- Email: [email protected]
Activation of PPARγ negatively regulates O-GlcNAcylation of Sp1
Biochemical and Biophysical Research Communications 372 (2008) 713–718
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Activation of PPARc negatively regulates O-GlcNAcylation of Sp1 Sung Soo Chung 1, Ji Hyun Kim 1, Ho Seon Park, Hye Hun Choi, Kyeong Won Lee, Young Min Cho, Hong Kyu Lee, Kyong Soo Park * Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Republic of Korea
a r t i c l e
i n f o
Article history: Received 27 April 2008 Available online 28 May 2008
Keywords: O-GlcNAcylation Thiazolidinediones PPARc Sp1
a b s t r a c t O-GlcNAcylation is a kind of post-translational modification and many nuclear and cytoplasmic proteins are O-GlcNAcylated. In this study, we demonstrated that thiazolidinediones (TZDs), which are used as insulin sensitizer, specifically inhibited the O-GlcNAcylation of Sp1 but did not affect the O-GlcNAcylation of the total proteins in cell culture systems and mouse models. This effect was mediated by peroxisome proliferator activated receptor c (PPARc) activation and probably by synthesis of a specific protein induced by PPARc activation. In addition, we demonstrated that the O-GlcNAcylation sites in the zinc-finger domain were involved in the transcriptional activation of Sp1 and that rosiglitazone, a member of TZDs, affected Sp1 transcriptional activity partially by regulating the O-GlcNAcylation level of these sites. Considering the role of hexosamine biosynthesis pathway in hyperglycemia-induced insulin resistance and Sp1 in the hyperglycemia-induced gene expression, the regulation of Sp1 O-GlcNAcylation by TZDs may help to explain the function of TZDs as a treatment for insulin resistance and diabetes. Ó 2008 Elsevier Inc. All rights reserved.
O-linked N-acetylglucosamine (O-GlcNAc) modification, OGlcNAcylation, occurs at the serine or threonine residues of many nuclear and cytoplasmic proteins [1]. A very small portion (2–5%) of the fructose-6-phosphate generated by glycolysis is converted to glucosamine-6-phosphate and serves as a substrate of O-GlcNAcylation [2]. Since chronic hyperglycemia increases cellular hexosamine flux, O-GlcNAcylation is thought to be closely related to glucose toxicity. The elevation of O-GlcNAcylation disrupts insulin signaling in adipocytes and muscle tissue [3–5]. The overexpression of OGT in muscle or adipose tissue induces diabetes in transgenic mice [6]. Hyperglycemia also increases O-GlcNAcylation in pancreatic b-cells and vascular tissues, suggesting the role of O-GlcNAcylation in the development of diabetes and diabetic complication [4,7]. Since numerous transcription factors are O-GlcNAcylated, O-GlcNAc modification may regulate the transcription of various genes in response to glucose levels. Sp1 is a ubiquitously expressed transcription factor and is known to be O-GlcNAcylated. Sp1 is involved in the transcription of various genes by interaction with other transcriptional regulators and chromatin remodeling factors [8,9]. In addition to O-GlcNAcylation, the transcriptional activity of Sp1 is also regulated by phosphorylation [10]. It has been reported that there are at least
* Corresponding author. Fax: +82 2 3676 8309. E-mail address: [email protected] (K.S. Park). 1 These two authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.05.096
nine O-GlcNAcylation sites in Sp1, mainly located in the amino terminal transcription activation domain [11,12]. Recently, five O-GlcNAcylation sites were discovered at the C-terminal zinc-finger DNA-binding domain [13]. O-GlcNAcylation in the activation domain of Sp1 inhibits its transcriptional activity [14]. On the other hand, other reports show that Sp1 activity is increased by glucosamine [15]. In addition, it has been demonstrated that O-GlcNAcylation increases Sp1 protein stability and that insulin modulates the O-GlcNAcylation and phosphorylation of Sp1 [16,17]. Sp1 mediates the hyperglycemia-induced transcriptional activation of several genes, such as plasminogen activator inhibitor 1 (PAI-1), resistin, and acetyl-CoA carboxylase [15,18–20]. Based on these observations, it will be interesting to see whether the O-GlcNAcylation of Sp1 is involved in the hyperglycemia-induced transcriptional activation of these genes. Thiazolidinediones (TZDs), including troglitazone and rosiglitazone, are used as anti-diabetic drugs and specific ligands of peroxisome proliferator activated receptor c (PPARc), a member of the nuclear receptor superfamily. PPARc plays a key role in adipocyte differentiation, regulates lipid and glucose homeostasis, and improves insulin sensitivity [21,22]. We have demonstrated that Sp1 is involved in the induction of the resistin gene which is related to diabetes by a high level of glucose in adipocytes [19]. In addition, TZDs repress resistin expression through the Sp1-binding site in the resistin gene promoter and Sp1 O-GlcNAcylation is reduced by TZDs [23]. In this study, we examined the regulation of Sp1 O-GlcNAcylation by TZDs in vitro and in vivo.
714
S.S. Chung et al. / Biochemical and Biophysical Research Communications 372 (2008) 713–718
Materials and methods Plasmids and antibodies. An expression vector for the GAL4-Sp1 fusion protein, in which human Sp1 (amino acids 83–778) was fused to the DNA-binding domain of the yeast transcription factor GAL4 (amino acids 1–147), was generated by insertion of the Sp1 cDNA into the pM vector and named pM-Sp1wt. Five O-GlcNAcylation sites in the Sp1 wild-type, Ser612, Thr640, Ser641, Ser698, and Ser702 were mutated to Ala by using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and the resulting construct was named pM-Sp1mt. Immunoprecipitation or immunoblot analyses were performed with the following antibodies; Sp1 (SC-59) (Santa Cruz Biotechnology, Santa Cruz, CA), O-GlcNAc (RL2) (Affinity BioReagents), (MMS-240R) (Covance, Berkeley, CA), and phosphoserine (p3430) (Sigma–Aldrich, Louis, MO). Cell culture and adipocyte differentiation. 3T3-L1 adipocytes were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% calf serum (CS) (Invitrogen, Carlsbad, CA). Differentiation was induced by the addition of 0.5 nM of 3-isobutyl-1-methylxanthine, 0.25 lM dexamethasone (Sigma–Aldrich), and 5 lg/ml insulin to the media for 48 h. The cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 1 lg/ml insulin for an additional 10 days with the media changed every other day. HepG2 cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen). Animals. C57BLKS/J-db/db 16-week-old male mice (n = 15) and their littermates, C57BL/6J mice (n = 5), were used for these experiments (SCL, Japan). The diabetic mice (n = 15) were divided into four groups, untreated (n = 6), rosiglitazone treated for 6 h (n = 3), rosiglitazone treated for 54 h (n = 3), and insulin treated (n = 3). A rosiglitazone treated group (30 mg/kg, GlaxoSmithKline) (6 h, n = 3) was sacrificed 6 h after the rosiglitazone treatment, and the other group (54 h, n = 3) were treated with rosiglitazone every 24 h and then sacrificed 54 h after the first treatment of rosiglitazone. For the insulin treated group, the diabetic mice (n = 3) were injected with insulin (5 U) every 6 h and sacrificed 54 h after the first injection of insulin. Immunoprecipitation and immunoblot analysis. Differentiated 3T3-L1 adipocytes were treated with troglitazone or rosiglitazone (10 lM) for 24 h in low glucose media (5.5 mM), and then cells were incubated with glucosamine (5 mM) and PUGNAc (50 lM) (TRC, North York, Ont., Canada), an inhibitor of OGA (N-acetyl-bD-glucosaminase) for an additional 24 h. Cells were harvested in the lysis buffer (20 mM Tris–HCl, pH 7.4, 5 mM Na2P2O7, 100 mM NaF, 2 mM Na3VO4, 1% NP-40, 1 lg/ml aprotinin, 1 lg/ml leupeptin, and 1 mM PMSF). Mouse liver and white adipose tissues were homogenized in the same lysis buffer. The lysates (1 mg) were incubated with 4 lg of anti-Sp1 antibody for 12 h at 4 °C. The samples were subjected to SDS–PAGE and immunoblot was performed with anti-Sp1, anti-O-GlcNAc or anti-phosphoserine antibodies. RNA preparation and Northern blot analysis. 3T3-L1 adipocytes differentiated for 10 days were incubated with TZDs for 48 h. Total RNAs were extracted using Trizol (Invitrogen) according to the manufacturer’s instructions. Total RNAs (10 lg) were separated by electrophoresis and transferred to a Nytranmembrane (NY13N; Schleicher & Schuell, Keene, NH). The membranes were hybridized with the [a-32P] labeled probes for OGT and GAPDH. Transient transfection and reporter assays. HepG2 cells were cultured in DMEM supplemented with 10% FBS. Cells in 12-well plates were transfected with a total of 0.4 lg of DNA per well: 0.2 lg of pGAL4-Luc (as a reporter plasmid), 0.1 lg of pM, pMSp1wt, or pM-Sp1mt, 0.05 lg of pSG5-PPARc, and 0.05 lg of pCMV-b-galactosidase using Lipofectamine Plus (Invitrogen). The cells were treated with rosiglitazone (48 h) and glucosamine plus
