Small Ubiquitin-like Modifier (SUMO) Protein-specific Protease 1 De-SUMOylates Sharp-1 Protein and Controls Adipocyte Differentiation*

Background: It is unknown whether SENP1 controls adipocyte differentiation. Results: SENP1 de-SUMOylates Sharp-1 and promotes PPARγ expression and adipogenesis. Conclusion: SENP1 regulates adipocyte differentiation. Significance: SENP1 is a novel regulator in adipocyte differentiation. Adipocyte differentiation is regulated by a transcriptional cascade that mainly includes CCAAT/enhancer-binding protein family members and the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). Here we show the defects in adipocyte differentiation as well as PPARγ expression in Senp1−/− mouse embryonic fibroblast cells induced by adipogenic stimuli. We further determine that SENP1 is a specific de-SUMOylation protease for Sharp-1, a repressor for PPARγ transcription and adipogenesis. SENP1 enhances adipogenesis through de-SUMOylation of Sharp-1, which then releases Sharp-1 repression of PPARγ expression and adipocyte differentiation. These results reveal SENP1 as a novel regulator in adipogenesis.

Adipocyte differentiation is regulated by a transcriptional cascade that mainly includes CCAAT/enhancer-binding protein (C/EBP) 2 family members (i.e. C/EBP␣, C/EBP␤, and C/EBP␦) and the nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥). This transcriptional cascade directs the extensive programming of gene expression required to convert preadipocytes to mature adipocytes (1)(2)(3). C/EBPs are early adipogenic transcription factors that are induced within hours of initiation of adipogenesis. C/EBP␤ directly binds to the PPAR␥2 promoter and activates PPAR␥2 expression (4). C/EBP␤ and C/EBP␦ also activate the expression of another principal adipogenic transcription factor, C/EBP␣ (5).
PPAR␥ mainly coordinates with C/EBP␣ in a positive feedback loop to promote the terminal differentiation of adipocytes (6).
It is well documented that the activity of C/EBPs and PPAR␥ is tightly controlled by distinct regulators in adipocyte differentiation. These regulators include transcriptional coactivators and corepressors, Wnt and FGF signaling, as well as protein posttranslational modification enzymes (3). Sharp-1, a basic helix-loop-helix transcription factor, is one of the transcriptional corepressors modulating C/EBPs and PPAR␥ activity. Sharp-1 binds to class B E-box sites with high affinity to repress the transcription of target genes and also associates with distinct corepressors, including HDAC1, SIRT1, and the histone methyltransferase G9a, to inhibit gene transcription (7). Taneja and co-workers (8) found that Sharp-1 interacts with and inhibits the transcriptional activity of both C/EBP␤ and C/EBP␣ by retaining HDAC1 and G9a at the C/EBP regulatory sites on the C/EBP␣ and PPAR␥2 promoters to inhibit their expression and, thus, adipogenesis, identifying Sharp-1 function as a negative regulator during adipogenesis.
In this study, Senp1 Ϫ/Ϫ MEF cells show defects in adipocyte differentiation and PPAR␥ expression induced by adipogenic stimuli. A mechanism study found that SENP1 de-SUMOylates Sharp-1 and releases Sharp-1 inhibition of PPAR␥ expression and adipogenesis. These results reveal a role of SENP1 in control of Sharp-1 activity and adipocyte differentiation.
Adipocyte Differentiation and Oil Red O Staining-MEF and 3T3-L1 cells were cultured in DMEM (Hyclone) containing 10% FBS (Invitrogen) and 1% antibiotics until reaching full confluence. Two days later (day 2), differentiation was induced by addition of insulin (5 g/ml, Sigma), dexamethasone (1 M, Sigma), isobutyl-1-methylxanthine (0.5 mM, Sigma), and rosiglitazone (1 M, Sigma). On day 2, the medium was replaced with the same medium containing 5 g/ml insulin and 1 M rosiglitazone. This medium was changed every 2 days until the end of differentiation. Oil Red O (Sigma) staining and quantification were performed as described previously (8).
Transfection-Plasmids were transfected into 293T cells using Lipofectamine TM 2000 (Invitrogen). MEF and 3T3-L1 cells were transfected with plasmids by electroporation with a NEPA21 system (NEPA GENE) according to the instructions of the manufacturer. Briefly, 1 ϫ 10 6 MEF and 3T3-L1 cells (100 l of Opti-MEM/10 g of a plasmid/cuvette) were electroporated under the most optimal conditions (140 V, 10 ms, twice and 275 V, 2 ms, twice, respectively). Cells were then plated on 6-well plates (3 ϫ 10 5 cells/well) and treated with differentiation mixture for analysis.
