Artigo Acesso aberto Revisado por pares

Klotho is a target gene of PPAR-γ

2008; Elsevier BV; Volume: 74; Issue: 6 Linguagem: Inglês

10.1038/ki.2008.244

ISSN

1523-1755

Autores

Hong Zhang, Yuanyuan Li, Yanbo Fan, Jing Wu, Beilei Zhao, Youfei Guan, Shu Chien, Nanping Wang,

Tópico(s)

Chronic Kidney Disease and Diabetes

Resumo

Klotho is an anti-aging gene whose expression is regulated by many stimuli. Here we examined the transcriptional regulation of the klotho gene by peroxisome proliferator-activated receptor-γ (PPAR-γ). The PPAR-γ agonists thiazolidinediones increased both klotho mRNA and protein expression in HEK293 cells and several renal epithelial cell lines. The induction was blocked by PPAR-γ antagonists or small-interfering RNA-mediated gene silencing of PPAR-γ, suggesting a PPAR-γ-dependent mechanism. Chromatin immuno-precipitation and gel shift assays found a noncanonical PPAR-responsive element within the 5′-flanking region of the human klotho gene with promoter–reporter assays further confirming transcriptional functionality. Moreover, thiazolidinediones or adenovirus-mediated overexpression of PPAR-γ increased klotho expression in mouse kidneys while renal klotho expression was attenuated in mice treated with PPAR-γ antagonists. These results demonstrate that klotho is a target gene of PPAR-γ. Klotho is an anti-aging gene whose expression is regulated by many stimuli. Here we examined the transcriptional regulation of the klotho gene by peroxisome proliferator-activated receptor-γ (PPAR-γ). The PPAR-γ agonists thiazolidinediones increased both klotho mRNA and protein expression in HEK293 cells and several renal epithelial cell lines. The induction was blocked by PPAR-γ antagonists or small-interfering RNA-mediated gene silencing of PPAR-γ, suggesting a PPAR-γ-dependent mechanism. Chromatin immuno-precipitation and gel shift assays found a noncanonical PPAR-responsive element within the 5′-flanking region of the human klotho gene with promoter–reporter assays further confirming transcriptional functionality. Moreover, thiazolidinediones or adenovirus-mediated overexpression of PPAR-γ increased klotho expression in mouse kidneys while renal klotho expression was attenuated in mice treated with PPAR-γ antagonists. These results demonstrate that klotho is a target gene of PPAR-γ. The klotho gene encodes a type I transmembrane protein that shares homology with β-glucosidase. Klotho deficiency in mice results in a phenotype resembling human aging, including arteriosclerosis, neural degeneration, defective hearing, skin and gonadal atrophy, ectopic calcification in various soft tissue, pulmonary emphysema, and greatly shortened lifespan.1.Kuro-o M. Matsumura Y. Aizawa H. et al.Mutation of the mouse klotho gene leads to a syndrome resembling ageing.Nature. 1997; 390: 45-51Crossref PubMed Scopus (2488) Google Scholar The klotho gene is predominantly expressed in the kidney and, to a lesser extent, in the brain and reproductive and endocrine organs. The expression of the klotho gene is regulated in response to various physiological and pathological conditions such as thyroid hormone, oxidative stress, long-term hypertension, chronic renal failure,2.Koh N. Fujimori T. Nishiguchi S. et al.Severely reduced production of Klotho in human chronic renal failure kidney.Biochem Biophys Res Com. 2001; 280: 1015-1020Crossref PubMed Scopus (361) Google Scholar and so on. To date, little has been known about the transcriptional mechanisms underlying the regulated expression of the klotho gene. Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated nuclear hormone receptor and transcription factors.3.Forman B.M. Chen J. Evans R.M. The peroxisome proliferator-activated receptors: ligands and activators.Ann NY Acad Sci. 1996; 804: 266-275Crossref PubMed Scopus (196) Google Scholar,4.Desvergne B. Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism.Endocr Rev. 1999; 20: 649-688Crossref PubMed Scopus (2620) Google Scholar Among the three isoforms α, β/δ, and γ, PPAR-γ is predominantly expressed in adipose tissues and, to a lesser extent, in other tissues including kidneys.5.Guan Y. Zhang Y. Breyer M.D. The role of PPARs in the transcriptional control of cellular processes.Drug News Perspect. 2002; 15: 147-154Crossref PubMed Scopus (59) Google Scholar,6.Guan Y. Hao C. Cha D.R. et al.Thiazolidinediones expand body fluid volume through PPARgamma stimulation of ENaC-mediated renal salt absorption.Nat Med. 2005; 11: 861-866Crossref PubMed Scopus (496) Google Scholar PPAR-γ is activated by various natural and synthetic ligands and plays important roles in adipogenesis 7.Farmer S.R. Regulation of PPARgamma activity during adipogenesis.Int J Obes (Lond). 2005; 29: S13-S16Crossref PubMed Scopus (307) Google Scholar and insulin sensitivity.8.Nehlin J.O. Mogensen J.P. Petterson I. et al.Selective PPAR agonists for the treatment of type 2 diabetes.Ann NY Acad Sci. 2006; 1067: 448-453Crossref PubMed Scopus (17) Google Scholar An earlier study showed that troglitazone, an insulin sensitizer and agonist for PPAR-γ, augmented the renal klotho mRNA expression in OLETF (Otsuka Long-Evans Tokushima Fatty) rats, which exhibit pathophysiological changes that resemble metabolic syndrome in human, including hypertension, obesity, severe hyperglycemia, and hypertriglyceridemia.9.Yamagishi T. Saito Y. Nakamura T. et al.Troglitazone improves endothelial function and augments renal klotho mRNA expression in Otsuka Long-Evans Tokushima Fatty (OLETF) rats with multiple atherogenic risk factors.Hypertension Res. 2001; 24: 705-709Crossref PubMed Scopus (45) Google Scholar It has also been recently described that klotho promotes adipocyte differentiation in cultured preadipocytes10.Chihara Y. Rakugi H. Ishikawa K. et al.Klotho protein promotes adipocyte differentiation.Endocrinology. 2006; 147: 3835-3842Crossref PubMed Scopus (68) Google Scholar and that, in transgenic mice, overexpression of klotho slows the aging process through induction of insulin resistance. However, the role of PPAR-γ in the regulation of the klotho gene expression remains unknown. Therefore, this study was performed to characterize the specific role of PPAR-γ in the induction of the klotho gene both in vitro and in vivo. To determine the effect of PPAR-γ on the expression of klotho gene, we treated 293 cells with PPAR-γ agonists troglitazone and ciglitazone, both being thiazolidinediones (TZDs), at various concentrations for different time periods. Klotho mRNA level was measured by quantitative reverse-transcriptase-PCR (qRT-PCR). As shown in Figure 1, both troglitazone and ciglitazone significantly increased the mRNA expression of klotho gene in 293 cells in a time- and dose-dependent manner. To ascertain whether TZDs also induce klotho gene expression in differentiated renal tubular epithelial cells, we further examined the effect of troglitazone in IMCD (inner medullary collecting ducts cells), MCT (proximal tubular epithelial cells),11.Haverty T.P. Kelly C.J. Hines W.H. et al.Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis.J Cell Biol. 1988; 107: 1359-1368Crossref PubMed Scopus (261) Google Scholar and MDCK (distal tubule cells)12.Ng K.H. Lim B.G. Wong K.P. Sulfate conjugating and transport functions of MDCK distal tubular cells.Kidney Int. 2003; 63: 976-986Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar,13.Gaush C.R. Hard W.L. Smith T.F. Characterization of an established line of canine kidney cells (MDCK).Proc Soc Exp Biol Med. 1966; 122: 931-935Crossref PubMed Scopus (245) Google Scholar cell lines. Northern analysis confirmed that klotho gene was expressed to different extent in all the three cell lines tested, and the expression was similarly induced by troglitazone (Figure 2b).Figure 2TZD-induced klotho expression is dependent on PPAR-γ activation. (a) HEK293 or renal cell lines were treated with or without GW9662 (20 μM) or BADGE (20 μM) for 1 h and exposed to DMSO or troglitazone (50 μM) for 24 h. Total RNA was extracted and subjected to northern blotting (the upper panel) and qRT-PCR (the