PUGNAc (24 h). Luciferase activities were determined according to the Luciferase Assay System Kit (Promega, Madison, WI). Relative transfection efficiency was determined by b-galactosidase activity. Statistical analysis. SPSS, version 12.0 (SPSS Inc., Chicago, IL), was used for statistic analysis. The data were expressed as means ± SE. Statistical significance was calculated using the Mann–Whitney Utest. A p-value of less than 0.05 denoted the presence of a statistically significant difference. Results TZDs reduced the level of O-GlcNAcylated Sp1 in 3T3-L1 adipocytes To easily detect O-GlcNAcylated Sp1 in the fully differentiated 3T3-L1 adipocytes, cells were treated with glucosamine for 24 h and then harvested for immunoprecipitation with anti-Sp1 antibodies. O-GlcNAcylated Sp1 was substantially increased by the treatment of glucosamine. In other hand, pretreatment with troglitazone or rosiglitazone, two thiazolidinediones (TZDs), repressed the level of O-GlcNAcylated Sp1 in the presence of glucosamine (Fig. 1A). To distinguish whether these TZDs inhibit the O-GlcNAcylation of Sp1 or TZDs enhance O-GlcNAc dissociation from Sp1 by O-GlcNAcase (OGA), PUGNAc, a specific OGA inhibitor, was added to the media simultaneously with glucosamine. In the presence of PUGNAc, the O-GlcNAcylated Sp1 level was still reduced by TZDs, suggesting that TZDs inhibit the O-GlcNAcylation of Sp1 (Fig. 1B). To determine whether TZDs modulate the general O-GlcNAcylation state of cellular proteins, the total O-GlcNAcylated proteins were detected by western blot analysis. The O-GlcNAcylation level of total proteins was significantly increased by glucosamine and PUGNAc, but was not affected by TZDs (Fig. 1C). In addition, the result of the Northern blot analysis showed that the mRNA level of the O-linked GlcNAc transferase (OGT) was not changed by the treatment with TZDs (Fig. 1D). Since neither the O-GlcNAcylation level of the total proteins nor the expression of OGT was significantly changed by the TZDs, the regulation of the OGlcNAcylation by the TZDs was probably limited to some specific proteins, including Sp1. Effect of TZDs on O-GlcNAcylation of Sp1 was PPARc dependent To determine whether PPARc was involved in the inhibition of Sp1 O-GlcNAcylation by the TZDs, cells were treated with troglitazone or rosiglitazone in the presence of GW9662, a specific PPARc antagonist. HepG2 cells were transfected with the expression vector of Sp1 linked to GAL4DBD (Fig. 2A) and immunoprecipitation was performed with anti-GAL4DBD antibody. Troglitazone or rosiglitazone did not change the Sp1 O-GlcNAcylation level when PPARc activity was inhibited by GW9662 (Fig. 2B). In addition, the effect on the O-GlcNAcylation of Sp1 by TZDs disappeared in the presence of cycloheximide, a specific protein translation inhibitor (Fig. 2B). O-GlcNAcylation of Sp1 increased its transcriptional activity Next, we tested the effect of O-GlcNAcylation on the function of Sp1. Treatment with PUGNAc or rosiglitazone did not change the Sp1 protein level in the nucleus (data not shown). In addition, electrophoretic mobility shift assay (EMSA) results showed that O-GlcNAcylation or rosiglitazone treatment hardly affected the DNAbinding affinity of endogenous Sp1 in HepG2 cells (data not shown). Recently, five sites of Sp1 O-GlcNAcylation were identified in the zinc-finger domain, Ser612, Thr640, Ser641, Ser698 and Ser702. We made a mutant form of Sp1 in which these sites were changed
S.S. Chung et al. / Biochemical and Biophysical Research Communications 372 (2008) 713–718
715
Sp1 wild-type. O-GlcNAcylation was increased more substantially in the GAL4-Sp1 wild-type than in the GAL4-Sp1 mutant. In addition, the O-GlcNAcylation level of the wild-type was reduced by rosiglitazone and a similar effect was shown in the mutant-type (Fig. 3B). There was a significant difference in the basal transcriptional activities between the Sp1 wild-type and the mutant-type (Fig. 3C). Treatment with rosiglitazone significantly inhibited the transcriptional activity of the Sp1 wild-type but only slightly affected the activity of the mutant form. Effect of rosiglitazone on the O-GlcNAcylation level of Sp1 in vivo The O-GlcNAcylation state of Sp1 was examined in normal (blood glucose level 165.4 ± 9.6 mg/dl and body weight about 30 g) and diabetic db/db (blood glucose level 569.7 ± 30.3 mg/dl and body weight about 59.8 g) mouse models. O-GlcNAcylated Sp1 was significantly increased in the livers of diabetic mice (Fig. 4A), but the Sp1 protein level was not changed (data not shown). When diabetic mice (db/db mice) were treated with rosiglitazone for 6 or 54 h, the blood glucose levels of the mice were decreased to 349.3 ± 30.9 mg/dl and 175.0 ± 20.1 mg/dl, respectively. Treatment with rosiglitazone for 54 h effectively reduced the levels of Sp1 O-GlcNAcylation in the liver (Fig. 4B). Similar results were obtained when white adipose and kidney tissues were used for immunoprecipitation (data not shown). Consistent with the results obtained in the cell culture systems, neither the O-GlcNAcylation level for total proteins nor the Sp1 expression was significantly changed by the TZDs (data not shown). We tested the effect of rosiglitazone on another important posttranslational modification of Sp1, phosphorylation at serine residues, and the phosphorylation state of Sp1 was not changed (Fig. 4C). Since rosiglitazone reduced the blood glucose level in the diabetic mice, we determined whether the effect of the rosiglitazone on the Sp1 O-GlcNAcylation was related to the reduction of blood glucose levels. We injected db/db mice with insulin to decrease their blood glucose levels (blood glucose level 201.3 ± 52.3 mg/dl and body weight about 60.7 g), which were similar to the levels shown in the rosiglitazone treated group. There was no difference in the Sp1 O-GlcNAcylation levels of the insulin injected mice compared to the untreated mice (data not shown). Discussion
Fig. 1. TZDs reduce the level of O-GlcNAcylated Sp1. (A) Differentiated 3T3-L1 adipocytes were treated with troglitazone (Tro) or rosiglitazone (Rosi) for 24 h and then glucosamine (5 mM) was added to the media. Cells were harvested 24 h after the addition of glucosamine and cellular proteins were subjected to immunoprecipitation with anti-Sp1 antibodies. The precipitated proteins were analyzed by immunoblotting with anti-O-GlcNAc and anti-Sp1 antibodies. (B) Cells were treated with TZDs as described above and PUGNAc (50 lM) was treated simultaneously with glucosamine. (C) Total cell lysates were subjected to western blot analysis. (D) Total RNA was isolated from the cells treated with TZDs and glucosamine. The mRNA levels of OGT and GAPDH were compared by Northern blot analysis.
