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SREBP-1c regulates glucose-stimulated hepatic clusterin expression
Biochemical and Biophysical Research Communications 408 (2011) 720–725
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SREBP-1c regulates glucose-stimulated hepatic clusterin expression Gukhan Kim a,1,2, Geun Hyang Kim a,c,1, Gyun-Sik Oh a,c,1, Jin Yoon a,c, Hae Won Kim a, Min-Seon Kim b, Seung-Whan Kim a,c,⇑ a
Department of Pharmacology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea c Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 20 April 2011 Available online 28 April 2011 Keywords: Clusterin Apolipoprotein J Glucose SREBP-1c E-box
a b s t r a c t Clusterin is a stress-response protein that is involved in diverse biological processes, including cell proliferation, apoptosis, tissue differentiation, inflammation, and lipid transport. Its expression is upregulated in a broad spectrum of diverse pathological states. Clusterin was recently reported to be associated with diabetes, metabolic syndrome, and their sequelae. However, the regulation of clusterin expression by metabolic signals was not addressed. In this study we evaluated the effects of glucose on hepatic clusterin expression. Interestingly, high glucose concentrations significantly increased clusterin expression in primary hepatocytes and hepatoma cell lines, but the conventional promoter region of the clusterin gene did not respond to glucose stimulation. In contrast, the first intronic region was transcriptionally activated by high glucose concentrations. We then defined a glucose response element (GlRE) of the clusterin gene, showing that it consists of two E-box motifs separated by five nucleotides and resembles carbohydrate response element (ChoRE). Unexpectedly, however, these E-box motifs were not activated by ChoRE binding protein (ChREBP), but were activated by sterol regulatory element binding protein-1c (SREBP-1c). Furthermore, we found that glucose induced recruitment of SREBP-1c to the E-box of the clusterin gene intronic region. Taken together, these results suggest that clusterin expression is increased by glucose stimulation, and SREBP-1c plays a crucial role in the metabolic regulation of clusterin. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Clusterin, also known as apolipoprotein J (Apo J), agingassociated protein 4 (AAG4), complement lysis inhibitor (CLI), testosterone-repressed prostate message 2 (TRPM-2), Ku70-binding protein 1 (KUB1), X-ray-induced protein 8 (XIP8), and sulfated glycoprotein-2 (SGP-2), was first identified as a glycoprotein that elicited clustering of suspended cells [1]. It is a 75–80 kDa disulfide-linked heterodimeric protein composed of a and b subunits that are generated by a post-translational cleavage of a single-chain precursor protein [2,3]. Clusterin has been proposed to be involved in a variety of important biological processes, including sperm maturation, tissue differentiation, tissue remodeling, membrane recycling, reverse lipid transport, cell–cell or cell–substrate interaction, promotion of erythrocyte aggregation, attenuation of complement
⇑ Corresponding author at: Department of Pharmacology, University of Ulsan College of Medicine, 388-1 PungNap-2dong, Songpa-gu, Seoul 138-736, Republic of Korea. Fax: +82 2 3010 2941. E-mail address: [email protected] (S.-W. Kim). 1 These authors contributed equally to this work. 2 Present address: Oregon Health and Science University, Portland, OR 97239-3098, United States. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.04.111
activity, cell proliferation, cell survival, and apoptosis [4]. It is expressed ubiquitously in many tissues. Clusterin transcripts are present at relatively high levels in the brain, ovary, testis, stomach, and liver; are less abundant in heart, spleen, lung, kidney, and breast; and are absent in T-lymphocytes [5,6]. Interestingly, clusterin expression is differentially regulated in many pathological conditions, including cancer, atherosclerosis, diabetes, and renal and neurodegenerative diseases [7,8]. A number of studies have shown that clusterin expression levels are associated with prostate cancer [9], gastric cancer [10], and breast cancer [11], as well as colon [12], cervical [13], and ovarian cancers [14]. There is also considerable evidence suggesting a relationship between clusterin and the metabolic diseases such as diabetes and atherosclerosis [15–19]. Type 2 diabetic subjects have higher serum clusterin levels than healthy individuals [17], and these elevated levels of clusterin are positively correlated with blood glucose levels. In addition, an analysis of single nucleotide polymorphisms (SNPs) in Japanese subjects revealed a significant association of clusterin gene polymorphisms with diabetes, serum lipid levels, and the progression of carotid atherosclerosis [19,20]. Given these associations of clusterin with metabolic diseases, it is reasonable to speculate that clusterin expression could be regulated by metabolic signals, such as nutrients. However, to date, no studies
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have addressed the metabolic regulation of clusterin expression. In this study, we sought to investigate whether glucose regulates clusterin expression in hepatocytes and elucidate the underlying regulatory mechanism at the transcriptional level. Our results indicate that glucose stimulation increases clusterin expression, and further show that sterol regulatory element binding protein-1c (SREBP-1c) is involved in glucose-stimulated transcriptional activation of the clusterin gene through tandem intronic E-box motifs.
2. Materials and methods 2.1. Isolation of primary hepatocytes and cell culture Primary hepatocytes were isolated from 10-week-old mice using the previously described collagenase perfusion method, with minor modifications [21]. Perfusion was performed with Mg2+/ Ca2+-free Hanks’ balanced salt solution containing 100 U/ml collagenase (Invitrogen, Carlsbad, CA) and 48 lg/ml trypsin inhibitor (Sigma, St. Louis, MO). Cells were plated onto collagen type I-coated culture dishes and maintained in Medium 199. Rat hepatoma FAO and human hepatoma HepG2 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified 5% CO2 incubator. 2.2. RNA preparation and quantitative real-time PCR (qRT-PCR) Total RNA was isolated from primary hepatocytes using the TRIzol reagent (Invitrogen). Purified total RNA was reverse-transcribed using M-MLV reverse transcriptase (Promega, Madison, WI), according to the manufacturer’s protocol. Quantitative gene expression analyses were performed on a Roche LightCycler 480 System (Roche, Basel, Switzerland) using SYBR Green PCR Master Mix. PCR primers were designed using Primer Express 3.0 software with the manufacturer’s default settings, and were validated for identical efficiencies. 18S rRNA was used as the internal control. Ratios of target genes to 18S rRNA expression levels were calculated using the 2DDC T method [22]. The oligonucleotide primers used for qRT-PCR were as follows: mClusterin, 50 -ATA AGG AGA TTC AGA ACG CC-30 and 50 -GCT CTG CGT TGG TTT TTT CTA TG-30 ; mSREBP-1c, 50 -AGC CAT GGA TTG CAC ATT TGA-30 and 50 -CAA ATA GGC CAG GGA AGT CA-30 ; 18s rRNA, 50 -CCG CGG TTC TAT TTT GTT GGT-30 and 50 -CTC TAG CGG CGC AAT ACG A-30 . The oligonucleotide primers used for conventional RT-PCR were as follows: mClusterin, 50 -AGG AGC TAA ACG ACT CGC T-30 and 50 -CTT TTC CTG CGG TAT TCC T-30 ; mSREBP-1c, 50 -GGC GCA TGG ATT GCA CAT TT-30 and 50 -GCA GGC TGT AGG ATG GTG A-30 ; 18s rRNA, 50 CGT CCC CCA ACT TCT TAG AG-30 and 50 -CAC CTA CGG AAA CCT TGT TAC-30 . 2.3. Preparation of cell lysates and Western blotting Protein extracts of hepatocytes were prepared in lysis buffer containing 20 mM Tris–Cl (pH 7.5), 100 mM KCl, 5 mM EDTA (pH 8.0), 10 mM Na4P2O7, 100 mM NaF, 2 mM Na2VO4, and 1% NP-40. Cells were incubated in lysis buffer for 1 h on ice. Cell debris was removed by centrifuging the lysates at 15,000g for 10 min. Protein concentrations were determined using the Bradford assay. For Western blot analysis, 50–100 lg of lysate protein was separated by electrophoresis on 7.5–10% polyacrylamide gels under denaturing conditions and then transferred to PVDF membranes (Millipore, Billerica, MA). Blots were incubated with antibodies to clusterin (1:1000 dilution; Santa Cruz), SREBP-1c (1:500 dilution; Santa Cruz), and b-actin (1:10,000 dilution; Sigma).