lower panel). (b) Northern analysis of klotho gene expression in IMCD, MCT, or MDCK cell lines. The bar graph represents the results of scanned densities of three northern blotting experiments using the NIH Image J after normalization with β-actin. (c) HEK293 cells were transfected with siRNA specific for PPAR-γ or GFP (40 nM) for 48 h before the exposure to troglitazone or DMSO. Relative mRNA levels of PPAR-γ and klotho were determined by using qRT-PCR. *P<0.05; **P<0.01.View Large Image Figure ViewerDownload (PPT) To examine whether the TZD-induced expression of klotho was mediated by PPAR-γ activation, cells were pretreated with a selective PPAR-γ antagonist GW9662 (20 μM for 1 h) prior to the incubation with troglitazone. Both qRT-PCR and northern blotting demonstrated that GW9662 blocked the troglitazone-induced increase of klotho mRNA (Figure 2a and b). Similarly, bisphenol-A diglycidyl ether (BADGE), another PPAR-γ antagonist, also reduced the induction of klotho expression by troglitazone (Figure 2a, lower panel). To further confirm the role of endogenous PPAR-γ on the expression of klotho mRNA, the cells were transfected with PPAR-γ-specific short interfering RNA (siRNA) or green fluorescence protein (GFP)-siRNA as a control. As shown in Figure 2c, the expression level of the endogenous PPAR-γ was reduced by approximately 90% compared with the control siRNA-transfected cells. Knockdown of PPAR-γ abrogated the trogilatazone-induced expression of klotho. Taken together, these results suggested that TZD-induced klotho expression was dependent on the activation of endogenous PPAR-γ. Sequence analysis of the 5′-flanking region of human klotho gene revealed no typical binding site for PPARs. However, two noncanonical PPAR-responsive element (PPRE) motifs were found, one at –3698 bp (GAACTCCTGACCT) and the other at -2760 bp (ACTGGATGAAGGA) upstream of the ATG start codon. The binding activities of these regions to PPAR-γ were examined by chromatin immunoprecipitation (ChIP) assay, which showed that the region harboring the distal PPRE could be immunoprecipitated by PPAR-γ antibody but not the control IgG (Figure 3a), indicating that PPAR-γ was able to bind to this region of the klotho gene in 293 cells. To further examine whether the noncanonical PPRE (–3698) motif binds PPAR-γ and whether the binding was affected by the PPAR-γ ligand, we performed EMSA (electrophoretic mobility shift assay) with the oligonucleotide probe. The results showed that the probe containing the region between -3698 and -3686 bp was shifted by the nuclear proteins. The DNA binding was increased by the troglitazone treatment and abolished by the unlabeled probes (Figure 3b). However, the oligonucleotide probe containing the PPRE-2 (–2760/-2746 bp) was not shifted by the nuclear extract (data no showed). In addition, the PPAR-γ antibody resulted in a super-shifted band (Figure 3b). Thus, these results indicated that PPAR-γ may directly bind to the noncanonical PPRE to activate the klotho gene. To further validate the transcriptional functionality of this noncanonical PPRE, we transfected the luciferase reporter-driven by 2 × KL-PPRE into human embryonic kidney (HEK) 293 (HEK293 cells; 293 cell line was used because it has a better transfection efficiency). As shown in Figure 3c, rosiglitazone increased the transcriptional activity of the reporter and the induced activity was abolished in the presence of GW9662. These results indicated that PPAR-γ activated klotho expression through a functional cis-element within the klotho gene. To determine whether klotho is a target gene of PPAR-γ in vivo, we have treated C57BL/6 mice with or without troglitazone (200 mg/kg/day) for 3 days and measured the klotho expression at both mRNA and protein levels. The qRT-PCR and northern blotting (Figure 4a) showed that the klotho mRNA level was significantly increased in the kidney of troglitazone-treated mice compared with the control group. Similarly, the protein level of klotho was also increased by troglitazone (Figure 4b). To