to alanines (Fig. 3A) and compared the O-GlcNAcylation state and the transcriptional activity of the mutant form to those of the
Our previous study showed the possibility that TZDs affected the O-GlcNAcylation level of Sp1. In this study, we demonstrated that the regulation of Sp1 O-GlcNAcylation by TZDs was mediated by PPARc activation. Probably there was an expression of an unknown gene induced by PPARc and its product may be involved in the inhibition of Sp1 O-GlcNAcylation. The reduction of O-GlcNAcylation by rosiglitazone was also observed in diabetic mice, as well as in the cell culture system. Treatment with rosiglitazone for 6 h did not change the O-GlcNAcylation level of Sp1 but longer exposure (54 h) significantly inhibited the modification, suggesting that the accumulation of an unknown protein(s) was required to exert the inhibition effect of rosiglitazone on the O-GlcNAcylation of Sp1. Even though we do not know the mechanism how TZDs regulates the Sp1 O-GlcNAcylation, it is possible that a protein having interaction with Sp1 modulates the O-GlcNAcylation of Sp1 and the expression of this protein is regulated by PPARc. In some cases, O-GlcNAcylation sites overlap with phosphorylation sites and these two modifications usually have a reciprocal relationship [24,25]. Therefore, we also cannot exclude the possibility that the phosphorylation of O-GlcNAcylation sites or neighboring regions regulates the O-GlcNAcylation of Sp1, and that PPARc modulates the phosphorylation level by changing the expression of a specific
716
S.S. Chung et al. / Biochemical and Biophysical Research Communications 372 (2008) 713–718
Fig. 2. PPARc is involved in the inhibition of Sp1 O-GlcNAcylation by TZDs. (A) A DNA fragment representing Sp1 (from amino acid 83 to 778) was ligated to GAL4DBD in pMvector. (B) HepG2 cells were transfected with pM-Sp1wt and treated with TZDs in the presence of GW9662 (10 lM) or cycloheximide (5 lM) for 24 h and then glucosamine and PUGNAc were added to the media for an additional 24 h. Cell lysates were immunoprecipitated with anti-GAL4 antibodies and immunoblot analysis was performed with anti-O-GlcNAc and anti-Sp1 antibodies.
Fig. 3. The O-GlcNAcylation sites of the zinc-finger domain of Sp1 are important for the transcriptional activity of Sp1. (A) Structure of the Sp1 wild-type (GAL4-Sp1wt) and mutant-type (GAL4-Sp1mt). (B) HepG2 cells were transfected with pM-Sp1 wt or pM-Sp1 mt and then treated with rosiglitazone, glucosamine, and PUGNAc as described in Fig. 1. Cell lysates were immunoprecipitated with anti-GAL4DBD antibodies and an immunoblot analysis was performed with anti-O-GlcNAc and anti-Sp1 antibodies. (C) HepG2 cells were transfected as described in the Materials and Methods. The luciferase activity of the cells transfected with pM in the absence of rosiglitazone was set to 1, and the other activities were presented as relative to that value. The bar graphs show the means ± SE of four independent experiments.*p < 0.05
S.S. Chung et al. / Biochemical and Biophysical Research Communications 372 (2008) 713–718
717
activation domain and zinc-finger domain were identified. We made mutations at the five O-GlcNAcylation sites at the zinc-finger domain since this domain is not only a DNA-binding region but also a region to interact with several transcriptional regulators, such as histone deacetylase 1 (HDAC1), silencing mediator for retinoid and thyroid receptor (SMRT), and nuclear receptor corepressor (NCoR) [9]. Our data showed that this region was a major part of the total O-GlcNAcylation sites and that O-GlcNAcylation at these sites was involved in its transcriptional activation. Rosiglitazone reduced O-GlcNAcylation at these sites and repressed the Sp1 transcriptional activity. Since we used GAL4 DNA-binding domainfused Sp1 constructs for these experiments, differences in the transcriptional activities of the Sp1 wild-type and mutant-type might be generated by changes in the interaction with other transcriptional regulators. Further investigation to determine the effect of the O-GlcNAcylation of this region on the interaction with corepressors or coactivators will be required to suggest a mechanism for the O-GlcNAcylation-induced transcriptional activation of Sp1. In addition, concerning our results and several previous reports, we can imagine that different O-GlcNAcylation sites may differently regulate Sp1 transcriptional activity. The hexosamine biosynthesis pathway plays a role in hyperglycemia-induced insulin resistance [26–28]. Glucosamine treatment induces insulin resistance in mice and increases the O-GlcNAcylation of skeletal muscle proteins [29,30]. Since Sp1 is involved in the hyperglycemia-induced expression of several genes, it may be possible that TZDs regulates the expression of these genes by modulating the Sp1 activity. In addition, although TZDs did not reduce the O-GlcNAcylation state of total proteins, it is possible that TZDs affect a few proteins including Sp1. Therefore the investigation of proteins whose O-GlcNAcylation is modulated by TZDs will help to explain the function of TZDs as a treatment for insulin resistance and diabetes. Acknowledgments This study was supported by a grant from the Innovative Research Institute for Cell Therapy, Republic of Korea (Project No. A062260) and a grant from Marine Biotechnology Program funded by Ministry of Land, Transport, and Maritime Affairs, Republic of Korea. References
Fig. 4. O-GlcNAcylation of Sp1 is reduced by rosiglitazone in vivo. (A) Liver proteins from normal mice (n = 5) and diabetic db/db mice (n = 6) were prepared and the protein lysates (1 mg) were immunoprecipitated with anti-Sp1 antibody. The intensity of an O-GlcNAc-Sp1 band was normalized by the intensity of the corresponding Sp1 band. The bar graphs show the means ± SE. *p < 0.05 vs normal mice. (B) Diabetic db/db mice were treated with rosiglitazone as described in the Materials and methods. The mean value of the control group (0 h, n = 6) was set to 1 and the other values (6 h, n = 3; 54 h, n = 3) were presented as relative to that. * p < 0.01 (C) Immunobloting was performed with anti-phosphoserine antibodies after the immunoprecipitation with anti-Sp1 antibodies. No significant differences were detected between the groups tested.