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2.4. Construction of luciferase reporter genes and luciferase assay The 2285/+430 and +2158/+4426 clusterin promoter regions used to construct promoter–luciferase reporter were amplified from human genomic DNA by PCR and were used as templates for the construction of other reporters. Deletion and site-specific mutations within the promoter were created using PCR cloning strategies. The 2285/+430 clusterin promoter and corresponding deletion constructs +230/+430 and +331/+430 were amplified using the unique forward primers 50 -CGG GGT ACC AAA CCC AGC TGT GTA AGT CCA TAA-30 (-2285), 50 -CGG GGT ACC GGC ATT CTT TGG GCG TGA GTC-30 (+230), and 50 -CGG GGT ACC CGC GGC GTC GCC AG-30 (+331), and the common reverse primer 50 -CCG CTC GAG CAT CCG TCC TGG TGT GGC TCT-30 (+430). The +2158/+4426 clusterin promoter was amplified using the primers 50 -CGG GGT ACC AAG TGG TTT AAG CCT TCT TAG G-30 (forward) and 50 -CCC CCC GGG AGA GAA CAG GAG ACC AT-30 (reverse). Two mutant promoters were prepared using the following primer pairs: M1, 50 -AGT GCT CAT CAG AGA CCC GTG AGA CCA CA-30 (forward) and 50 -GGT CTC TGA TGA GCA CTG CCC ACT GAG C-30 (reverse); and M2, 50 -GGT CAG AGA TGG TCA AGA CCA CAG CCT TC-30 (forward) and 50 -CTT GAC CAT CTC TGA CCC CAG CTG CCC A-30 (reverse). The authenticity of plasmid constructs was confirmed by DNA sequencing. For luciferase reporter assay, HepG2 cells were cultured in DMEM supplemented with 10% FBS. Transfections were performed in 24-well plates using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. pActin-bgal plasmid (50 ng) was included in each transfection experiment to control for the efficiency of transfection. Luciferase activity was measured using a luminometer (Centro LB 960, Berthold Technologies), and values were normalized to b-galactosidase activity. 2.5. Chromatin immunoprecipitation (ChIP) ChIP assays were performed as described previously, with minor modifications [23]. Briefly, HepG2 cells were transfected with SREBP-1c expression plasmids and incubated overnight. After stimulation with 25 mM glucose for 6 h, cells were fixed with 1% formaldehyde for 15 min at room temperature. The cells were washed with cold phosphate-buffered saline (PBS) and resuspended in lysis buffer containing 50 mM Tris–HCl (pH 8.0), 1% SDS, and 10 mM EDTA. Soluble chromatin was prepared by sonication and immunoprecipitated with antibodies against SREBP-1c (sc-13551; Santa Cruz Biotechnology) or preimmune IgG. The final DNA extractions were analyzed by qRT-PCR using two primer pairs: +3093/+3113 (50 -TTC TGG CTG GCT TTG TCT CTC T-30 ) and +3149/+3166 (50 TGC CCA CTG AGC CCT GAA-30 ), encompassing the first intronic Ebox-containing region; and +915/+936 (50 -TTC TGC CTC CTA ATG CAT CTG A-30 ) and +956/+975 (50 -AGG CCT GGT GGA TCT TGT GT30 ), encompassing a control region containing no E-boxes. 2.6. Statistical analysis Results are expressed as means ± standard errors (SEs). Statistical significance was assessed using unpaired Student’s two-tailed t-tests. Differences with a P-value <0.05 were considered statistically significant. 3. Results 3.1. High glucose concentration increases clusterin mRNA and protein levels The expressions of many metabolic enzymes and related factors are regulated by metabolic signals, such as nutrients. Clusterin is
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Fig. 1. High glucose increased hepatic clusterin expression. (A) Clusterin mRNA levels were determined by conventional RT-PCR analysis. Mouse primary hepatocytes were incubated in 5.5 mM glucose DMEM for 12 h and then stimulated with 25 mM glucose for 12 h. SREBP-1c and 18S rRNA were used as a known glucose-stimulated gene and a loading control, respectively. (B) Clusterin and SREBP-1c mRNA levels were determined by qRT-PCR. Mouse primary hepatocytes were stimulated with 25 mM glucose as described in (A). (C) Western blot analysis of clusterin protein in rat hepatoma FAO, human hepatoma HepG2, and mouse primary hepatocytes. Cells were incubated in 5.5 mM glucose DMEM for 12 h and then stimulated with 25 mM glucose DMEM for 24 h. ⁄P < 0.005.
differentially regulated in metabolic disease states and has been proposed to have metabolic functions. To address the relevance of clusterin to metabolism, we analyzed the effects of glucose concentration on hepatic clusterin expression. Conventional RT-PCR analyses showed that exposure of primary hepatocytes to high glucose induced an increase in clusterin transcript levels (Fig. 1A). SREBP-1c, which is positively regulated by glucose at the transcriptional level [24], was used as a positive control for hepatic glucose response in these experiments. These glucose-induced increases in clusterin and SREBP-1c mRNA expression were confirmed by qRTPCR (Fig. 1B). Consistent with the increases in clusterin at the mRNA level, clusterin protein levels were also increased by high glucose in both primary hepatocytes and hepatoma cell lines (Fig. 1C). These results suggest that clusterin expression in hepatocytes is regulated by the important nutrient, glucose, and indicate that clusterin may serve a metabolic function in the response to nutrient states. 3.2. Tandem E-boxes in the first intron are responsible for glucose stimulation of clusterin transcription To define the glucose response element (GlRE) in the clusterin promoter, we first analyzed the genomic sequence of the human clusterin gene using TransFac Search software and Genomatix program (Fig. 2A). The transcription factors carbohydrate response element binding protein (ChREBP), liver X receptor (LXR), and SREBP-1c are well known to mediate nutritional signals. Prediction software showed no LXR response elements (LXRE) in the upstream region of the clusterin gene, but did indicate the presence of sterol regulatory element (SRE)/E-box motifs and carbohydrate response elements (ChoREs) in the conventional promoter region and the first intronic region. Therefore, based on the locations of SRE/E-box and ChoRE, we constructed luciferase reporters containing conventional promoter regions or the first intronic region. Although the upstream conventional promoter region contained numerous SRE/E-box motifs and ChoREs, the transcriptional activity of this region was not activated by high glucose in hepatoma cell lines (Fig. 2B and C). Interestingly, glucose significantly stimulated the activity of a reporter driven by the first intronic region
containing a ChoRE. These results were reproduced in both rat hepatoma FAO and human hepatoma HepG2 cell lines (Fig. 2B and C). The ChoRE in the first intron of the clusterin gene consists of two Ebox motifs separated by five nucleotides. To confirm the role of these E-box motifs in the glucose-stimulated transcription of clusterin, we generated two mutant reporters, M1 and M2, in which each of the two E-box motifs was individually mutated in the context of the wild-type +2158/+4426 sequence (Fig. 2D). In both cases, mutation of the E-box motif abrogated glucose-stimulated transcription of the reporter gene (Fig. 2E). These results indicate that the putative ChoRE, consisting of two E-box motifs, is responsible for glucose-stimulated transcription of clusterin in hepatocytes. 3.3. Clusterin transcription by glucose is mediated by SREBP-1c Next, we sought to identify the transcription factor responsible for mediating transcription of the clusterin gene in response to glucose stimulation. It is well known that ChREBP binds to the ChoRE and activates transcription of ChoRE-containing target genes. Therefore, we tested the ability of ChREBP to activate the clusterin promoter. The basic helix-loop-helix leucine zipper (bHLH-LZ) factor Max-like protein X (Mlx)-b or -c was cotransfected with ChREBP because ChREBP requires heterodimerization with Mlx for transcriptional activity. Unexpectedly, ChREBP/Mlx did not activate either the upstream promoter or the intronic ChoRE-containing promoter (Fig. 3A). An acetyl-CoA carboxylase (ACC) ChoREcontaining reporter used as a positive control was substantially activated by ChREBP under the same conditions (Fig. 3A). On the other hand, it is well known that SREBP-1c binds to SRE or E-box [25], and SREBP-1c transcription is activated by glucose [24]. Accordingly, we investigated the effects of SREBP-1c on clusterin transcription by cotransfecting HepG2 cells with SREBP-1c expression plasmids and wild-type or mutated promoter-reporter constructs. Notably, SREBP-1c stimulated the transcriptional activity of the +2158/+4426 intronic reporter gene, but showed no significant effects on other reporter genes, similar to the effects of high glucose (Fig. 3B). Furthermore, this activation of the clusterin promoter by SREBP-1c was abolished by both M1 and M2 mutations
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Fig. 2. Identification of the glucose response element in the clusterin gene. (A) Representation of putative SREBP-1c and ChREBP binding sites in the human clusterin gene. Four reporter genes were constructed based on the locations of these binding sites. (B and C) Glucose responses of each reporter gene were determined in FAO (B) and HepG2 (C) cell lines by assaying the activity of luciferase reporter constructs. Six hours after transfection, cells were incubated in 5.5 mM glucose DMEM for 6 h, and then treated with 25 mM glucose for 12 h. (D) M1 and M2 mutant promoter reporters were constructed by mutation of each of two tandem E-box motifs in the first intron of the clusterin gene. (E) Glucose stimulation of the +2158/+4426 reporter was abrogated by M1 or M2 mutation in HepG2 cells.