examine whether different PPAR-γ agonist elicited the same effect in vivo, mice were also treated with rosiglitazone. The results showed that rosiglitazone at a lower dose (20 mg/kg/day) elicited a similar induction of klotho gene expression in the kidneys. Importantly, treatment with BADGE, a PPAR-γ antagonist, significantly reduced the TZD-induced expression, suggesting that PPAR-γ activation is required for the klotho induction by TZD in vivo (Figure 4c). To further investigate whether PPAR-γ activation is sufficient to induce the klotho expression in vivo, a constitutively active PPAR-γ was adenovirally infected into the mouse kidney. As shown by qRT-PCR and western blotting, Ad-PPAR-γ but not Ad-GFP infection achieved the overexpression of PPAR-γ in mouse kidneys, which subsequently resulted in a marked increase in klotho at mRNA and protein levels. The immunohistochemical studies also demonstrated that intra-renal infection of Ad-PPRA-γ induced overexpresion of PPAR-γ, which was stained positive in the nuclei for the HA antibody. Klotho expression was clearly induced and predominantly shown as membrane-associated staining (Figure 5c). The sections were stained negative when the primary antibody was omitted (figure not shown). Therefore, the results suggest that PPAR-γ activation is sufficient to induce the klotho expression in situ. In the present study, we have established a novel transcriptional mechanism that controls the expression of klotho, an important anti-aging hormone, by showing that klotho is a target gene of PPAR-γ in the cultured kidney cells as well as in mouse kidneys in vivo. This conclusion is supported by both the loss-of-function and the gain-of-function approaches: TZD induction of klotho was attenuated in the presence of specific antagonists GW9662 and BADGE both in vitro and in vivo or the knockdown of endogenous PPAR-γ by using the specific siRNA; conversely, the pharmacological TZD agonists and the ligand-independent constitutive activation resulted in the induction of klotho. Moreover, we have identified a functional noncanonical PPRE motif within the 5′-flanking region of the klotho gene that is recognized by PPAR-γ and may mediate the inductive effect of PPAR-γ. A previous study described that administration of troglitazone for 10 weeks significantly reduced systolic blood pressure, plasma glucose, and triglyceride levels, while augmenting endothelium-dependent aortic relaxation and renal klotho mRNA expression in OLETF rats.14.Yamagishi T. Saito Y. Nakamura T. et al.Troglitazone improves endothelial function and augments renal klotho mRNA expression in otsuka long-evans tokushima fatty (OLETF) rats with multiple atherogenic risk factors.Hyperten Res. 2001; 24: 705-709Crossref PubMed Scopus (0) Google Scholar However, it remained unclear whether the augmented expression of klotho mRNA was due to a direct effect of PPAR-γ activation or an indirect effect secondary to the improved metabolism. A recent report showed that in 3T3-L1 preadipocytes a TZD agonist, pioglitazone, temporarily increased the klotho mRNA expression and that recombinant klotho protein accelerated adipocyte differentiation, suggesting a link between klotho and adipogenesis.10.Chihara Y. Rakugi H. Ishikawa K. et al.Klotho protein promotes adipocyte differentiation.Endocrinology. 2006; 147: 3835-3842Crossref PubMed Scopus (68) Google Scholar Here, our study has provided the first evidence for a specific and direct role of PPAR-γ in the transcriptional control of klotho gene expression. PPAR-γ is a key transcription factor controlling adipogenesis and insulin sensitivity. PPAR-γ dimerizes with retinoid X receptor and activates the gene expression by binding to the cognate PPRE within the regulatory region of the target genes. Although there has been no apparent consensus PPRE found within the regulatory region of the klotho gene, noncanonical motifs for PPAR-γ have been identified in many PPAR-γ target genes.15.Lemay D.G. Hwang D.H. Genome-wide identification of peroxisome proliferator response elements using integrated computational genomics.J Lipid Res. 2006; 47: 1583-1587Crossref PubMed Scopus (110) Google Scholar,16.Hevener A.L. Olefsky J.M. Reichart D. et al.Macrophage PPARgamma is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones.J Clin Invest. 