kinase or phosphatase. In addition, since insulin treatment did not affect the level of Sp1 O-GlcNAcylation in the mouse model, we could exclude the possibility that reduced blood glucose level by rosiglitazone was the major reason for the inhibition of Sp1OGlcNAcylation. Although several reports indicated the possibility of multiple OGlcNAcylation sites in Sp1, only a few residues in the N-terminal
[1] G.W. Hart, Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins, Annu. Rev. Biochem. 66 (1997) 315–335. [2] G. Boehmelt, I. Fialka, G. Brothers, M.D. McGinley, S.D. Patterson, R. Mo, C.C. Hui, S. Chung, L.A. Huber, T.W. Mak, N.N. Iscove, Cloning and characterization of the murine glucosamine-6-phosphate acetyltransferase EMeg32. Differential expression and intracellular membrane association, J. Biol. Chem. 275 (2000) 12821–12832. [3] L. Wells, K. Vosseller, G.W. Hart, A role for N-acetylglucosamine as a nutrient sensor and mediator of insulin resistance, Cell Mol. Life Sci. 60 (2003) 222–228. [4] M.G. Buse, Hexosamines, insulin resistance, and the complications of diabetes: current status, Am. J. Physiol. Endocrinol. Metab. 290 (2006) E1–E8. [5] G.W. Hart, M.P. Housley, C. Slawson, Cycling of O-linked beta-Nacetylglucosamine on nucleocytoplasmic proteins, Nature 446 (2007) 1017– 1022. [6] D.A. McClain, W.A. Lubas, R.C. Cooksey, M. Hazel, G.J. Parker, D.C. Love, J.A. Hanover, Altered glycan-dependent signaling induces insulin resistance and hyperleptinemia, Proc. Natl. Acad. Sci. USA 99 (2002) 10695–10699. [7] K. Liu, A.J. Paterson, E. Chin, J.E. Kudlow, Glucose stimulates protein modification by O-linked GlcNAc in pancreatic beta cells: linkage of O-linked GlcNAc to beta cell death, Proc. Natl. Acad. Sci. USA 97 (2000) 2820–2825. [8] A. Doetzlhofer, H. Rotheneder, G. Lagger, M. Koranda, V. Kurtev, G. Brosch, E. Wintersberger, C. Seiser, Histone deacetylase 1 can repress transcription by binding to Sp1, Mol. Cell Biol. 19 (1999) 5504–5511. [9] J.A. Lee, D.C. Suh, J.E. Kang, M.H. Kim, H. Park, M.N. Lee, J.M. Kim, B.N. Jeon, H.E. Roh, M.Y. Yu, K.Y. Choi, K.Y. Kim, M.W. Hur, Transcriptional activity of Sp1 is regulated by molecular interactions between the zinc finger DNA binding domain and the inhibitory domain with corepressors, and this interaction is modulated by MEK, J. Biol. Chem. 280 (2005) 28061–28071.
718
S.S. Chung et al. / Biochemical and Biophysical Research Communications 372 (2008) 713–718
[10] P. Bouwman, S. Philipsen, Regulation of the activity of Sp1-related transcription factors, Mol. Cell Endocrinol. 195 (2002) 27–38. [11] S.P. Jackson, R. Tjian, O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation, Cell 55 (1988) 125– 133. [12] M.D. Roos, K. Su, J.R. Baker, J.E. Kudlow, O glycosylation of an Sp1-derived peptide blocks known Sp1 protein interactions, Mol. Cell Biol. 17 (1997) 6472– 6480. [13] G. Majumdar, A. Harrington, J. Hungerford, A. Martinez-Hernandez, I.C. Gerling, R. Raghow, S. Solomon, Insulin dynamically regulates calmodulin gene expression by sequential o-glycosylation and phosphorylation of sp1 and its subcellular compartmentalization in liver cells, J. Biol. Chem. 281 (2006) 3642– 3650. [14] X. Yang, K. Su, M.D. Roos, Q. Chang, A.J. Paterson, J.E. Kudlow, O-linkage of Nacetylglucosamine to Sp1 activation domain inhibits its transcriptional capability, Proc. Natl. Acad. Sci. USA 98 (2001) 6611–6616. [15] H.J. Goldberg, C.I. Whiteside, I.G. Fantus, The hexosamine pathway regulates the plasminogen activator inhibitor-1 gene promoter and Sp1 transcriptional activation through protein kinase C-beta I and -delta, J. Biol. Chem. 277 (2002) 33833–33841. [16] I. Han, J.E. Kudlow, Reduced O glycosylation of Sp1 is associated with increased proteasome susceptibility, Mol. Cell Biol. 17 (1997) 2550–2558. [17] K. Su, M.D. Roos, X. Yang, I. Han, A.J. Paterson, J.E. Kudlow, An N-terminal region of Sp1 targets its proteasome-dependent degradation in vitro, J. Biol. Chem. 274 (1999) 15194–15202. [18] H.J. Goldberg, J. Scholey, I.G. Fantus, Glucosamine activates the plasminogen activator inhibitor 1 gene promoter through Sp1 DNA binding sites in glomerular mesangial cells002C, Diabetes 49 (2000) 863–871. [19] S.S. Chung, H.H. Choi, K.W. Kim, Y.M. Cho, H.K. Lee, K.S. Park, Regulation of human resistin gene expression in cell systems: an important role of stimulatory protein 1 interaction with a common promoter polymorphic site, Diabetologia 48 (2005) 1150–1158.
[20] S. Daniel, K.H. Kim, Sp1 mediates glucose activation of the acetyl-CoA carboxylase promoter, J. Biol. Chem. 271 (1996) 1385–1392. [21] M. Lehrke, M.A. Lazar, The many faces of PPARgamma, Cell 123 (2005) 993– 999. [22] S.M. Rangwala, M.A. Lazar, Peroxisome proliferator-activated receptor gamma in diabetes and metabolism, Trends Pharmacol. Sci. 25 (2004) 331–336. [23] S.S. Chung, H.H. Choi, Y.M. Cho, H.K. Lee, K.S. Park, Sp1 mediates repression of the resistin gene by PPARgamma agonists in 3T3-L1 adipocytes, Biochem. Biophys. Res. Commun. 348 (2006) 253–258. [24] F.I. Comer, G.W. Hart, O-glycosylation of nuclear and cytosolic proteins. Dynamic interplay between O-GlcNAc and O-phosphate, J. Biol. Chem. 275 (2000) 29179–29182. [25] F.I. Comer, G.W. Hart, Reciprocity between O-GlcNAc and O-phosphate on the carboxyl terminal domain of RNA polymerase II, Biochemistry 40 (2001) 7845–7852. [26] L.F. Hebert Jr., M.C. Daniels, J. Zhou, E.D. Crook, R.L. Turner, S.T. Simmons, J.L. Neidigh, J.S. Zhu, A.D. Baron, D.A. McClain, Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance, J. Clin. Invest. 98 (1996) 930–936. [27] D.A. McClain, E.D. Crook, Hexosamines and insulin resistance, Diabetes 45 (1996) 1003–1009. [28] A.D. Baron, J.S. Zhu, J.H. Zhu, H. Weldon, L. Maianu, W.T. Garvey, Glucosamine induces insulin resistance in vivo by affecting GLUT 4 translocation in skeletal muscle. Implications for glucose toxicity, J Clin Invest 96 (1995) 2792–2801. [29] H. Yki-Jarvinen, A. Virkamaki, M.C. Daniels, D. McClain, W.K. Gottschalk, Insulin and glucosamine infusions increase O-linked N-acetyl-glucosamine in skeletal muscle proteins in vivo, Metabolism 47 (1998) 449–455. [30] L. Rossetti, M. Hawkins, W. Chen, J. Gindi, N. Barzilai, In vivo glucosamine infusion induces insulin resistance in normoglycemic but not in hyperglycemic conscious rats, J. Clin. Invest. 96 (1995) 132–140.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Activation of PPARc negatively regulates O-GlcNAcylation of Sp1 Sung Soo Chung 1, Ji Hyun Kim 1, Ho Seon Park, Hye Hun Choi, Kyeong Won Lee, Young Min Cho, Hong Kyu Lee, Kyong Soo Park * Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Republic of Korea
a r t i c l e
i n f o
Article history: Received 27 April 2008 Available online 28 May 2008
Keywords: O-GlcNAcylation Thiazolidinediones PPARc Sp1
a b s t r a c t O-GlcNAcylation is a kind of post-translational modification and many nuclear and cytoplasmic proteins are O-GlcNAcylated. In this study, we demonstrated that thiazolidinediones (TZDs), which are used as insulin sensitizer, specifically inhibited the O-GlcNAcylation of Sp1 but did not affect the O-GlcNAcylation of the total proteins in cell culture systems and mouse models. This effect was mediated by peroxisome proliferator activated receptor c (PPARc) activation and probably by synthesis of a specific protein induced by PPARc activation. In addition, we demonstrated that the O-GlcNAcylation sites in the zinc-finger domain were involved in the transcriptional activation of Sp1 and that rosiglitazone, a member of TZDs, affected Sp1 transcriptional activity partially by regulating the O-GlcNAcylation level of these sites. Considering the role of hexosamine biosynthesis pathway in hyperglycemia-induced insulin resistance and Sp1 in the hyperglycemia-induced gene expression, the regulation of Sp1 O-GlcNAcylation by TZDs may help to explain the function of TZDs as a treatment for insulin resistance and diabetes. Ó 2008 Elsevier Inc. All rights reserved.