Immunoprecipitation-Cells were collected 48 h after transfection and lysed in the presence of 10 mM N-ethylmaleimide using ice-cold radioimmune precipitation assay buffer (50 mM Tris-HCl (pH 7.4), 400 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM PMSF, and protease inhibitors (Roche)). Cell lysis was performed for 30 min on ice, and the DNA in the sample was sheared with a 22-gauge needle. After centrifugation at 20,000 ϫ g for 10 min at 4°C, the supernatants were added to the appropriate antibody coupled to 20 l of protein A/G-Sepharose beads (Amersham Biosciences). The bead suspensions were rotated for 3 h at 4°C. The beads were then washed five times with radioimmune precipitation assay buffer. The immunoprecipitates were treated with 30 l of 2% SDS solution containing 5% ␤-mercaptoethanol and analyzed by Western blotting.
Luciferase Assay-293T cells in a 24-well plate were transiently transfected with expression plasmids. The cells were incubated for 24 h before luciferase was assayed using the Dual-Luciferase reporter assay system (Promega). Luc-PPAR␥ was used for construction of the mouse PPAR␥ promoter (Ϫ628ϳϩ32 bp), which was ligated between the KpnI and XhoI sites to plasmid pGL3.0-Basic. Renilla luciferase activity was used as an internal control.
Statistical Analysis-Error bars indicate mean Ϯ S.D. Statistical analysis was performed using Student's t test, and p Ͻ 0.01 was considered to be statistically significant.

SENP1 Deficiency Decreases Adipogenesis in Senp1
Ϫ/Ϫ MEF Cells-To determine whether SENP1 is involved in the regulation of adipogenesis, we first monitored the expression of SENP1 during adipogenesis. MEF cells were induced to differentiate by dexamethasone, isobutyl-1-methylxanthine, insulin, and rosiglitazone (referred to as "DMIR"). Analysis of the messenger RNA of SENP1 showed that SENP1 expression increased in the early stage (peak at day 2 after induction) and then went down to normal levels ( Fig. 1A) during differentiation induced by DMIR. This result indicates that SENP1 might be involved in the regulation of the initiation event of adipogenesis. We further compared the adipogenic ability of Senp1 ϩ/ϩ and Senp1 Ϫ/Ϫ MEF cells. Senp1 ϩ/ϩ and Senp1 Ϫ/Ϫ MEF cells were induced by the adipocyte differentiation stimulant DMIR for 8 days. The differentiated adipocytes were stained with Oil Red O. As shown in Fig. 1, B and C, there was less red staining in Senp1 Ϫ/Ϫ MEF cells than that in the Senp1 ϩ/ϩ control. The lipid droplet formation in Senp1 Ϫ/Ϫ MEF cells was also much less than that in Senp1 ϩ/ϩ MEF cells. These data suggest an essential role of SENP1 in adipocyte differentiation.
Adipogenically Related Genes Are Down-regulated in Senp1 Ϫ/Ϫ MEF Cells during Adipocyte Differentiation-To understand the molecular basis in SENP1 regulation of adipogenesis, we analyzed the expression of adipogenically related genes in Senp1 ϩ/ϩ and Senp1 Ϫ/Ϫ MEF cells induced by DMIR. The expression of C/EBP␣/␤ and PPAR␥, the master regulators and transcription factors during adipogenesis, was down-regulated significantly in Senp1 Ϫ/Ϫ MEF cells compared with Senp1 ϩ/ϩ MEFs (Fig. 2, A-C). We also examined the expression of aP2, adiponectin, and LPL, which are indicators of the differentiated adipocyte and PPAR␥ target genes, and found that SENP1 deficiency significantly reduced the expression of these genes during adipogenesis (Fig. 2, D-F). These data reveal an essential role of SENP1 in the regulation of C/EBP␣/␤ and PPAR␥ expression in adipocyte differentiation.
SENP1 Is a Positive Regulator of PPAR␥ Transcription-Because SENP1 functions in the initiation of adipogenesis, we determined PPAR␥ expression in MEF cells at the first day of induction by DMIR. As shown in Fig. 3A, PPAR␥ mRNA increased abruptly in Senp1 ϩ/ϩ MEFs after induction. However, only a mild increase in PPAR␥ expression was detected in the induced Senp1 Ϫ/Ϫ cells, suggesting that SENP1 is essential for PPAR␥ expression in the initiation stage of adipocyte differentiation. By using PPAR␥ promoter-driven luciferase, we confirmed the role of SENP1 in promoting PPAR␥ transcription (Fig. 3B). Importantly, the SENP1 catalytic mutant could not activate PPAR␥ expression, suggesting that de-SUMOylation activity is essential for SENP1 regulation of PPAR␥ transcription. To further demonstrate the de-SUMOylation activity of SENP1 in the regulation of the expression of adipogenically related genes, we generated SENP1 wild-type-or SENP1 catalytic mutant-transfected Senp1 Ϫ/Ϫ MEF cells. SENP1 upregulated the expression of PPAR␥ and its targets aP2, adiponectin, and LPL, as shown in SENP1-transfected cells. However, the mutation of the catalytic domain abolished the SENP1 induction of these genes (Fig. 3C). These data suggest that SENP1 is a positive regulator of PPAR␥ expression through de-SUMOylation.