2007; 117: 1658-1669Crossref PubMed Scopus (383) Google Scholar They are often located at a distal region upstream of the transcription initiative site.17.Degenhardt T. Matilainen M. Herzig K.H. et al.The insulin-like growth factor-binding protein 1 gene is a primary target of peroxisome proliferator-activated receptors.J Biol Chem. 2006; 281: 39607-39619Crossref PubMed Scopus (47) Google Scholar By using ChIP, gel shift, and supershift assays, we have identified a novel PPRE motif that can be recognized by PPAR-γ. Moreover, the reporter assay showed that the noncanonical PPRE is functionally activated by rosiglitazone and the activation is abolished by the PPAR-γ antagonist GW0742. These results, taken together, support the concept that klotho is a target gene of PPAR-γ. However, the lack of larger segments of the 5′-flanking region upstream of the human and mouse klotho genes limits understanding of the interactive role of this motif with other regulatory elements. The klotho gene encodes a single-pass transmembrane protein that is detectable in a limited number of tissues including kidney and brain. In kidney, klotho is expressed at a high level in convoluted tubules as well as in other tubular epithelia such as inner medullary collecting duct cells.1.Kuro-o M. Matsumura Y. Aizawa H. et al.Mutation of the mouse klotho gene leads to a syndrome resembling ageing.Nature. 1997; 390: 45-51Crossref PubMed Scopus (2488) Google Scholar,18.Mitobe M. Yoshida T. Sugiura H. et al.Oxidative stress decreases klotho expression in a mouse kidney cell line.Nephron Exp Nephrol. 2005; 101: e67-e74Crossref PubMed Scopus (136) Google Scholar Besides using HEK293, we also examined the regulation of klotho by PPAR-γ in several other renal cell lines including MDCK, MCT, and IMCD. These cell lines are known to exhibit many features for distal tubules, proximal tubules, and inner medullary collecting ducts, respectively.10.Chihara Y. Rakugi H. Ishikawa K. et al.Klotho protein promotes adipocyte differentiation.Endocrinology. 2006; 147: 3835-3842Crossref PubMed Scopus (68) Google Scholar,11.Haverty T.P. Kelly C.J. Hines W.H. et al.Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis.J Cell Biol. 1988; 107: 1359-1368Crossref PubMed Scopus (261) Google Scholar,12.Ng K.H. Lim B.G. Wong K.P. Sulfate conjugating and transport functions of MDCK distal tubular cells.Kidney Int. 2003; 63: 976-986Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar,13.Gaush C.R. Hard W.L. Smith T.F. Characterization of an established line of canine kidney cells (MDCK).Proc Soc Exp Biol Med. 1966; 122: 931-935Crossref PubMed Scopus (245) Google Scholar In this study, we found that TZDs induce klotho expression both in HEK293 cells and, more importantly, several differentiated renal tissues across a number of species, implying that the PPAR-γ induction of renal klotho represents an evolutionarily conserved mechanism and may have an important physiological significance. It has been reported that gene expression of klotho was reduced in patients with chronic renal failure.2.Koh N. Fujimori T. Nishiguchi S. et al.Severely reduced production of Klotho in human chronic renal failure kidney.Biochem Biophys Res Com. 2001; 280: 1015-1020Crossref PubMed Scopus (361) Google Scholar The downregulation of the klotho gene in the kidney could exacerbate ischemic renal failure and of the renal damage induced by angiotensin II.19.Sugiura H. Yoshida T. Tsuchiya K. et al.Klotho reduces apoptosis in experimental ischaemic acute renal failure.Nephrol Dialysis Transplant. 2005; 20: 2636-2645Crossref PubMed Scopus (134) Google Scholar,20.Negri A.L. The klotho gene: a gene predominantly expressed in the kidney is a fundamental regulator of aging and calcium/phosphorus metabolism.J Nephrol. 