O-linked N-acetylglucosamine (O-GlcNAc) modification, OGlcNAcylation, occurs at the serine or threonine residues of many nuclear and cytoplasmic proteins [1]. A very small portion (2–5%) of the fructose-6-phosphate generated by glycolysis is converted to glucosamine-6-phosphate and serves as a substrate of O-GlcNAcylation [2]. Since chronic hyperglycemia increases cellular hexosamine flux, O-GlcNAcylation is thought to be closely related to glucose toxicity. The elevation of O-GlcNAcylation disrupts insulin signaling in adipocytes and muscle tissue [3–5]. The overexpression of OGT in muscle or adipose tissue induces diabetes in transgenic mice [6]. Hyperglycemia also increases O-GlcNAcylation in pancreatic b-cells and vascular tissues, suggesting the role of O-GlcNAcylation in the development of diabetes and diabetic complication [4,7]. Since numerous transcription factors are O-GlcNAcylated, O-GlcNAc modification may regulate the transcription of various genes in response to glucose levels. Sp1 is a ubiquitously expressed transcription factor and is known to be O-GlcNAcylated. Sp1 is involved in the transcription of various genes by interaction with other transcriptional regulators and chromatin remodeling factors [8,9]. In addition to O-GlcNAcylation, the transcriptional activity of Sp1 is also regulated by phosphorylation [10]. It has been reported that there are at least
* Corresponding author. Fax: +82 2 3676 8309. E-mail address: [email protected] (K.S. Park). 1 These two authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.05.096
nine O-GlcNAcylation sites in Sp1, mainly located in the amino terminal transcription activation domain [11,12]. Recently, five O-GlcNAcylation sites were discovered at the C-terminal zinc-finger DNA-binding domain [13]. O-GlcNAcylation in the activation domain of Sp1 inhibits its transcriptional activity [14]. On the other hand, other reports show that Sp1 activity is increased by glucosamine [15]. In addition, it has been demonstrated that O-GlcNAcylation increases Sp1 protein stability and that insulin modulates the O-GlcNAcylation and phosphorylation of Sp1 [16,17]. Sp1 mediates the hyperglycemia-induced transcriptional activation of several genes, such as plasminogen activator inhibitor 1 (PAI-1), resistin, and acetyl-CoA carboxylase [15,18–20]. Based on these observations, it will be interesting to see whether the O-GlcNAcylation of Sp1 is involved in the hyperglycemia-induced transcriptional activation of these genes. Thiazolidinediones (TZDs), including troglitazone and rosiglitazone, are used as anti-diabetic drugs and specific ligands of peroxisome proliferator activated receptor c (PPARc), a member of the nuclear receptor superfamily. PPARc plays a key role in adipocyte differentiation, regulates lipid and glucose homeostasis, and improves insulin sensitivity [21,22]. We have demonstrated that Sp1 is involved in the induction of the resistin gene which is related to diabetes by a high level of glucose in adipocytes [19]. In addition, TZDs repress resistin expression through the Sp1-binding site in the resistin gene promoter and Sp1 O-GlcNAcylation is reduced by TZDs [23]. In this study, we examined the regulation of Sp1 O-GlcNAcylation by TZDs in vitro and in vivo.
714
S.S. Chung et al. / Biochemical and Biophysical Research Communications 372 (2008) 713–718
Materials and methods Plasmids and antibodies. An expression vector for the GAL4-Sp1 fusion protein, in which human Sp1 (amino acids 83–778) was fused to the DNA-binding domain of the yeast transcription factor GAL4 (amino acids 1–147), was generated by insertion of the Sp1 cDNA into the pM vector and named pM-Sp1wt. Five O-GlcNAcylation sites in the Sp1 wild-type, Ser612, Thr640, Ser641, Ser698, and Ser702 were mutated to Ala by using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and the resulting construct was named pM-Sp1mt. Immunoprecipitation or immunoblot analyses were performed with the following antibodies; Sp1 (SC-59) (Santa Cruz Biotechnology, Santa Cruz, CA), O-GlcNAc (RL2) (Affinity BioReagents), (MMS-240R) (Covance, Berkeley, CA), and phosphoserine (p3430) (Sigma–Aldrich, Louis, MO). Cell culture and adipocyte differentiation. 3T3-L1 adipocytes were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% calf serum (CS) (Invitrogen, Carlsbad, CA). Differentiation was induced by the addition of 0.5 nM of 3-isobutyl-1-methylxanthine, 0.25 lM dexamethasone (Sigma–Aldrich), and 5 lg/ml insulin to the media for 48 h. The cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 1 lg/ml insulin for an additional 10 days with the media changed every other day. HepG2 cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen). Animals. C57BLKS/J-db/db 16-week-old male mice (n = 15) and their littermates, C57BL/6J mice (n = 5), were used for these experiments (SCL, Japan). The diabetic mice (n = 15) were divided into four groups, untreated (n = 6), rosiglitazone treated for 6 h (n = 3), rosiglitazone treated for 54 h (n = 3), and insulin treated (n = 3). A rosiglitazone treated group (30 mg/kg, GlaxoSmithKline) (6 h, n = 3) was sacrificed 6 h after the rosiglitazone treatment, and the other group (54 h, n = 3) were treated with rosiglitazone every 24 h and then sacrificed 54 h after the first treatment of rosiglitazone. For the insulin treated group, the diabetic mice (n = 3) were injected with insulin (5 U) every 6 h and sacrificed 54 h after the first injection of insulin. Immunoprecipitation and immunoblot analysis. Differentiated 3T3-L1 adipocytes were treated with troglitazone or rosiglitazone (10 lM) for 24 h in low glucose media (5.5 mM), and then cells were incubated with glucosamine (5 mM) and PUGNAc (50 lM) (TRC, North York, Ont., Canada), an inhibitor of OGA (N-acetyl-bD-glucosaminase) for an additional 24 h. Cells were harvested in the lysis buffer (20 mM Tris–HCl, pH 7.4, 5 mM Na2P2O7, 100 mM NaF, 2 mM Na3VO4, 1% NP-40, 1 lg/ml aprotinin, 1 lg/ml leupeptin, and 1 mM PMSF). Mouse liver and white adipose tissues were homogenized in the same lysis buffer. The lysates (1 mg) were incubated with 4 lg of anti-Sp1 antibody for 12 h at 4 °C. The samples were subjected to SDS–PAGE and immunoblot was performed with anti-Sp1, anti-O-GlcNAc or anti-phosphoserine antibodies. RNA preparation and Northern blot analysis. 3T3-L1 adipocytes differentiated for 10 days were incubated with TZDs for 48 h. Total RNAs were extracted using Trizol (Invitrogen) according to the manufacturer’s instructions. Total RNAs (10 lg) were separated by electrophoresis and transferred to a Nytranmembrane (NY13N; Schleicher & Schuell, Keene, NH). The membranes were hybridized with the [a-32P] labeled probes for OGT and GAPDH. Transient transfection and reporter assays. HepG2 cells were cultured in DMEM supplemented with 10% FBS. Cells in 12-well plates were transfected with a total of 0.4 lg of DNA per well: 0.2 lg of pGAL4-Luc (as a reporter plasmid), 0.1 lg of pM, pMSp1wt, or pM-Sp1mt, 0.05 lg of pSG5-PPARc, and 0.05 lg of pCMV-b-galactosidase using Lipofectamine Plus (Invitrogen). The cells were treated with rosiglitazone (48 h) and glucosamine plus
