no change was detected in the ADRP PPRE (Figure 5C), which still supports the possibility that SUMOylation affects the binding of PPARγ to the PPREs. Therefore these results suggest that the SUMOylation status of PPARγ does not affect the DNA-binding affinity of PPARγ itself, and may affect its binding to the PPREs in the longer context of promoters. We still do not know how de-SUMOylation of PPARγ selectively regulates the expression of PPARγ target genes. One possible mechanism is that SUMOylation of PPARγ modulates the interaction between PPARγ and the other proteins that bind to the promoter regions, which can also explain the difference shown in the EMSA and ChIP results. Further studies are required to confirm this idea.

A very small fraction of PPARγ is SUMOylated in cells and it is hard to explain how the small amount of the SUMOylated form derives a relatively large effect. Practically, this is a common question for many SUMOylated proteins. It may be possible that SUMOylated PPARγ sequesters RXRα, a unique partner of PPARγ for dimerization, from un-SUMOylated PPARγ or affects the binding of the un-SUMOylated form through an unknown mechanism. In addition, as RXRα can also be SUMOylated and SUMOylation represses its transcriptional activity [33], the effect of SENP2 would be expected to increase if SENP2 also de-SUMOylates RXRα. We tested this possibility in the transient transfection experiments, and the results showed that SENP2 can also remove SUMO from RXRα–SUMO conjugates (results not shown).

In the present study, overexpression of SENP2 enhances the transcriptional levels of FABP3 and CD36 genes in the absence and presence of rosiglitazone (Figure 3B); however, SENP2 overexpression had little effect when PPARγ is overexpressed (results not shown). Furthermore, overexpression of SENP2 in adipocytes, where PPARγ is abundantly expressed, barely affects the expression of PPARγ target genes such as aP2 and adiponectin (results not shown). These results suggest that regulation of the PPARγ transcriptional activity by SUMOylation is more important in tissues or cells where the expression level of PPARγ is quite low. Similarly, it will be possible to manage PPARγ activity by regulating SUMOylation/de-SUMOylation states without affecting protein levels of PPARγ in muscle, liver, islet and vascular cells, which have a relatively small amount of PPARγ. For example, we have already shown that the PPARγ K107R mutant inhibits cell migration and caspase 3 activity more efficiently than wild-type in vascular smooth muscle cells [13]. In conclusion, PPARγ transcriptional activity may be selectively regulated by the SUMOylation status of PPARγ in myotubes and probably in other tissues.

AUTHOR CONTRIBUTION

Sung Soo Chung and Kyong Soo Park designed the study. Byung Yong Ahn, Min Kim and Jun Ho Kho performed the experiments. Sung Soo Chung, Byung Yong Ahn, Hye Seung Jung and Kyong Soo Park collected and analysed the data. Sung Soo Chung, Byung Yong Ahn and Kyong Soo Park wrote the paper.

FUNDING

This work was supported by 21C Frontier Functional Proteomics Project [grant number FPR08A1-070]; MarineBio21, Ministry of Maritime Affairs and Fisheries, Korea; and the WCU (World Class University) project of the MEST (Korean Ministry of Education, Science and Technology)/KOSEF (Korea Science and Engineering Foundation) [grant number R31-2008-000-10103-0].

Abbreviations: ADRP, adipose differentiation-related protein; aP2, adipocyte fatty-acid-binding protein 2; CD36, fatty acid translocase; ChIP, chromatin immunoprecipitation; DMEM, Dulbecco's modified Eagle's medium; DN, dominant negative; EMSA, electrophoretic mobility-shift assay; FABP3, fatty-acid-binding protein 3; FBS, fetal bovine serum; GFP, green fluorescent protein; HA, haemagglutinin; MOI, multiplicity of infection; NCoR1, nuclear receptor co-repressor 1; PPAR, peroxisome-proliferator-activated receptor; PPRE, PPAR-response element; RXRα, retinoid X receptor α; siRNA, small interfering RNA; SUMO, small ubiquitin-like modifier; SENP, SUMO1/sentrin/SMT3 (suppressor of mif two 3 homologue 1)-specific peptidase; TZD, thiazolidinedione

References

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