2005; 18: 654-658PubMed Google Scholar Moreover, a recent study demonstrated that klotho may serve as a potential renoprotective humoral factor by reducing mitochondrial oxidative stress.21.Haruna Y. Kashihara N. Satoh M. et al.Amelioration of progressive renal injury by genetic manipulation of Klotho gene.Proc Natl Acad Sci USA. 2007; 104: 2331-2336Crossref PubMed Scopus (197) Google Scholar Up to one-third diabetic patients suffer end-stage renal failure due to diabetic nephropathies. Clinical studies have demonstrated that TZDs ameliorate diabetic nephropathy and many of the favorable effects of TZDs occur through both PPAR-γ-dependent and -independent mechanisms.22.Panchapakesan U. Chen X.M. Pollock C.A. Drug insight: thiazolidinediones and diabetic nephropathy—relevance to renoprotection.Nat Clin Pract Nephrol. 2005; 1: 33-43Crossref PubMed Scopus (44) Google Scholar,23.Zhang Y. Guan Y. PPAR-gamma agonists and diabetic nephropathy.Curr Diab Rep. 2005; 5: 470-475Crossref PubMed Scopus (17) Google Scholar,24.Guan Y. Peroxisome proliferator-activated receptor family and its relationship to renal complications of the metabolic syndrome.J Am Soc Nephrol. 2004; 15: 2801-2815Crossref PubMed Scopus (145) Google Scholar Given our results that klotho is a renal target of PPAR-γ, it is suggested that the upregulation of klotho expression by PPAR-γ may contribute to the protective effects of TZDs in renal complications of the type II diabetes. These findings may have important pathophysiological significance. Intriguingly, an interaction between klotho and insulin resistance has been recently uncovered. Koro-o and colleagues found that klotho functions as a humoral factor that signals suppression of intracellular insulin/IGF1 signaling, which may contribute to its anti-aging properties.25.Kurosu H. Yamamoto M. Clark J.D. et al.Suppression of aging in mice by the hormone Klotho.Science. 2005; 309: 1829-1833Crossref PubMed Scopus (1225) Google Scholar,26.Bartke A. Long-lived Klotho mice: new insights into the roles of IGF-1 and insulin-in aging.Trends Endocrinol Metab. 2006; 17: 33-35Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar Since PPAR-γ is an important therapeutic target for the treatment of insulin resistance and type II diabetes, the functional role of klotho regulation by PPAR-γ warrants further investigation. In conclusion, our study demonstrates a novel transcriptional mechanism by which the TZD insulin sensitizers regulate the klotho expression. These results suggest an important link between PPAR-γ and klotho, the renal-derived hormone. Human embryonic kidney 293, mouse proximal tubular cells (MCT), Madin–Darby canine renal distal tubular cells (MDCK), and inner medullary collecting ducts cells (IMCD) (ATCC, Rockville, MD, USA) were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS). Troglitazone (Parke-Davis Pharmaceuticals, Detroit, MI, USA), ciglitazone, rosiglitazone, BADGE, and GW9662 (Cayman Chemical, Ann Arbor, MI, USA) were dissolved in dimethyl sulfoxide (DMSO). Total RNA was isolated from HEK293 cells or mouse kidneys using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). For qRT-PCR, cDNA was generated from 2 μg RNA by using Superscript II reverse transcriptase and oligo-dT primer (Invitrogen). PCR reactions were performed with the use of specific primers (Table 1) on Opticon continuous fluorescence detection system (MJ Research Inc., Waltham, MA, USA) with SYBR green fluorescence (Molecular Probes, Eugene, OR, USA). Gene expression was quantified by using the comparative CT method, normalized to β-actin and expressed as fold induction of control. For northern blotting, 20 μg of RNA for each sample was separated on a 1.2% agarose-formaldehyde gel, transferred to nitrocellulose, and hybridized with α-32P-dCTP-labeled cDNA probes for klotho or β-actin.27.Wang N. Verna L. Chen N.G. et al.Constitutive activation of peroxisome proliferator-activated receptor-gamma suppresses pro-inflammatory adhesion molecules in human vascular endothelial