PUGNAc (24 h). Luciferase activities were determined according to the Luciferase Assay System Kit (Promega, Madison, WI). Relative transfection efficiency was determined by b-galactosidase activity. Statistical analysis. SPSS, version 12.0 (SPSS Inc., Chicago, IL), was used for statistic analysis. The data were expressed as means ± SE. Statistical significance was calculated using the Mann–Whitney Utest. A p-value of less than 0.05 denoted the presence of a statistically significant difference. Results TZDs reduced the level of O-GlcNAcylated Sp1 in 3T3-L1 adipocytes To easily detect O-GlcNAcylated Sp1 in the fully differentiated 3T3-L1 adipocytes, cells were treated with glucosamine for 24 h and then harvested for immunoprecipitation with anti-Sp1 antibodies. O-GlcNAcylated Sp1 was substantially increased by the treatment of glucosamine. In other hand, pretreatment with troglitazone or rosiglitazone, two thiazolidinediones (TZDs), repressed the level of O-GlcNAcylated Sp1 in the presence of glucosamine (Fig. 1A). To distinguish whether these TZDs inhibit the O-GlcNAcylation of Sp1 or TZDs enhance O-GlcNAc dissociation from Sp1 by O-GlcNAcase (OGA), PUGNAc, a specific OGA inhibitor, was added to the media simultaneously with glucosamine. In the presence of PUGNAc, the O-GlcNAcylated Sp1 level was still reduced by TZDs, suggesting that TZDs inhibit the O-GlcNAcylation of Sp1 (Fig. 1B). To determine whether TZDs modulate the general O-GlcNAcylation state of cellular proteins, the total O-GlcNAcylated proteins were detected by western blot analysis. The O-GlcNAcylation level of total proteins was significantly increased by glucosamine and PUGNAc, but was not affected by TZDs (Fig. 1C). In addition, the result of the Northern blot analysis showed that the mRNA level of the O-linked GlcNAc transferase (OGT) was not changed by the treatment with TZDs (Fig. 1D). Since neither the O-GlcNAcylation level of the total proteins nor the expression of OGT was significantly changed by the TZDs, the regulation of the OGlcNAcylation by the TZDs was probably limited to some specific proteins, including Sp1. Effect of TZDs on O-GlcNAcylation of Sp1 was PPARc dependent To determine whether PPARc was involved in the inhibition of Sp1 O-GlcNAcylation by the TZDs, cells were treated with troglitazone or rosiglitazone in the presence of GW9662, a specific PPARc antagonist. HepG2 cells were transfected with the expression vector of Sp1 linked to GAL4DBD (Fig. 2A) and immunoprecipitation was performed with anti-GAL4DBD antibody. Troglitazone or rosiglitazone did not change the Sp1 O-GlcNAcylation level when PPARc activity was inhibited by GW9662 (Fig. 2B). In addition, the effect on the O-GlcNAcylation of Sp1 by TZDs disappeared in the presence of cycloheximide, a specific protein translation inhibitor (Fig. 2B). O-GlcNAcylation of Sp1 increased its transcriptional activity Next, we tested the effect of O-GlcNAcylation on the function of Sp1. Treatment with PUGNAc or rosiglitazone did not change the Sp1 protein level in the nucleus (data not shown). In addition, electrophoretic mobility shift assay (EMSA) results showed that O-GlcNAcylation or rosiglitazone treatment hardly affected the DNAbinding affinity of endogenous Sp1 in HepG2 cells (data not shown). Recently, five sites of Sp1 O-GlcNAcylation were identified in the zinc-finger domain, Ser612, Thr640, Ser641, Ser698 and Ser702. We made a mutant form of Sp1 in which these sites were changed
S.S. Chung et al. / Biochemical and Biophysical Research Communications 372 (2008) 713–718
715
Sp1 wild-type. O-GlcNAcylation was increased more substantially in the GAL4-Sp1 wild-type than in the GAL4-Sp1 mutant. In addition, the O-GlcNAcylation level of the wild-type was reduced by rosiglitazone and a similar effect was shown in the mutant-type (Fig. 3B). There was a significant difference in the basal transcriptional activities between the Sp1 wild-type and the mutant-type (Fig. 3C). Treatment with rosiglitazone significantly inhibited the transcriptional activity of the Sp1 wild-type but only slightly affected the activity of the mutant form. Effect of rosiglitazone on the O-GlcNAcylation level of Sp1 in vivo The O-GlcNAcylation state of Sp1 was examined in normal (blood glucose level 165.4 ± 9.6 mg/dl and body weight about 30 g) and diabetic db/db (blood glucose level 569.7 ± 30.3 mg/dl and body weight about 59.8 g) mouse models. O-GlcNAcylated Sp1 was significantly increased in the livers of diabetic mice (Fig. 4A), but the Sp1 protein level was not changed (data not shown). When diabetic mice (db/db mice) were treated with rosiglitazone for 6 or 54 h, the blood glucose levels of the mice were decreased to 349.3 ± 30.9 mg/dl and 175.0 ± 20.1 mg/dl, respectively. Treatment with rosiglitazone for 54 h effectively reduced the levels of Sp1 O-GlcNAcylation in the liver (Fig. 4B). Similar results were obtained when white adipose and kidney tissues were used for immunoprecipitation (data not shown). Consistent with the results obtained in the cell culture systems, neither the O-GlcNAcylation level for total proteins nor the Sp1 expression was significantly changed by the TZDs (data not shown). We tested the effect of rosiglitazone on another important posttranslational modification of Sp1, phosphorylation at serine residues, and the phosphorylation state of Sp1 was not changed (Fig. 4C). Since rosiglitazone reduced the blood glucose level in the diabetic mice, we determined whether the effect of the rosiglitazone on the Sp1 O-GlcNAcylation was related to the reduction of blood glucose levels. We injected db/db mice with insulin to decrease their blood glucose levels (blood glucose level 201.3 ± 52.3 mg/dl and body weight about 60.7 g), which were similar to the levels shown in the rosiglitazone treated group. There was no difference in the Sp1 O-GlcNAcylation levels of the insulin injected mice compared to the untreated mice (data not shown). Discussion
Fig. 1. TZDs reduce the level of O-GlcNAcylated Sp1. (A) Differentiated 3T3-L1 adipocytes were treated with troglitazone (Tro) or rosiglitazone (Rosi) for 24 h and then glucosamine (5 mM) was added to the media. Cells were harvested 24 h after the addition of glucosamine and cellular proteins were subjected to immunoprecipitation with anti-Sp1 antibodies. The precipitated proteins were analyzed by immunoblotting with anti-O-GlcNAc and anti-Sp1 antibodies. (B) Cells were treated with TZDs as described above and PUGNAc (50 lM) was treated simultaneously with glucosamine. (C) Total cell lysates were subjected to western blot analysis. (D) Total RNA was isolated from the cells treated with TZDs and glucosamine. The mRNA levels of OGT and GAPDH were compared by Northern blot analysis.