SENP1 De-SUMOylates Sharp-1-At the initiation of adipogenesis, C/EBP binds directly to the PPAR␥ promoter and induces PPAR␥. PPAR␥ also turns on the expression of C/EBP and then further induces PPAR␥ expression. This self-reinforcing regulatory loop is critical for PPAR␥ function in the initia-   tion of adipogenesis (1-3). However, this regulation is modulated by negative regulators such as Sharp-1, a member of the transcriptional repressor subfamily of basic helix-loop-helix transcription factors (7). Sharp-1 has been shown to be a corepressor to retain HDAC1 and G9a on the C/EBP␣ and PPAR␥2 promoter to inhibit PPAR␥ expression in adipogenesis (8). Sharp-1 has been reported to be a SUMOylated protein (21). Interestingly, Sharp-1 SUMOylation was decreased in NIH3T3-L1 cells treated with DMIR (Fig. 4A), which coincides with the up-regulation of SENP1 expression. Therefore, we proposed that SENP1 could de-SUMOylate Sharp-1, which would be essential to activate PPAR␥ expression and adipogenesis. We first tested whether SENP1 could bind to Sharp-1. As shown in Fig. 4B, Sharp-1 pulled down SENP1 from 293T cell cotransfected with Sharp-1 and SENP1. We then tested whether SENP1 could de-SUMOylate Sharp-1 in a cotransfection system. SUMOylated Sharp-1 was readily detected in the cotransfection of Sharp-1 and SUMO-1. This SUMOylated band disappeared after overexpression of the SENP1 wild type but not the catalytic mutant (Fig. 4C). To further determine the specificity of SENP1 in the de-SUMOylation of Sharp-1, we immunoprecipitated the endogenous SUMO-conjugated proteins from Senp1 ϩ/ϩ or Senp1 Ϫ/Ϫ MEF cells and detected the SUMOylated Sharp with an anti-Sharp-1 antibody. As shown in Fig. 4D, SUMO-Sharp-1 in Senp1 Ϫ/Ϫ cells was shown to be much more than that in Senp1 ϩ/ϩ cells, suggesting that SENP1 deficiency results in the accumulation of SUMOylated Sharp-1.
SUMOylation Contributes to Sharp-1 Repression of PPAR␥ Transcription-To test whether SUMOylation could modulate Sharp-1 activity in PPAR␥ expression, we compared the effect of Sharp-1 or the Sharp-1 SUMOylation mutant on PPAR␥ transcription. As shown in Fig. 5A, SUMOylationdeficient Sharp-1 showed much less repressive activity in PPAR␥ transcription than the Sharp-1 wild type, suggesting that SUMOylation could enhance Sharp-1 repression of PPAR␥ expression. This phenotype was further confirmed by coexpression of SENP1, which reduced the Sharp-1 repression of PPAR␥ luciferase activity (Fig. 5, A and B). Importantly, the SENP1 catalytic mutant could not affect Sharp-1 repression (Fig. 5B), suggesting that SENP1 regulation of PPAR␥ transcription is through de-SUMOylation of Sharp-1.