cells.J Biol Chem. 2002; 277: 34176-34181Crossref PubMed Scopus (193) Google ScholarTable 1Sequences for primers and probes usedGeneAccession no.SequencesPrimers for PCR Human klothoNM_0047955′-gcccacatactggatggtatcaa-3′5′-actgcactcagtacacacggtga-3′ Mouse klothoNM_0138235′-acaaagaagtggccgagaga-3′5′-cctgactgggagagttgagc-3′ Human PPAR-γNM_1387125′-tcatggcaattgaatgtcgt-3′5′-ccaacagcttctccttctcg-3′ β-ActinEF6070935′-atctggcaccacaccttc-3′5′-agccaggtccagacgca-3′Primers for ChIP assay Klotho PPRE-15′-gccaccatgttggtgaattt-3′5′-tcacacctgtaatcccagca-3′ Klotho PPRE-25′-agtgcatctcctgctccatt-3′5′-cttgctgtgctttgaacagg-3′Probes for EMSAaComplementary sequences are omitted. Consensus PPRE (Aco oxidase)5′-ggggaccaggacaaaggtcacgttcgggagct-3′ Klotho PPRE-15′-ctcgaactcctgaccttag-3′ Klotho PPRE-25′-ggcactggatgaaggaatg-3′siRNAaComplementary sequences are omitted. GFP-siRNA5′-ggcuacguccaggagcgcacc-3′ PPAR-γ-siRNA5′-gagugggaguggucuuccauu-3′ChIP, chromatin immunoprecipitation, EMSA, electrophoretic mobility shift assay; GFP, green fluorescence protein; PPAR-γ, peroxisome proliferator-activated receptor-γ; PPRE, PPAR-responsive element; siRNA, small interfering RNA.a Complementary sequences are omitted. Open table in a new tab ChIP, chromatin immunoprecipitation, EMSA, electrophoretic mobility shift assay; GFP, green fluorescence protein; PPAR-γ, peroxisome proliferator-activated receptor-γ; PPRE, PPAR-responsive element; siRNA, small interfering RNA. Protein samples were extracted from mouse kidneys as previously described, with modifications.28.Li S.A. Watanabe M. Yamada H. et al.Immunohistochemical localization of Klotho protein in brain, kidney, and reproductive organs of mice.Cell Struct Funct. 2004; 29: 91-99Crossref PubMed Scopus (266) Google Scholar Briefly, mice were killed and the kidneys were homogenized in the homogenizing buffer (0.25 M sucrose, 2 mM EDTA (ethylenediaminetetraacetic acid), 10 mM EGTA (ethyleneglycol tetraacetate), 10 mM 2-mercaptoethanol, and 20 mM Tris-HCl, pH7.5). The homogenates were centrifuged at 12,000 r.p.m. for 10 min at 4°C, and the supernatant was centrifuged at 100,000 r.p.m. for 3 h at 4°C. The pellets were resuspended in the homogenizing buffer, supplemental with 1% Triton X-100 and 4% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate). Protein samples were separated on 10% SDS-PAGE (sodium dodecil sulfate-polyacrylamide gel electrophoresis), transferred to nitrocellulose and probed by using a rat monoclonal antibody against klotho (1:1000) or rabbit antibody against α-tubulin, followed by detection with horseradish peroxidase-labeled secondary antibodies and the ECL chemiluminescence kit (Amersham, Piscataway, NJ, USA).29.Wang N. Verna L. Hardy S. et al.c-Jun triggers apoptosis in human vascular endothelial cells.Circ Res. 1999; 85: 387-393Crossref PubMed Scopus (73) Google Scholar Short interfering RNA specific for GFP, human PPAR-γ, or human klotho30.de Oliveira R.M. Klotho RNAi induces premature senescence of human cells via a p53/p21 dependent pathway.FEBS Lett. 2006; 580: 5753-5758Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar were synthesized (see Table 1 for the sequences), annealed, and transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen) as previously described.31.Toyofuku T. Yoshida J. Sugimoto T. et al.FARP2 triggers signals for Sema3A-mediated axonal repulsion.Nat Neurosci. 2005; 8: 1712-1719Crossref PubMed Scopus (183) Google Scholar Cells were coinfected with Ad-PPAR-γ, which expresses a mouse PPAR-γ1 fused to a minimal VP16 transactivation domain and tagged with an HA-epitope, and Ad-tTA that encodes a tetracycline-responsive transactivator in the presence or absence of tetracycline (0.1 μg/ml) for 24 h, as previously described.27.Wang N. Verna L. Chen N.G. et al.Constitutive activation of peroxisome proliferator-activated receptor-gamma suppresses pro-inflammatory adhesion molecules in human vascular endothelial cells.J Biol Chem. 2002; 277: 34176-34181Crossref PubMed Scopus (193) Google Scholar For ChIP assays, cells were