(Fig. 3C). Finally, a ChIP analysis showed that SREBP-1c protein was recruited to E-box motifs of the clusterin intronic region in glucose-stimulated HepG2 cells (Fig. 3D). Taken together, these results suggest that SREBP-1c regulates the transcription of clusterin in response to glucose stimulation.
4. Discussion The present study was initiated to explore the possibility that clusterin expression is regulated by nutrient signals. Three major findings arose from this study. First, high glucose increases clusterin expression in hepatocytes. Second, SREBP-1c is directly involved in the transcriptional activation of clusterin by glucose. Third, this glucose-induced activation process is mediated through tandem Ebox motifs in the first intron of the clusterin gene. The transcription factors ChREBP, SREBP-1c, and LXR are important in the nutritional regulation of hepatic genes. SREBP1c and LXR are known to mediate insulin signaling and stimulate lipogenesis. On the other hand, glucose signaling is thought to be transduced through ChREBP and to regulate transcription of ChoRE-containing target genes, such as liver pyruvate kinase
(L-PK), ACC, and fatty acid synthase (FAS). However, whether these transcription factors act as glucose sensors has been a subject of controversy. Mitro et al. reported that D-glucose and Dglucose-6-phosphate are direct agonists of LXR [26]. In contrast, Denechaud et al. demonstrated that LXR is not required for the induction of glucose-regulated genes in the liver and that glucose is required for ChREBP activity [27]. Recently, Anthonisen et al. showed that hepatic LXR is O-GlcNAcylated in refed and streptozotocin-induced diabetic mice concomitant with an increase in SREBP-1c expression, implying that LXR is a glucose sensor [28]. On the other hand, glucose also activates the SREBP-1c promoter independently of insulin [24]. Interestingly, we found in the current study that glucose-regulated expression of clusterin was not mediated by ChREBP or LXR, but rather by SREBP-1c. The GlRE of the clusterin promoter is composed of two E-box motifs separated by five nucleotides, a structure similar to that of ChoRE. Thus, additional studies will be required to determine the mechanism responsible for discriminating between the SREBP-1c response element and ChREBP response element. It is noteworthy that the relative induction of different ChoREs by SREBP-1c correlates with SREBP-1c binding affinity examined by electrophoretic mobility shift assays, in which SREBP-1c showed the
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Fig. 3. SREBP-1c activated clusterin transcription through E-box motifs in the first intron. (A) ChREBP/Mlx did not stimulate the transcriptional activity of clusterin promoters in HepG2 cells. Clusterin promoter-luciferase reporter constructs were cotransfected with ChREBP and Mlxb or Mlxc. Six hours after transfection, cells were incubated in 25 mM DMEM for 24 h and lysed for assay of luciferase activity. An ACC ChoRE reporter was used as a positive control. (B and C) Effects of SREBP-1c on transcription were determined by luciferase assay in cells cotransfected with SREBP-1c expression plasmids and various wild-type or mutant clusterin promoter–luciferase reporter constructs. (D) ChIP analyses were performed using anti-SREBP-1c antibodies or preimmune IgG. Recruitment of SREBP-1c to E-boxes of the clusterin gene was measured by qRT-PCR. ⁄ P < 0.005.
highest affinity for the S14 ChoRE and the lowest affinity for the LPK ChoRE [29]. The clusterin gene is a single, nine-exon gene that is transcribed into three mRNA isoforms, termed isoform 1, isoform 2, and isoform 11036 [30,31]. It is translated into several protein forms [31,32], although it is not yet clear how each transcript is related to the diverse clusterin protein forms. Notwithstanding these uncertainties, the wide variation in clusterin expression levels in different tissues implies that clusterin expression is tightly regulated. Hormones and growth factors are well-known regulators of clusterin gene expression [33,34], and clusterin expression is also affected by oncogenes [35]. However, despite considerable evidence for a link between clusterin and metabolic diseases such as diabetes and atherosclerosis, the metabolic regulation of clusterin expression has not been previously studied. To our knowledge, this is the first study demonstrating nutrient-regulated clusterin expression. This study not only strengthens the relevance of clusterin in metabolic regulation, but also elucidates a novel pathway of glucose signaling. Clusterin has been shown to be involved in diverse pathological states, including cancer, Alzheimer disease, and cardiovascular disease, all of which could be caused by metabolic disturbances. Therefore, modulation of this regulation module could be exploited to prevent or treat clusterin-related diseases. Disclosure statement None declared. Acknowledgments This work was supported by grants from the National Research Foundation of Korea funded by the Korean Government (2006-
0050225 and 2009-0084114); the Korea Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084335); and by intramural grants (2007-352 and 2009352) from the Asan Institute for Life Sciences, Seoul, Korea. References [1] I.B. Fritz, K. Burdzy, B. Setchell, O. Blaschuk, Ram rete testis fluid contains a protein (clusterin) which influences cell–cell interactions in vitro, Biol. Reprod. 28 (1983) 1173–1188. [2] M.R. Wilson, S.B. Easterbrook-Smith, Clusterin is a secreted mammalian chaperone, Trends Biochem. Sci. 25 (2000) 95–98. [3] B.F. Burkey, H.V. deSilva, J.A. Harmony, Intracellular processing of apolipoprotein J precursor to the mature heterodimer, J. Lipid Res. 32 (1991) 1039–1048. [4] S. Bettuzzi, Introduction, Adv. Cancer Res. 104 (2009) 1–8 (Chapter 1). [5] H.V. de Silva, J.A. Harmony, W.D. Stuart, C.M. Gil, J. Robbins, Apolipoprotein J: structure and tissue distribution, Biochemistry 29 (1990) 5380–5389. [6] T.C. Jordan-Starck, S.D. Lund, D.P. Witte, B.J. Aronow, C.A. Ley, W.D. Stuart, D.K. Swertfeger, L.R. Clayton, S.F. Sells, B. Paigen, Mouse apolipoprotein J: characterization of a gene implicated in atherosclerosis, J. Lipid Res. 35 (1994) 194–210. [7] M.E. Rosenberg, J. Silkensen, Clusterin: physiologic and pathophysiologic considerations, Int. J. Biochem. Cell Biol. 27 (1995) 633–645. [8] F. Rizzi, S. Bettuzzi, Targeting clusterin in prostate cancer, J. Physiol. Pharmacol. 59 (Suppl. 9) (2008) 265–274. [9] H. Miyake, M. Muramaki, J. Furukawa, T. Kurahashi, M. Fujisawa, Serum level of clusterin and its density in men with prostate cancer as novel biomarkers reflecting disease extension, Urology 75 (2010) 454–459. [10] J. Bi, A.L. Guo, Y.R. Lai, B. Li, J.M. Zhong, H.Q. Wu, Z. Xie, Y.L. He, Z.L. Lv, S.H. Lau, Q. Wang, X.H. Huang, L.J. Zhang, J.M. Wen, X.Y. Guan, Overexpression of clusterin correlates with tumor progression, metastasis in gastric cancer: a study on tissue microarrays, Neoplasma 57 (2010) 191–197. [11] L. Flanagan, L. Whyte, N. Chatterjee, M. Tenniswood, Effects of clusterin overexpression on metastatic progression and therapy in breast cancer, BMC Cancer 10 (2010) 107. [12] S. Pucci, E. Bonanno, F. Sesti, P. Mazzarelli, A. Mauriello, F. Ricci, G.B. Zoccai, F. Rulli, G. Galata, L.G. Spagnoli, Clusterin in stool: a new biomarker for colon cancer screening?, Am J. Gastroenterol. 104 (2009) 2807–2815.