Deficiency in SUMOylation Reduces Sharp-1 Suppression of Adipogenesis-To determine whether SUMOylation has an effect on Sharp-1 regulation of adipogenesis, we generated Sharp-1 wild-type or Sharp-1 SUMOylation mutant-transfected 3T3-L1 cell lines. We first measured the expression of aP2 and PPAR␥ in these cells on day 2 after DMIR induction. Overexpression of Sharp-1 markedly reduced the expression of these genes compared with the vector control. However, Sharp-1 repression of these genes was reduced significantly in Sharp-1 SUMOylation mutant cells (Fig. 6A). We then stained these cells with Oil Red O on day 6 after DMIR induction. The Sharp-1 wild-type cells showed less red staining compared with the vector. However, the Sharp-1 SUMOylation mutant had more red color than the Sharp-1 wild type. The lipid droplet formation in the Sharp-1 cells was also much less than that in the vector cells and in Sharp-1 mutant cells were more than in the Sharp-1 wild type (Fig. 6B). To further confirm the contribution of Sharp-1 SUMOylation in SENP1 regulation of adipogenesis, we stably transfected the Sharp-1 wild type and the Sharp-1 SUMOylation mutant into Senp1 Ϫ/Ϫ MEF cells and compared their ability for adipogenesis. As shown in Fig. 6C, the Sharp-1-Senp1 Ϫ/Ϫ cells showed less red staining compared with the vector-Senp1 Ϫ/Ϫ cells. However, Sharp-1m-Senp1 Ϫ/Ϫ cells had more red color than Sharp-1-Senp1 Ϫ/Ϫ cells. The lipid droplet formation in the Sharp-1-Senp1 Ϫ/Ϫ cells was also much less than in the vector-Senp1 Ϫ/Ϫ cells. However, in Sharp-1 mutant cells, it was more than in Sharp-1-Senp1 Ϫ/Ϫ MEF cells. These data suggest that SUMOylation contributes to Sharp-1 repression of adipogenesis.

DISCUSSION
In this study, we reveal the role of SENP1 as a novel positive regulator in adipogenesis on the basis of the following evidence. First, we observed a phenotype of Senp1 Ϫ/Ϫ MEF cells showing defects in adipogenesis. Second, SENP1 can enhance the expression of PPAR␥, a master regulator of adipogenesis. Third, SENP1 can de-SUMOylate Sharp-1, a repressor for PPAR␥ transcription as well as for adipogenesis. Therefore, we propose a model in which SENP1 enhances adipogenesis through de-SUMOylation of Sharp-1, which then releases Sharp-1 repression of PPAR␥ function as well as adipocyte differentiation.
It is well documented that adipogenesis is controlled by a tightly regulated transcriptional cascade where the transcription factors activate or repress the expression of each other in a sequential manner. C/EBP and PPAR␥, as master transcription factors in adipogenesis, play a critical role in regulation of the expression of adipocyte differentiation-related genes. Adipogenic stimuli activate C/EBP, which directly binds to the PPAR␥ promoter and induces PPAR␥. PPAR␥ also turns on the expression of C/EBP and then further induces PPAR␥ expression. This self-reinforcing regulatory loop is critical for PPAR␥ function in adipogenesis. Recently, many regulators have been identified that control C/EBP and PPAR␥ activity. Sharp-1, a member of the transcriptional repressor subfamily of basic helix-loop-helix transcription factors, has been shown to be one of the negative regulators in control of C/EBP and PPAR␥ activity. Taneja and co-workers (8) reported that Sharp-1, as a corepressor, inhibits C/EBP activity by retaining HDAC1 and G9a on the C/EBP␣ and PPAR␥2 promoter to inhibit PPAR␥ expression and adipogenesis. Furthermore, Sharp-1 has been shown to be a SUMOylated protein. However, it is unknown whether SUMOylation contributes to Sharp-1 suppression of adipogenesis. In this study, we show that SUMOylation enhances Sharp-1 repression of PPAR␥ expression and adipocyte differen-  tiation. SENP1, as a de-SUMOylation protease, reduces Sharp-1 inhibition of PPAR␥ expression.
SUMOylation has emerged as a novel regulatory mechanism in adipogenesis. C/EBP and PPAR␥ are SUMOylated proteins. Dutchak et al. (22) reported that FGF21 knockout mice have a marked increase in the SUMOylation of PPAR␥ and decreased body fat, indicating that SUMOylation of PPAR␥ modulates adipogenesis. Liu et al. (23) found that PIAS1 promotes C/EBP␤ SUMOylation and inhibits adipogenesis. Chung et al. (24) found that a de-SUMOylation protease, SENP2, can directly modulate C/EBP␤ SUMOylation and its stability and, consequently, is essential for adipogenesis. Here we provide more evidence for SUMOylation in the regulation of adipogenesis by showing that SENP1, another member of SENP family, is also essential for adipogenesis. SENP1 de-SUMOylates Sharp-1, not C/EBP␤, a target of SENP2 in adipogenesis. Interestingly, Chung et al. (24) didn't detect any change of SENP1 expression in 3T3-L1 induced by DMI. However, in our study, we clearly demonstrated the increase of SENP1 in MEF cells when induced by DMIR. It is unknown whether the discrepancy results from the addition of rosiglitazone to our adipogenic stimuli. Overall, we reveal that SENP1 deconjugates SUMOylated Sharp-1, releases Sharp-1 suppression of PPAR␥ expression, and, thus, promotes adipocyte differentiation.