crosslinked with formaldehyde, and sonicated and immunoprecipitated with the antibody against PPAR-γ or control IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The eluted DNA and the input control were detected by PCR amplification (30 cycles) using the specific primer sets spanning the putative PPRE within the 5′-flanking region of the human klotho gene (0ENSG00000133116, see Table 1 for sequences).32.Zhang H. Sun L. Liang J. et al.The catalytic subunit of the proteasome is engaged in the entire process of estrogen receptor-regulated transcription.EMBO J. 2006; 25: 4223-4233Crossref PubMed Scopus (71) Google Scholar For EMSA, 5 μg of the nuclear extracts were incubated with the [γ-32P]ATP-labeled double-strand oligonucleotides and poly (dI-dC) for 30 min on ice, and then resolved on a 4% polyacrylamide gel. The unlabeled probe was included 100 M in excess in cold competition assays.33.Wang N. Verna L. Hardy S. et al.Adenovirus-mediated overexpression of c-Jun and c-Fos induces intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human endothelial cells.Arterioscler Thromb Vasc Biol. 1999; 19: 2078-2084Crossref PubMed Scopus (66) Google Scholar To construct the KL-PPRE-luciferase reporter plasmid, we synthesized complementary oligonucleotides containing two repeats of the putative klotho PPRE (the motifs are in italic) (5′-ATAGGTACCGTCTCGAACTCCTGACCTTAGGTGGTCTCGAGTCTCGAACTCTGACCTTAGGTGGTCTAAGCTTGG-3′) and flanking sites for KpnI and HindIII. The double strands were annealed and subcloned to pGL3-Enhancer (Promega, Madison, WI, USA). The KL-PPRE-Luc plasmid (1 μg/well) was transfected together with the β-galactosidase expression plasmid into subconfluent 293 cells with the use of Lipofectamine 2000 (Invitrogen). Twenty-four hours later, the cells were treated with DMSO, rosiglitazone, or GW9662. Cell lysates were harvested 24 h later to measure luciferase and β-galactosidase activities. Animal protocols were approved by the Peking University Health Science Center, complying with the Guideline for Care and Use of Laboratory Animals. C57BL/6 mice weighting 25–30 g were given troglitazone (200 mg/kg/day), rosiglitazone (20 mg/kg/day), or BADGE (10 mg/kg/day) by oral gavage. Alternatively, intra-renal adenoviral infection was performed under pentobarbital anesthetization. Ad-PPAR-γ or Ad-GFP, together with Ad-tTA was injected into kidney at 1 × 109 PFU. Three days after the troglitazone treatment or the adenoviral infection, the mice were killed and the kidneys were immediately excised to extract the RNA and protein, or frozen-embedded in optimum cutting temperature compound for immunohistochemical study. Cryostat sections (6 μm) of frozen kidneys were fixed in 4% paraformaldehyde. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide. Sections were reacted with the primary antibodies against klotho (rat anti-mouse klotho monoclonal antibody; R&D, Minneapolis, MN, USA) or HA (mouse anti-HA monoclonal antibody; Sigma-Aldrich, St Louis, MO, USA). After washing, the sections were incubated with secondary antibodies (biotinylated rabbit anti-rat or anti-mouse IgG), and then with avidin–biotin–peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Sections were visualized with diaminobenzidine substrate and counterstained with hematoxylin.34.Qin X. Tian J. Zhang P. et al.Laminar shear stress up-regulates the expression of stearoyl-CoA desaturase-1 in vascular endothelial cells.Cardiovasc Res. 2007; 74: 506-514Crossref PubMed Scopus (38) Google Scholar Statistical analyses were performed by one-way ANOVA (analysis of variance) or Student t-test. Data are expressed as mean±s.e.m. The P-values less than 0.05 were considered statistically significant. All authors of this paper have nothing to disclose. This study was supported, in part, by the Ministry of Education (PCSIRT) and grants from National Science Foundation of China no. 30470810, 30670848, and 30600231), the Ministry of Science and Technology (2006CB503900 and 2006CB943503).

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