G. Kim et al. / Biochemical and Biophysical Research Communications 408 (2011) 720–725 [13] H. Watari, T. Kanuma, Y. Ohta, M.K. Hassan, T. Mitamura, M. Hosaka, T. Minegishi, N. Sakuragi, Clusterin expression inversely correlates with chemosensitivity and predicts poor survival in patients with locally advanced cervical cancer treated with cisplatin-based neoadjuvant chemotherapy and radical hysterectomy, Pathol. Oncol. Res. 16 (2010) 345–352. [14] G.F. Yang, X.M. Li, D. Xie, Overexpression of clusterin in ovarian cancer is correlated with impaired survival, Int. J. Gynecol. Cancer 19 (2009) 1342–1346. [15] B.F. Burkey, W.D. Stuart, J.A. Harmony, Hepatic apolipoprotein J is secreted as a lipoprotein, J. Lipid Res. 33 (1992) 1517–1526. [16] I.P. Trougakos, M. Poulakou, M. Stathatos, A. Chalikia, A. Melidonis, E.S. Gonos, Serum levels of the senescence biomarker clusterin/apolipoprotein J increase significantly in diabetes type II and during development of coronary heart disease or at myocardial infarction, Exp. Gerontol. 37 (2002) 1175– 1187. [17] T. Kujiraoka, H. Hattori, Y. Miwa, M. Ishihara, T. Ueno, J. Ishii, M. Tsuji, T. Iwasaki, Y. Sasaguri, T. Fujioka, S. Saito, M. Tsushima, T. Maruyama, I.P. Miller, N.E. Miller, T. Egashira, Serum apolipoprotein J in health, coronary heart disease and type 2 diabetes mellitus, J. Atheroscler. Thromb. 13 (2006) 314– 322. [18] A.N. Hoofnagle, M. Wu, A.K. Gosmanova, J.O. Becker, E.M. Wijsman, J.D. Brunzell, S.E. Kahn, R.H. Knopp, T.J. Lyons, J.W. Heinecke, Low clusterin levels in high-density lipoprotein associate with insulin resistance, obesity, and dyslipoproteinemia, Arterioscler. Thromb. Vasc. Biol. 30 (2010) 2528–2534. [19] M. Daimon, T. Oizumi, S. Karasawa, W. Kaino, K. Takase, K. Tada, Y. Jimbu, K. Wada, W. Kameda, S. Susa, M. Muramatsu, I. Kubota, S. Kawata, T. Kato, Association of the clusterin gene polymorphisms with type 2 diabetes mellitus, Metabolism, 2010. [20] Y. Miwa, S. Takiuchi, K. Kamide, M. Yoshii, T. Horio, C. Tanaka, M. Banno, T. Miyata, T. Sasaguri, Y. Kawano, Insertion/deletion polymorphism in clusterin gene influences serum lipid levels and carotid intima-media thickness in hypertensive Japanese females, Biochem. Biophys. Res. Commun. 331 (2005) 1587–1593. [21] J.E. Klaunig, P.J. Goldblatt, D.E. Hinton, M.M. Lipsky, J. Chacko, B.F. Trump, Mouse liver cell culture. I. Hepatocyte isolation, In Vitro 17 (1981) 913–925. [22] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2DDC T method, Methods 25 (2001) 402–408. [23] V. Orlando, H. Strutt, R. Paro, Analysis of chromatin structure by in vivo formaldehyde cross-linking, Methods 11 (1997) 205–214.
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[24] A.H. Hasty, H. Shimano, N. Yahagi, M. Amemiya-Kudo, S. Perrey, T. Yoshikawa, J. Osuga, H. Okazaki, Y. Tamura, Y. Iizuka, F. Shionoiri, K. Ohashi, K. Harada, T. Gotoda, R. Nagai, S. Ishibashi, N. Yamada, Sterol regulatory element-binding protein-1 is regulated by glucose at the transcriptional level, J. Biol. Chem. 275 (2000) 31069–31077. [25] J.B. Kim, G.D. Spotts, Y.D. Halvorsen, H.M. Shih, T. Ellenberger, H.C. Towle, B.M. Spiegelman, Dual DNA binding specificity of ADD1/SREBP1 controlled by a single amino acid in the basic helix–loop–helix domain, Mol. Cell Biol. 15 (1995) 2582–2588. [26] N. Mitro, P.A. Mak, L. Vargas, C. Godio, E. Hampton, V. Molteni, A. Kreusch, E. Saez, The nuclear receptor LXR is a glucose sensor, Nature 445 (2007) 219– 223. [27] P.D. Denechaud, P. Bossard, J.M. Lobaccaro, L. Millatt, B. Staels, J. Girard, C. Postic, ChREBP, but not LXRs, is required for the induction of glucose-regulated genes in mouse liver, J. Clin. Invest. 118 (2008) 956–964. [28] E.H. Anthonisen, L. Berven, S. Holm, M. Nygard, H.I. Nebb, L.M. GrønningWang, Nuclear receptor liver X receptor is O-GlcNAc-modified in response to glucose, J. Biol. Chem. 285 (2010) 1607–1615. [29] S.H. Koo, A.K. Dutcher, H.C. Towle, Glucose and insulin function through two distinct transcription factors to stimulate expression of lipogenic enzyme genes in liver, J. Biol. Chem. 276 (2001) 9437–9445. [30] C. Lee, L. Atanelov, B. Modrek, Y. Xing, ASAP: the alternative splicing annotation project, Nucleic Acids Res. 31 (2003) 101–105. [31] F. Rizzi, M. Coletta, S. Bettuzzi, Clusterin (CLU): from one gene and two transcripts to many proteins, Adv. Cancer Res. 104 (2009) 9–23 (Chapter 2). [32] R.M. Moretti, M.M. Marelli, S. Mai, A. Cariboni, M. Scaltriti, S. Bettuzzi, P. Limonta, Clusterin isoforms differentially affect growth and motility of prostate cells: possible implications in prostate tumorigenesis, Cancer Res. 67 (2007) 10325–10333. [33] C. Gutacker, G. Klock, P. Diel, C. Koch-Brandt, Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells, Biochem. J. 339 (Pt 3) (1999) 759–766. [34] D.R. Cochrane, Z. Wang, M. Muramaki, M.E. Gleave, C.C. Nelson, Differential regulation of clusterin and its isoforms by androgens in prostate cells, J. Biol. Chem. 282 (2007) 2278–2287. [35] A. Sala, S. Bettuzzi, S. Pucci, O. Chayka, M. Dews, A. Thomas-Tikhonenko, Regulation of CLU gene expression by oncogenes and epigenetic factors implications for tumorigenesis, Adv. Cancer Res. 105 (2009) 115–132.