to alanines (Fig. 3A) and compared the O-GlcNAcylation state and the transcriptional activity of the mutant form to those of the
Our previous study showed the possibility that TZDs affected the O-GlcNAcylation level of Sp1. In this study, we demonstrated that the regulation of Sp1 O-GlcNAcylation by TZDs was mediated by PPARc activation. Probably there was an expression of an unknown gene induced by PPARc and its product may be involved in the inhibition of Sp1 O-GlcNAcylation. The reduction of O-GlcNAcylation by rosiglitazone was also observed in diabetic mice, as well as in the cell culture system. Treatment with rosiglitazone for 6 h did not change the O-GlcNAcylation level of Sp1 but longer exposure (54 h) significantly inhibited the modification, suggesting that the accumulation of an unknown protein(s) was required to exert the inhibition effect of rosiglitazone on the O-GlcNAcylation of Sp1. Even though we do not know the mechanism how TZDs regulates the Sp1 O-GlcNAcylation, it is possible that a protein having interaction with Sp1 modulates the O-GlcNAcylation of Sp1 and the expression of this protein is regulated by PPARc. In some cases, O-GlcNAcylation sites overlap with phosphorylation sites and these two modifications usually have a reciprocal relationship [24,25]. Therefore, we also cannot exclude the possibility that the phosphorylation of O-GlcNAcylation sites or neighboring regions regulates the O-GlcNAcylation of Sp1, and that PPARc modulates the phosphorylation level by changing the expression of a specific
716
S.S. Chung et al. / Biochemical and Biophysical Research Communications 372 (2008) 713–718
Fig. 2. PPARc is involved in the inhibition of Sp1 O-GlcNAcylation by TZDs. (A) A DNA fragment representing Sp1 (from amino acid 83 to 778) was ligated to GAL4DBD in pMvector. (B) HepG2 cells were transfected with pM-Sp1wt and treated with TZDs in the presence of GW9662 (10 lM) or cycloheximide (5 lM) for 24 h and then glucosamine and PUGNAc were added to the media for an additional 24 h. Cell lysates were immunoprecipitated with anti-GAL4 antibodies and immunoblot analysis was performed with anti-O-GlcNAc and anti-Sp1 antibodies.
Fig. 3. The O-GlcNAcylation sites of the zinc-finger domain of Sp1 are important for the transcriptional activity of Sp1. (A) Structure of the Sp1 wild-type (GAL4-Sp1wt) and mutant-type (GAL4-Sp1mt). (B) HepG2 cells were transfected with pM-Sp1 wt or pM-Sp1 mt and then treated with rosiglitazone, glucosamine, and PUGNAc as described in Fig. 1. Cell lysates were immunoprecipitated with anti-GAL4DBD antibodies and an immunoblot analysis was performed with anti-O-GlcNAc and anti-Sp1 antibodies. (C) HepG2 cells were transfected as described in the Materials and Methods. The luciferase activity of the cells transfected with pM in the absence of rosiglitazone was set to 1, and the other activities were presented as relative to that value. The bar graphs show the means ± SE of four independent experiments.*p < 0.05
S.S. Chung et al. / Biochemical and Biophysical Research Communications 372 (2008) 713–718
717
activation domain and zinc-finger domain were identified. We made mutations at the five O-GlcNAcylation sites at the zinc-finger domain since this domain is not only a DNA-binding region but also a region to interact with several transcriptional regulators, such as histone deacetylase 1 (HDAC1), silencing mediator for retinoid and thyroid receptor (SMRT), and nuclear receptor corepressor (NCoR) [9]. Our data showed that this region was a major part of the total O-GlcNAcylation sites and that O-GlcNAcylation at these sites was involved in its transcriptional activation. Rosiglitazone reduced O-GlcNAcylation at these sites and repressed the Sp1 transcriptional activity. Since we used GAL4 DNA-binding domainfused Sp1 constructs for these experiments, differences in the transcriptional activities of the Sp1 wild-type and mutant-type might be generated by changes in the interaction with other transcriptional regulators. Further investigation to determine the effect of the O-GlcNAcylation of this region on the interaction with corepressors or coactivators will be required to suggest a mechanism for the O-GlcNAcylation-induced transcriptional activation of Sp1. In addition, concerning our results and several previous reports, we can imagine that different O-GlcNAcylation sites may differently regulate Sp1 transcriptional activity. The hexosamine biosynthesis pathway plays a role in hyperglycemia-induced insulin resistance [26–28]. Glucosamine treatment induces insulin resistance in mice and increases the O-GlcNAcylation of skeletal muscle proteins [29,30]. Since Sp1 is involved in the hyperglycemia-induced expression of several genes, it may be possible that TZDs regulates the expression of these genes by modulating the Sp1 activity. In addition, although TZDs did not reduce the O-GlcNAcylation state of total proteins, it is possible that TZDs affect a few proteins including Sp1. Therefore the investigation of proteins whose O-GlcNAcylation is modulated by TZDs will help to explain the function of TZDs as a treatment for insulin resistance and diabetes. Acknowledgments This study was supported by a grant from the Innovative Research Institute for Cell Therapy, Republic of Korea (Project No. A062260) and a grant from Marine Biotechnology Program funded by Ministry of Land, Transport, and Maritime Affairs, Republic of Korea. References
Fig. 4. O-GlcNAcylation of Sp1 is reduced by rosiglitazone in vivo. (A) Liver proteins from normal mice (n = 5) and diabetic db/db mice (n = 6) were prepared and the protein lysates (1 mg) were immunoprecipitated with anti-Sp1 antibody. The intensity of an O-GlcNAc-Sp1 band was normalized by the intensity of the corresponding Sp1 band. The bar graphs show the means ± SE. *p < 0.05 vs normal mice. (B) Diabetic db/db mice were treated with rosiglitazone as described in the Materials and methods. The mean value of the control group (0 h, n = 6) was set to 1 and the other values (6 h, n = 3; 54 h, n = 3) were presented as relative to that. * p < 0.01 (C) Immunobloting was performed with anti-phosphoserine antibodies after the immunoprecipitation with anti-Sp1 antibodies. No significant differences were detected between the groups tested.