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SREBP-1c regulates glucose-stimulated hepatic clusterin expression Gukhan Kim a,1,2, Geun Hyang Kim a,c,1, Gyun-Sik Oh a,c,1, Jin Yoon a,c, Hae Won Kim a, Min-Seon Kim b, Seung-Whan Kim a,c,⇑ a
Department of Pharmacology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea c Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 20 April 2011 Available online 28 April 2011 Keywords: Clusterin Apolipoprotein J Glucose SREBP-1c E-box
a b s t r a c t Clusterin is a stress-response protein that is involved in diverse biological processes, including cell proliferation, apoptosis, tissue differentiation, inflammation, and lipid transport. Its expression is upregulated in a broad spectrum of diverse pathological states. Clusterin was recently reported to be associated with diabetes, metabolic syndrome, and their sequelae. However, the regulation of clusterin expression by metabolic signals was not addressed. In this study we evaluated the effects of glucose on hepatic clusterin expression. Interestingly, high glucose concentrations significantly increased clusterin expression in primary hepatocytes and hepatoma cell lines, but the conventional promoter region of the clusterin gene did not respond to glucose stimulation. In contrast, the first intronic region was transcriptionally activated by high glucose concentrations. We then defined a glucose response element (GlRE) of the clusterin gene, showing that it consists of two E-box motifs separated by five nucleotides and resembles carbohydrate response element (ChoRE). Unexpectedly, however, these E-box motifs were not activated by ChoRE binding protein (ChREBP), but were activated by sterol regulatory element binding protein-1c (SREBP-1c). Furthermore, we found that glucose induced recruitment of SREBP-1c to the E-box of the clusterin gene intronic region. Taken together, these results suggest that clusterin expression is increased by glucose stimulation, and SREBP-1c plays a crucial role in the metabolic regulation of clusterin. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Clusterin, also known as apolipoprotein J (Apo J), agingassociated protein 4 (AAG4), complement lysis inhibitor (CLI), testosterone-repressed prostate message 2 (TRPM-2), Ku70-binding protein 1 (KUB1), X-ray-induced protein 8 (XIP8), and sulfated glycoprotein-2 (SGP-2), was first identified as a glycoprotein that elicited clustering of suspended cells [1]. It is a 75–80 kDa disulfide-linked heterodimeric protein composed of a and b subunits that are generated by a post-translational cleavage of a single-chain precursor protein [2,3]. Clusterin has been proposed to be involved in a variety of important biological processes, including sperm maturation, tissue differentiation, tissue remodeling, membrane recycling, reverse lipid transport, cell–cell or cell–substrate interaction, promotion of erythrocyte aggregation, attenuation of complement
⇑ Corresponding author at: Department of Pharmacology, University of Ulsan College of Medicine, 388-1 PungNap-2dong, Songpa-gu, Seoul 138-736, Republic of Korea. Fax: +82 2 3010 2941. E-mail address: [email protected] (S.-W. Kim). 1 These authors contributed equally to this work. 2 Present address: Oregon Health and Science University, Portland, OR 97239-3098, United States. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.04.111
activity, cell proliferation, cell survival, and apoptosis [4]. It is expressed ubiquitously in many tissues. Clusterin transcripts are present at relatively high levels in the brain, ovary, testis, stomach, and liver; are less abundant in heart, spleen, lung, kidney, and breast; and are absent in T-lymphocytes [5,6]. Interestingly, clusterin expression is differentially regulated in many pathological conditions, including cancer, atherosclerosis, diabetes, and renal and neurodegenerative diseases [7,8]. A number of studies have shown that clusterin expression levels are associated with prostate cancer [9], gastric cancer [10], and breast cancer [11], as well as colon [12], cervical [13], and ovarian cancers [14]. There is also considerable evidence suggesting a relationship between clusterin and the metabolic diseases such as diabetes and atherosclerosis [15–19]. Type 2 diabetic subjects have higher serum clusterin levels than healthy individuals [17], and these elevated levels of clusterin are positively correlated with blood glucose levels. In addition, an analysis of single nucleotide polymorphisms (SNPs) in Japanese subjects revealed a significant association of clusterin gene polymorphisms with diabetes, serum lipid levels, and the progression of carotid atherosclerosis [19,20]. Given these associations of clusterin with metabolic diseases, it is reasonable to speculate that clusterin expression could be regulated by metabolic signals, such as nutrients. However, to date, no studies
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have addressed the metabolic regulation of clusterin expression. In this study, we sought to investigate whether glucose regulates clusterin expression in hepatocytes and elucidate the underlying regulatory mechanism at the transcriptional level. Our results indicate that glucose stimulation increases clusterin expression, and further show that sterol regulatory element binding protein-1c (SREBP-1c) is involved in glucose-stimulated transcriptional activation of the clusterin gene through tandem intronic E-box motifs.
2. Materials and methods 2.1. Isolation of primary hepatocytes and cell culture Primary hepatocytes were isolated from 10-week-old mice using the previously described collagenase perfusion method, with minor modifications [21]. Perfusion was performed with Mg2+/ Ca2+-free Hanks’ balanced salt solution containing 100 U/ml collagenase (Invitrogen, Carlsbad, CA) and 48 lg/ml trypsin inhibitor (Sigma, St. Louis, MO). Cells were plated onto collagen type I-coated culture dishes and maintained in Medium 199. Rat hepatoma FAO and human hepatoma HepG2 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified 5% CO2 incubator. 2.2. RNA preparation and quantitative real-time PCR (qRT-PCR) Total RNA was isolated from primary hepatocytes using the TRIzol reagent (Invitrogen). Purified total RNA was reverse-transcribed using M-MLV reverse transcriptase (Promega, Madison, WI), according to the manufacturer’s protocol. Quantitative gene expression analyses were performed on a Roche LightCycler 480 System (Roche, Basel, Switzerland) using SYBR Green PCR Master Mix. PCR primers were designed using Primer Express 3.0 software with the manufacturer’s default settings, and were validated for identical efficiencies. 18S rRNA was used as the internal control. Ratios of target genes to 18S rRNA expression levels were calculated using the 2DDC T method [22]. The oligonucleotide primers used for qRT-PCR were as follows: mClusterin, 50 -ATA AGG AGA TTC AGA ACG CC-30 and 50 -GCT CTG CGT TGG TTT TTT CTA TG-30 ; mSREBP-1c, 50 -AGC CAT GGA TTG CAC ATT TGA-30 and 50 -CAA ATA GGC CAG GGA AGT CA-30 ; 18s rRNA, 50 -CCG CGG TTC TAT TTT GTT GGT-30 and 50 -CTC TAG CGG CGC AAT ACG A-30 . The oligonucleotide primers used for conventional RT-PCR were as follows: mClusterin, 50 -AGG AGC TAA ACG ACT CGC T-30 and 50 -CTT TTC CTG CGG TAT TCC T-30 ; mSREBP-1c, 50 -GGC GCA TGG ATT GCA CAT TT-30 and 50 -GCA GGC TGT AGG ATG GTG A-30 ; 18s rRNA, 50 CGT CCC CCA ACT TCT TAG AG-30 and 50 -CAC CTA CGG AAA CCT TGT TAC-30 . 2.3. Preparation of cell lysates and Western blotting Protein extracts of hepatocytes were prepared in lysis buffer containing 20 mM Tris–Cl (pH 7.5), 100 mM KCl, 5 mM EDTA (pH 8.0), 10 mM Na4P2O7, 100 mM NaF, 2 mM Na2VO4, and 1% NP-40. Cells were incubated in lysis buffer for 1 h on ice. Cell debris was removed by centrifuging the lysates at 15,000g for 10 min. Protein concentrations were determined using the Bradford assay. For Western blot analysis, 50–100 lg of lysate protein was separated by electrophoresis on 7.5–10% polyacrylamide gels under denaturing conditions and then transferred to PVDF membranes (Millipore, Billerica, MA). Blots were incubated with antibodies to clusterin (1:1000 dilution; Santa Cruz), SREBP-1c (1:500 dilution; Santa Cruz), and b-actin (1:10,000 dilution; Sigma).