kinase or phosphatase. In addition, since insulin treatment did not affect the level of Sp1 O-GlcNAcylation in the mouse model, we could exclude the possibility that reduced blood glucose level by rosiglitazone was the major reason for the inhibition of Sp1OGlcNAcylation. Although several reports indicated the possibility of multiple OGlcNAcylation sites in Sp1, only a few residues in the N-terminal
[1] G.W. Hart, Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins, Annu. Rev. Biochem. 66 (1997) 315–335. [2] G. Boehmelt, I. Fialka, G. Brothers, M.D. McGinley, S.D. Patterson, R. Mo, C.C. Hui, S. Chung, L.A. Huber, T.W. Mak, N.N. Iscove, Cloning and characterization of the murine glucosamine-6-phosphate acetyltransferase EMeg32. Differential expression and intracellular membrane association, J. Biol. Chem. 275 (2000) 12821–12832. [3] L. Wells, K. Vosseller, G.W. Hart, A role for N-acetylglucosamine as a nutrient sensor and mediator of insulin resistance, Cell Mol. Life Sci. 60 (2003) 222–228. [4] M.G. Buse, Hexosamines, insulin resistance, and the complications of diabetes: current status, Am. J. Physiol. Endocrinol. Metab. 290 (2006) E1–E8. [5] G.W. Hart, M.P. Housley, C. Slawson, Cycling of O-linked beta-Nacetylglucosamine on nucleocytoplasmic proteins, Nature 446 (2007) 1017– 1022. [6] D.A. McClain, W.A. Lubas, R.C. Cooksey, M. Hazel, G.J. Parker, D.C. Love, J.A. Hanover, Altered glycan-dependent signaling induces insulin resistance and hyperleptinemia, Proc. Natl. Acad. Sci. USA 99 (2002) 10695–10699. [7] K. Liu, A.J. Paterson, E. Chin, J.E. Kudlow, Glucose stimulates protein modification by O-linked GlcNAc in pancreatic beta cells: linkage of O-linked GlcNAc to beta cell death, Proc. Natl. Acad. Sci. USA 97 (2000) 2820–2825. [8] A. Doetzlhofer, H. Rotheneder, G. Lagger, M. Koranda, V. Kurtev, G. Brosch, E. Wintersberger, C. Seiser, Histone deacetylase 1 can repress transcription by binding to Sp1, Mol. Cell Biol. 19 (1999) 5504–5511. [9] J.A. Lee, D.C. Suh, J.E. Kang, M.H. Kim, H. Park, M.N. Lee, J.M. Kim, B.N. Jeon, H.E. Roh, M.Y. Yu, K.Y. Choi, K.Y. Kim, M.W. Hur, Transcriptional activity of Sp1 is regulated by molecular interactions between the zinc finger DNA binding domain and the inhibitory domain with corepressors, and this interaction is modulated by MEK, J. Biol. Chem. 280 (2005) 28061–28071.
718
S.S. Chung et al. / Biochemical and Biophysical Research Communications 372 (2008) 713–718
[10] P. Bouwman, S. Philipsen, Regulation of the activity of Sp1-related transcription factors, Mol. Cell Endocrinol. 195 (2002) 27–38. [11] S.P. Jackson, R. Tjian, O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation, Cell 55 (1988) 125– 133. [12] M.D. Roos, K. Su, J.R. Baker, J.E. Kudlow, O glycosylation of an Sp1-derived peptide blocks known Sp1 protein interactions, Mol. Cell Biol. 17 (1997) 6472– 6480. [13] G. Majumdar, A. Harrington, J. Hungerford, A. Martinez-Hernandez, I.C. Gerling, R. Raghow, S. Solomon, Insulin dynamically regulates calmodulin gene expression by sequential o-glycosylation and phosphorylation of sp1 and its subcellular compartmentalization in liver cells, J. Biol. Chem. 281 (2006) 3642– 3650. [14] X. Yang, K. Su, M.D. Roos, Q. Chang, A.J. Paterson, J.E. Kudlow, O-linkage of Nacetylglucosamine to Sp1 activation domain inhibits its transcriptional capability, Proc. Natl. Acad. Sci. USA 98 (2001) 6611–6616. [15] H.J. Goldberg, C.I. Whiteside, I.G. Fantus, The hexosamine pathway regulates the plasminogen activator inhibitor-1 gene promoter and Sp1 transcriptional activation through protein kinase C-beta I and -delta, J. Biol. Chem. 277 (2002) 33833–33841. [16] I. Han, J.E. Kudlow, Reduced O glycosylation of Sp1 is associated with increased proteasome susceptibility, Mol. Cell Biol. 17 (1997) 2550–2558. [17] K. Su, M.D. Roos, X. Yang, I. Han, A.J. Paterson, J.E. Kudlow, An N-terminal region of Sp1 targets its proteasome-dependent degradation in vitro, J. Biol. Chem. 274 (1999) 15194–15202. [18] H.J. Goldberg, J. Scholey, I.G. Fantus, Glucosamine activates the plasminogen activator inhibitor 1 gene promoter through Sp1 DNA binding sites in glomerular mesangial cells002C, Diabetes 49 (2000) 863–871. [19] S.S. Chung, H.H. Choi, K.W. Kim, Y.M. Cho, H.K. Lee, K.S. Park, Regulation of human resistin gene expression in cell systems: an important role of stimulatory protein 1 interaction with a common promoter polymorphic site, Diabetologia 48 (2005) 1150–1158.
[20] S. Daniel, K.H. Kim, Sp1 mediates glucose activation of the acetyl-CoA carboxylase promoter, J. Biol. Chem. 271 (1996) 1385–1392. [21] M. Lehrke, M.A. Lazar, The many faces of PPARgamma, Cell 123 (2005) 993– 999. [22] S.M. Rangwala, M.A. Lazar, Peroxisome proliferator-activated receptor gamma in diabetes and metabolism, Trends Pharmacol. Sci. 25 (2004) 331–336. [23] S.S. Chung, H.H. Choi, Y.M. Cho, H.K. Lee, K.S. Park, Sp1 mediates repression of the resistin gene by PPARgamma agonists in 3T3-L1 adipocytes, Biochem. Biophys. Res. Commun. 348 (2006) 253–258. [24] F.I. Comer, G.W. Hart, O-glycosylation of nuclear and cytosolic proteins. Dynamic interplay between O-GlcNAc and O-phosphate, J. Biol. Chem. 275 (2000) 29179–29182. [25] F.I. Comer, G.W. Hart, Reciprocity between O-GlcNAc and O-phosphate on the carboxyl terminal domain of RNA polymerase II, Biochemistry 40 (2001) 7845–7852. [26] L.F. Hebert Jr., M.C. Daniels, J. Zhou, E.D. Crook, R.L. Turner, S.T. Simmons, J.L. Neidigh, J.S. Zhu, A.D. Baron, D.A. McClain, Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance, J. Clin. Invest. 98 (1996) 930–936. [27] D.A. McClain, E.D. Crook, Hexosamines and insulin resistance, Diabetes 45 (1996) 1003–1009. [28] A.D. Baron, J.S. Zhu, J.H. Zhu, H. Weldon, L. Maianu, W.T. Garvey, Glucosamine induces insulin resistance in vivo by affecting GLUT 4 translocation in skeletal muscle. Implications for glucose toxicity, J Clin Invest 96 (1995) 2792–2801. [29] H. Yki-Jarvinen, A. Virkamaki, M.C. Daniels, D. McClain, W.K. Gottschalk, Insulin and glucosamine infusions increase O-linked N-acetyl-glucosamine in skeletal muscle proteins in vivo, Metabolism 47 (1998) 449–455. [30] L. Rossetti, M. Hawkins, W. Chen, J. Gindi, N. Barzilai, In vivo glucosamine infusion induces insulin resistance in normoglycemic but not in hyperglycemic conscious rats, J. Clin. Invest. 96 (1995) 132–140.