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2.4. Construction of luciferase reporter genes and luciferase assay The 2285/+430 and +2158/+4426 clusterin promoter regions used to construct promoter–luciferase reporter were amplified from human genomic DNA by PCR and were used as templates for the construction of other reporters. Deletion and site-specific mutations within the promoter were created using PCR cloning strategies. The 2285/+430 clusterin promoter and corresponding deletion constructs +230/+430 and +331/+430 were amplified using the unique forward primers 50 -CGG GGT ACC AAA CCC AGC TGT GTA AGT CCA TAA-30 (-2285), 50 -CGG GGT ACC GGC ATT CTT TGG GCG TGA GTC-30 (+230), and 50 -CGG GGT ACC CGC GGC GTC GCC AG-30 (+331), and the common reverse primer 50 -CCG CTC GAG CAT CCG TCC TGG TGT GGC TCT-30 (+430). The +2158/+4426 clusterin promoter was amplified using the primers 50 -CGG GGT ACC AAG TGG TTT AAG CCT TCT TAG G-30 (forward) and 50 -CCC CCC GGG AGA GAA CAG GAG ACC AT-30 (reverse). Two mutant promoters were prepared using the following primer pairs: M1, 50 -AGT GCT CAT CAG AGA CCC GTG AGA CCA CA-30 (forward) and 50 -GGT CTC TGA TGA GCA CTG CCC ACT GAG C-30 (reverse); and M2, 50 -GGT CAG AGA TGG TCA AGA CCA CAG CCT TC-30 (forward) and 50 -CTT GAC CAT CTC TGA CCC CAG CTG CCC A-30 (reverse). The authenticity of plasmid constructs was confirmed by DNA sequencing. For luciferase reporter assay, HepG2 cells were cultured in DMEM supplemented with 10% FBS. Transfections were performed in 24-well plates using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. pActin-bgal plasmid (50 ng) was included in each transfection experiment to control for the efficiency of transfection. Luciferase activity was measured using a luminometer (Centro LB 960, Berthold Technologies), and values were normalized to b-galactosidase activity. 2.5. Chromatin immunoprecipitation (ChIP) ChIP assays were performed as described previously, with minor modifications [23]. Briefly, HepG2 cells were transfected with SREBP-1c expression plasmids and incubated overnight. After stimulation with 25 mM glucose for 6 h, cells were fixed with 1% formaldehyde for 15 min at room temperature. The cells were washed with cold phosphate-buffered saline (PBS) and resuspended in lysis buffer containing 50 mM Tris–HCl (pH 8.0), 1% SDS, and 10 mM EDTA. Soluble chromatin was prepared by sonication and immunoprecipitated with antibodies against SREBP-1c (sc-13551; Santa Cruz Biotechnology) or preimmune IgG. The final DNA extractions were analyzed by qRT-PCR using two primer pairs: +3093/+3113 (50 -TTC TGG CTG GCT TTG TCT CTC T-30 ) and +3149/+3166 (50 TGC CCA CTG AGC CCT GAA-30 ), encompassing the first intronic Ebox-containing region; and +915/+936 (50 -TTC TGC CTC CTA ATG CAT CTG A-30 ) and +956/+975 (50 -AGG CCT GGT GGA TCT TGT GT30 ), encompassing a control region containing no E-boxes. 2.6. Statistical analysis Results are expressed as means ± standard errors (SEs). Statistical significance was assessed using unpaired Student’s two-tailed t-tests. Differences with a P-value <0.05 were considered statistically significant. 3. Results 3.1. High glucose concentration increases clusterin mRNA and protein levels The expressions of many metabolic enzymes and related factors are regulated by metabolic signals, such as nutrients. Clusterin is
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Fig. 1. High glucose increased hepatic clusterin expression. (A) Clusterin mRNA levels were determined by conventional RT-PCR analysis. Mouse primary hepatocytes were incubated in 5.5 mM glucose DMEM for 12 h and then stimulated with 25 mM glucose for 12 h. SREBP-1c and 18S rRNA were used as a known glucose-stimulated gene and a loading control, respectively. (B) Clusterin and SREBP-1c mRNA levels were determined by qRT-PCR. Mouse primary hepatocytes were stimulated with 25 mM glucose as described in (A). (C) Western blot analysis of clusterin protein in rat hepatoma FAO, human hepatoma HepG2, and mouse primary hepatocytes. Cells were incubated in 5.5 mM glucose DMEM for 12 h and then stimulated with 25 mM glucose DMEM for 24 h. ⁄P < 0.005.
differentially regulated in metabolic disease states and has been proposed to have metabolic functions. To address the relevance of clusterin to metabolism, we analyzed the effects of glucose concentration on hepatic clusterin expression. Conventional RT-PCR analyses showed that exposure of primary hepatocytes to high glucose induced an increase in clusterin transcript levels (Fig. 1A). SREBP-1c, which is positively regulated by glucose at the transcriptional level [24], was used as a positive control for hepatic glucose response in these experiments. These glucose-induced increases in clusterin and SREBP-1c mRNA expression were confirmed by qRTPCR (Fig. 1B). Consistent with the increases in clusterin at the mRNA level, clusterin protein levels were also increased by high glucose in both primary hepatocytes and hepatoma cell lines (Fig. 1C). These results suggest that clusterin expression in hepatocytes is regulated by the important nutrient, glucose, and indicate that clusterin may serve a metabolic function in the response to nutrient states. 3.2. Tandem E-boxes in the first intron are responsible for glucose stimulation of clusterin transcription To define the glucose response element (GlRE) in the clusterin promoter, we first analyzed the genomic sequence of the human clusterin gene using TransFac Search software and Genomatix program (Fig. 2A). The transcription factors carbohydrate response element binding protein (ChREBP), liver X receptor (LXR), and SREBP-1c are well known to mediate nutritional signals. Prediction software showed no LXR response elements (LXRE) in the upstream region of the clusterin gene, but did indicate the presence of sterol regulatory element (SRE)/E-box motifs and carbohydrate response elements (ChoREs) in the conventional promoter region and the first intronic region. Therefore, based on the locations of SRE/E-box and ChoRE, we constructed luciferase reporters containing conventional promoter regions or the first intronic region. Although the upstream conventional promoter region contained numerous SRE/E-box motifs and ChoREs, the transcriptional activity of this region was not activated by high glucose in hepatoma cell lines (Fig. 2B and C). Interestingly, glucose significantly stimulated the activity of a reporter driven by the first intronic region
containing a ChoRE. These results were reproduced in both rat hepatoma FAO and human hepatoma HepG2 cell lines (Fig. 2B and C). The ChoRE in the first intron of the clusterin gene consists of two Ebox motifs separated by five nucleotides. To confirm the role of these E-box motifs in the glucose-stimulated transcription of clusterin, we generated two mutant reporters, M1 and M2, in which each of the two E-box motifs was individually mutated in the context of the wild-type +2158/+4426 sequence (Fig. 2D). In both cases, mutation of the E-box motif abrogated glucose-stimulated transcription of the reporter gene (Fig. 2E). These results indicate that the putative ChoRE, consisting of two E-box motifs, is responsible for glucose-stimulated transcription of clusterin in hepatocytes. 3.3. Clusterin transcription by glucose is mediated by SREBP-1c Next, we sought to identify the transcription factor responsible for mediating transcription of the clusterin gene in response to glucose stimulation. It is well known that ChREBP binds to the ChoRE and activates transcription of ChoRE-containing target genes. Therefore, we tested the ability of ChREBP to activate the clusterin promoter. The basic helix-loop-helix leucine zipper (bHLH-LZ) factor Max-like protein X (Mlx)-b or -c was cotransfected with ChREBP because ChREBP requires heterodimerization with Mlx for transcriptional activity. Unexpectedly, ChREBP/Mlx did not activate either the upstream promoter or the intronic ChoRE-containing promoter (Fig. 3A). An acetyl-CoA carboxylase (ACC) ChoREcontaining reporter used as a positive control was substantially activated by ChREBP under the same conditions (Fig. 3A). On the other hand, it is well known that SREBP-1c binds to SRE or E-box [25], and SREBP-1c transcription is activated by glucose [24]. Accordingly, we investigated the effects of SREBP-1c on clusterin transcription by cotransfecting HepG2 cells with SREBP-1c expression plasmids and wild-type or mutated promoter-reporter constructs. Notably, SREBP-1c stimulated the transcriptional activity of the +2158/+4426 intronic reporter gene, but showed no significant effects on other reporter genes, similar to the effects of high glucose (Fig. 3B). Furthermore, this activation of the clusterin promoter by SREBP-1c was abolished by both M1 and M2 mutations
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Fig. 2. Identification of the glucose response element in the clusterin gene. (A) Representation of putative SREBP-1c and ChREBP binding sites in the human clusterin gene. Four reporter genes were constructed based on the locations of these binding sites. (B and C) Glucose responses of each reporter gene were determined in FAO (B) and HepG2 (C) cell lines by assaying the activity of luciferase reporter constructs. Six hours after transfection, cells were incubated in 5.5 mM glucose DMEM for 6 h, and then treated with 25 mM glucose for 12 h. (D) M1 and M2 mutant promoter reporters were constructed by mutation of each of two tandem E-box motifs in the first intron of the clusterin gene. (E) Glucose stimulation of the +2158/+4426 reporter was abrogated by M1 or M2 mutation in HepG2 cells.
(Fig. 3C). Finally, a ChIP analysis showed that SREBP-1c protein was recruited to E-box motifs of the clusterin intronic region in glucose-stimulated HepG2 cells (Fig. 3D). Taken together, these results suggest that SREBP-1c regulates the transcription of clusterin in response to glucose stimulation.
4. Discussion The present study was initiated to explore the possibility that clusterin expression is regulated by nutrient signals. Three major findings arose from this study. First, high glucose increases clusterin expression in hepatocytes. Second, SREBP-1c is directly involved in the transcriptional activation of clusterin by glucose. Third, this glucose-induced activation process is mediated through tandem Ebox motifs in the first intron of the clusterin gene. The transcription factors ChREBP, SREBP-1c, and LXR are important in the nutritional regulation of hepatic genes. SREBP1c and LXR are known to mediate insulin signaling and stimulate lipogenesis. On the other hand, glucose signaling is thought to be transduced through ChREBP and to regulate transcription of ChoRE-containing target genes, such as liver pyruvate kinase
(L-PK), ACC, and fatty acid synthase (FAS). However, whether these transcription factors act as glucose sensors has been a subject of controversy. Mitro et al. reported that D-glucose and Dglucose-6-phosphate are direct agonists of LXR [26]. In contrast, Denechaud et al. demonstrated that LXR is not required for the induction of glucose-regulated genes in the liver and that glucose is required for ChREBP activity [27]. Recently, Anthonisen et al. showed that hepatic LXR is O-GlcNAcylated in refed and streptozotocin-induced diabetic mice concomitant with an increase in SREBP-1c expression, implying that LXR is a glucose sensor [28]. On the other hand, glucose also activates the SREBP-1c promoter independently of insulin [24]. Interestingly, we found in the current study that glucose-regulated expression of clusterin was not mediated by ChREBP or LXR, but rather by SREBP-1c. The GlRE of the clusterin promoter is composed of two E-box motifs separated by five nucleotides, a structure similar to that of ChoRE. Thus, additional studies will be required to determine the mechanism responsible for discriminating between the SREBP-1c response element and ChREBP response element. It is noteworthy that the relative induction of different ChoREs by SREBP-1c correlates with SREBP-1c binding affinity examined by electrophoretic mobility shift assays, in which SREBP-1c showed the
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Fig. 3. SREBP-1c activated clusterin transcription through E-box motifs in the first intron. (A) ChREBP/Mlx did not stimulate the transcriptional activity of clusterin promoters in HepG2 cells. Clusterin promoter-luciferase reporter constructs were cotransfected with ChREBP and Mlxb or Mlxc. Six hours after transfection, cells were incubated in 25 mM DMEM for 24 h and lysed for assay of luciferase activity. An ACC ChoRE reporter was used as a positive control. (B and C) Effects of SREBP-1c on transcription were determined by luciferase assay in cells cotransfected with SREBP-1c expression plasmids and various wild-type or mutant clusterin promoter–luciferase reporter constructs. (D) ChIP analyses were performed using anti-SREBP-1c antibodies or preimmune IgG. Recruitment of SREBP-1c to E-boxes of the clusterin gene was measured by qRT-PCR. ⁄ P < 0.005.
highest affinity for the S14 ChoRE and the lowest affinity for the LPK ChoRE [29]. The clusterin gene is a single, nine-exon gene that is transcribed into three mRNA isoforms, termed isoform 1, isoform 2, and isoform 11036 [30,31]. It is translated into several protein forms [31,32], although it is not yet clear how each transcript is related to the diverse clusterin protein forms. Notwithstanding these uncertainties, the wide variation in clusterin expression levels in different tissues implies that clusterin expression is tightly regulated. Hormones and growth factors are well-known regulators of clusterin gene expression [33,34], and clusterin expression is also affected by oncogenes [35]. However, despite considerable evidence for a link between clusterin and metabolic diseases such as diabetes and atherosclerosis, the metabolic regulation of clusterin expression has not been previously studied. To our knowledge, this is the first study demonstrating nutrient-regulated clusterin expression. This study not only strengthens the relevance of clusterin in metabolic regulation, but also elucidates a novel pathway of glucose signaling. Clusterin has been shown to be involved in diverse pathological states, including cancer, Alzheimer disease, and cardiovascular disease, all of which could be caused by metabolic disturbances. Therefore, modulation of this regulation module could be exploited to prevent or treat clusterin-related diseases. Disclosure statement None declared. Acknowledgments This work was supported by grants from the National Research Foundation of Korea funded by the Korean Government (2006-
0050225 and 2009-0084114); the Korea Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084335); and by intramural grants (2007-352 and 2009352) from the Asan Institute for Life Sciences, Seoul, Korea. References [1] I.B. Fritz, K. Burdzy, B. Setchell, O. Blaschuk, Ram rete testis fluid contains a protein (clusterin) which influences cell–cell interactions in vitro, Biol. Reprod. 28 (1983) 1173–1188. [2] M.R. Wilson, S.B. Easterbrook-Smith, Clusterin is a secreted mammalian chaperone, Trends Biochem. Sci. 25 (2000) 95–98. [3] B.F. Burkey, H.V. deSilva, J.A. Harmony, Intracellular processing of apolipoprotein J precursor to the mature heterodimer, J. Lipid Res. 32 (1991) 1039–1048. [4] S. Bettuzzi, Introduction, Adv. Cancer Res. 104 (2009) 1–8 (Chapter 1). [5] H.V. de Silva, J.A. Harmony, W.D. Stuart, C.M. Gil, J. Robbins, Apolipoprotein J: structure and tissue distribution, Biochemistry 29 (1990) 5380–5389. [6] T.C. Jordan-Starck, S.D. Lund, D.P. Witte, B.J. Aronow, C.A. Ley, W.D. Stuart, D.K. Swertfeger, L.R. Clayton, S.F. Sells, B. Paigen, Mouse apolipoprotein J: characterization of a gene implicated in atherosclerosis, J. Lipid Res. 35 (1994) 194–210. [7] M.E. Rosenberg, J. Silkensen, Clusterin: physiologic and pathophysiologic considerations, Int. J. Biochem. Cell Biol. 27 (1995) 633–645. [8] F. Rizzi, S. Bettuzzi, Targeting clusterin in prostate cancer, J. Physiol. Pharmacol. 59 (Suppl. 9) (2008) 265–274. [9] H. Miyake, M. Muramaki, J. Furukawa, T. Kurahashi, M. Fujisawa, Serum level of clusterin and its density in men with prostate cancer as novel biomarkers reflecting disease extension, Urology 75 (2010) 454–459. [10] J. Bi, A.L. Guo, Y.R. Lai, B. Li, J.M. Zhong, H.Q. Wu, Z. Xie, Y.L. He, Z.L. Lv, S.H. Lau, Q. Wang, X.H. Huang, L.J. Zhang, J.M. Wen, X.Y. Guan, Overexpression of clusterin correlates with tumor progression, metastasis in gastric cancer: a study on tissue microarrays, Neoplasma 57 (2010) 191–197. [11] L. Flanagan, L. Whyte, N. Chatterjee, M. Tenniswood, Effects of clusterin overexpression on metastatic progression and therapy in breast cancer, BMC Cancer 10 (2010) 107. [12] S. Pucci, E. Bonanno, F. Sesti, P. Mazzarelli, A. Mauriello, F. Ricci, G.B. Zoccai, F. Rulli, G. Galata, L.G. Spagnoli, Clusterin in stool: a new biomarker for colon cancer screening?, Am J. Gastroenterol. 104 (2009) 2807–2815.
G. Kim et al. / Biochemical and Biophysical Research Communications 408 (2011) 720–725 [13] H. Watari, T. Kanuma, Y. Ohta, M.K. Hassan, T. Mitamura, M. Hosaka, T. Minegishi, N. Sakuragi, Clusterin expression inversely correlates with chemosensitivity and predicts poor survival in patients with locally advanced cervical cancer treated with cisplatin-based neoadjuvant chemotherapy and radical hysterectomy, Pathol. Oncol. Res. 16 (2010) 345–352. [14] G.F. Yang, X.M. Li, D. Xie, Overexpression of clusterin in ovarian cancer is correlated with impaired survival, Int. J. Gynecol. Cancer 19 (2009) 1342–1346. [15] B.F. Burkey, W.D. Stuart, J.A. Harmony, Hepatic apolipoprotein J is secreted as a lipoprotein, J. Lipid Res. 33 (1992) 1517–1526. [16] I.P. Trougakos, M. Poulakou, M. Stathatos, A. Chalikia, A. Melidonis, E.S. Gonos, Serum levels of the senescence biomarker clusterin/apolipoprotein J increase significantly in diabetes type II and during development of coronary heart disease or at myocardial infarction, Exp. Gerontol. 37 (2002) 1175– 1187. [17] T. Kujiraoka, H. Hattori, Y. Miwa, M. Ishihara, T. Ueno, J. Ishii, M. Tsuji, T. Iwasaki, Y. Sasaguri, T. Fujioka, S. Saito, M. Tsushima, T. Maruyama, I.P. Miller, N.E. Miller, T. Egashira, Serum apolipoprotein J in health, coronary heart disease and type 2 diabetes mellitus, J. Atheroscler. Thromb. 13 (2006) 314– 322. [18] A.N. Hoofnagle, M. Wu, A.K. Gosmanova, J.O. Becker, E.M. Wijsman, J.D. Brunzell, S.E. Kahn, R.H. Knopp, T.J. Lyons, J.W. Heinecke, Low clusterin levels in high-density lipoprotein associate with insulin resistance, obesity, and dyslipoproteinemia, Arterioscler. Thromb. Vasc. Biol. 30 (2010) 2528–2534. [19] M. Daimon, T. Oizumi, S. Karasawa, W. Kaino, K. Takase, K. Tada, Y. Jimbu, K. Wada, W. Kameda, S. Susa, M. Muramatsu, I. Kubota, S. Kawata, T. Kato, Association of the clusterin gene polymorphisms with type 2 diabetes mellitus, Metabolism, 2010. [20] Y. Miwa, S. Takiuchi, K. Kamide, M. Yoshii, T. Horio, C. Tanaka, M. Banno, T. Miyata, T. Sasaguri, Y. Kawano, Insertion/deletion polymorphism in clusterin gene influences serum lipid levels and carotid intima-media thickness in hypertensive Japanese females, Biochem. Biophys. Res. Commun. 331 (2005) 1587–1593. [21] J.E. Klaunig, P.J. Goldblatt, D.E. Hinton, M.M. Lipsky, J. Chacko, B.F. Trump, Mouse liver cell culture. I. Hepatocyte isolation, In Vitro 17 (1981) 913–925. [22] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2DDC T method, Methods 25 (2001) 402–408. [23] V. Orlando, H. Strutt, R. Paro, Analysis of chromatin structure by in vivo formaldehyde cross-linking, Methods 11 (1997) 205–214.
725
[24] A.H. Hasty, H. Shimano, N. Yahagi, M. Amemiya-Kudo, S. Perrey, T. Yoshikawa, J. Osuga, H. Okazaki, Y. Tamura, Y. Iizuka, F. Shionoiri, K. Ohashi, K. Harada, T. Gotoda, R. Nagai, S. Ishibashi, N. Yamada, Sterol regulatory element-binding protein-1 is regulated by glucose at the transcriptional level, J. Biol. Chem. 275 (2000) 31069–31077. [25] J.B. Kim, G.D. Spotts, Y.D. Halvorsen, H.M. Shih, T. Ellenberger, H.C. Towle, B.M. Spiegelman, Dual DNA binding specificity of ADD1/SREBP1 controlled by a single amino acid in the basic helix–loop–helix domain, Mol. Cell Biol. 15 (1995) 2582–2588. [26] N. Mitro, P.A. Mak, L. Vargas, C. Godio, E. Hampton, V. Molteni, A. Kreusch, E. Saez, The nuclear receptor LXR is a glucose sensor, Nature 445 (2007) 219– 223. [27] P.D. Denechaud, P. Bossard, J.M. Lobaccaro, L. Millatt, B. Staels, J. Girard, C. Postic, ChREBP, but not LXRs, is required for the induction of glucose-regulated genes in mouse liver, J. Clin. Invest. 118 (2008) 956–964. [28] E.H. Anthonisen, L. Berven, S. Holm, M. Nygard, H.I. Nebb, L.M. GrønningWang, Nuclear receptor liver X receptor is O-GlcNAc-modified in response to glucose, J. Biol. Chem. 285 (2010) 1607–1615. [29] S.H. Koo, A.K. Dutcher, H.C. Towle, Glucose and insulin function through two distinct transcription factors to stimulate expression of lipogenic enzyme genes in liver, J. Biol. Chem. 276 (2001) 9437–9445. [30] C. Lee, L. Atanelov, B. Modrek, Y. Xing, ASAP: the alternative splicing annotation project, Nucleic Acids Res. 31 (2003) 101–105. [31] F. Rizzi, M. Coletta, S. Bettuzzi, Clusterin (CLU): from one gene and two transcripts to many proteins, Adv. Cancer Res. 104 (2009) 9–23 (Chapter 2). [32] R.M. Moretti, M.M. Marelli, S. Mai, A. Cariboni, M. Scaltriti, S. Bettuzzi, P. Limonta, Clusterin isoforms differentially affect growth and motility of prostate cells: possible implications in prostate tumorigenesis, Cancer Res. 67 (2007) 10325–10333. [33] C. Gutacker, G. Klock, P. Diel, C. Koch-Brandt, Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells, Biochem. J. 339 (Pt 3) (1999) 759–766. [34] D.R. Cochrane, Z. Wang, M. Muramaki, M.E. Gleave, C.C. Nelson, Differential regulation of clusterin and its isoforms by androgens in prostate cells, J. Biol. Chem. 282 (2007) 2278–2287. [35] A. Sala, S. Bettuzzi, S. Pucci, O. Chayka, M. Dews, A. Thomas-Tikhonenko, Regulation of CLU gene expression by oncogenes and epigenetic factors implications for tumorigenesis, Adv. Cancer Res. 105 (2009) 115–132.