Role of Krüppel-like Factor 15 (KLF15) in Transcriptional Regulation of Adipogenesis
2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês
10.1074/jbc.m410515200
ISSN1083-351X
AutoresToshiyuki Mori, Hiroshi Sakaue, Haruhisa Iguchi, Hideyuki Gomi, Yuko Okada, Yasuhiro Takashima, Kyoko Nakamura, Takehiro Nakamura, Toshimasa Yamauchi, Naoto Kubota, Takashi Kadowaki, Yasushi Matsuki, Wataru Ogawa, Ryuji Hiramatsu, Masato Kasuga,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoKrüppel-like zinc finger transcription factors (KLFs) play diverse roles during cell differentiation and development in mammals. We have now shown by microarray analysis that expression of the KLF15 gene is markedly up-regulated during the differentiation of 3T3-L1 preadipocytes into adipocytes. Inhibition of the function of KLF15, either by expression of a dominant negative mutant or by RNA interference, both reduced the expression of peroxisome proliferator-activated receptor γ (PPARγ) and blocked adipogenesis in 3T3-L1 preadipocytes exposed to inducers of adipocyte differentiation. However, the dominant negative mutant of KLF15 did not affect the expression of CCAAT/enhancer-binding protein β (C/EBPβ) elicited by inducers of differentiation in 3T3-L1 preadipocytes. In addition, ectopic expression of KLF15 in NIH 3T3 or C2C12 cells triggered both lipid accumulation and the expression of PPARγ in the presence of inducers of adipocyte differentiation. Ectopic expression of C/EBPβ, C/EBPδ, or C/EBPα in NIH 3T3 cells also elicited the expression of KLF15 in the presence of inducers of adipocyte differentiation. Moreover, KLF15 and C/EBPα acted synergistically to increase the activity of the PPARγ2 gene promoter in 3T3-L1 adipocytes. Our observations thus demonstrate that KLF15 plays an essential role in adipogenesis in 3T3-L1 cells through its regulation of PPAR γ expression. Krüppel-like zinc finger transcription factors (KLFs) play diverse roles during cell differentiation and development in mammals. We have now shown by microarray analysis that expression of the KLF15 gene is markedly up-regulated during the differentiation of 3T3-L1 preadipocytes into adipocytes. Inhibition of the function of KLF15, either by expression of a dominant negative mutant or by RNA interference, both reduced the expression of peroxisome proliferator-activated receptor γ (PPARγ) and blocked adipogenesis in 3T3-L1 preadipocytes exposed to inducers of adipocyte differentiation. However, the dominant negative mutant of KLF15 did not affect the expression of CCAAT/enhancer-binding protein β (C/EBPβ) elicited by inducers of differentiation in 3T3-L1 preadipocytes. In addition, ectopic expression of KLF15 in NIH 3T3 or C2C12 cells triggered both lipid accumulation and the expression of PPARγ in the presence of inducers of adipocyte differentiation. Ectopic expression of C/EBPβ, C/EBPδ, or C/EBPα in NIH 3T3 cells also elicited the expression of KLF15 in the presence of inducers of adipocyte differentiation. Moreover, KLF15 and C/EBPα acted synergistically to increase the activity of the PPARγ2 gene promoter in 3T3-L1 adipocytes. Our observations thus demonstrate that KLF15 plays an essential role in adipogenesis in 3T3-L1 cells through its regulation of PPAR γ expression. The amount of adipose tissue in the body is an important determinant of energy homeostasis in animals and is altered in various physiological or pathological conditions (1.Evans R.M. Barish G.D. Wang Y.-X. Nat. Med. 2004; 10: 355-361Crossref PubMed Scopus (1257) Google Scholar). An increase in adipose tissue mass can arise through increases in cell size, cell number, or both (2.Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1037) Google Scholar). The size of adipocytes varies markedly and reflects largely the amount of stored triglyceride, whereas the number of adipocytes is thought to increase as a result of the proliferation of preadipocytes and their subsequent differentiation into mature adipocytes.Murine preadipose cell lines, such as 3T3-L1 and 3T3-F442A, have been studied extensively to elucidate the mechanisms of growth and differentiation of preadipocytes (2.Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1037) Google Scholar, 3.Hwang C.S. Loftus T.M. Mandrup S. Lane M.D. Annu. Rev. Cell Dev. Biol. 1997; 13: 231-259Crossref PubMed Scopus (207) Google Scholar). In response to exposure to appropriate hormonal inducers (such as agents that increase the intracellular concentration of cyclic AMP, agonists of the insulin-like growth factor-1 receptor, glucocorticoids, and fetal bovine serum), these cells first undergo several rounds of mitosis, known as clonal expansion, and then become quiescent again, express adipocyte-specific proteins, and acquire biochemical and morphological characteristics of mature adipocytes (2.Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1037) Google Scholar, 3.Hwang C.S. Loftus T.M. Mandrup S. Lane M.D. Annu. Rev. Cell Dev. Biol. 1997; 13: 231-259Crossref PubMed Scopus (207) Google Scholar).Both the proliferation and differentiation of preadipocytes are characterized by marked changes in the pattern of gene expression that are achieved by the sequential induction of transcription factors. Preadipocytes exposed to inducers of differentiation thus manifest an early and transient increase in the expression of the transcription factors CCAAT/enhancer-binding protein β (C/EBPβ) 1The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; PPARγ, peroxisome proliferator-activated receptor γ; KLF, Krüppel-like zinc finger transcription factor; CREB, cyclic AMP response element-binding protein; IBMX, isobutylmethylxanthine; MEF, mouse embryonic fibroblast; RT, reverse transcription; GFP, green fluorescent protein; RNAi, RNA interference; siRNA, small interfering RNA.1The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; PPARγ, peroxisome proliferator-activated receptor γ; KLF, Krüppel-like zinc finger transcription factor; CREB, cyclic AMP response element-binding protein; IBMX, isobutylmethylxanthine; MEF, mouse embryonic fibroblast; RT, reverse transcription; GFP, green fluorescent protein; RNAi, RNA interference; siRNA, small interfering RNA. and C/EBPδ, which in turn appear to contribute to cell proliferation as well as to the subsequent increase in the expression of C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ) (4.Lane M.D. Tang Q.Q. Jiang M.S. Biochem. Biophys. Res. Commun. 1999; 266: 677-683Crossref PubMed Scopus (238) Google Scholar, 5.Zhang J.W. Tang Q.Q. Vinson C. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 43-47Crossref PubMed Scopus (151) Google Scholar). The latter two proteins are thought to act synergistically in the transcriptional activation of a variety of adipocyte-specific genes, with each also reciprocally activating the expression of the other (2.Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1037) Google Scholar, 3.Hwang C.S. Loftus T.M. Mandrup S. Lane M.D. Annu. Rev. Cell Dev. Biol. 1997; 13: 231-259Crossref PubMed Scopus (207) Google Scholar, 4.Lane M.D. Tang Q.Q. Jiang M.S. Biochem. Biophys. Res. Commun. 1999; 266: 677-683Crossref PubMed Scopus (238) Google Scholar).Krüppel-like zinc finger transcription factors (KLFs) are DNA-binding transcriptional regulators that contain the C2H2 zinc finger motif and play diverse roles in the regulation of cell proliferation, cell differentiation, and development (6.Dang D.T. Pevsner J. Yang V.W. Int. J. Biochem. Cell Biol. 2000; 32: 1103-1121Crossref PubMed Scopus (361) Google Scholar, 7.Black A.R. Black J.D. Azizkhan-Clifford J. J. Cell. Physiol. 2001; 188: 143-160Crossref PubMed Scopus (884) Google Scholar). All 16 members of the KLF family identified to date bind to GC-rich sequences including GC boxes and GT boxes (also known as CACCC boxes) (7.Black A.R. Black J.D. Azizkhan-Clifford J. J. Cell. Physiol. 2001; 188: 143-160Crossref PubMed Scopus (884) Google Scholar, 8.Bieker J.J. J. Biol. Chem. 2001; 276: 34355-34358Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). Certain KLF proteins have been implicated in adipogenesis. KLF2 has thus been shown to negatively regulate adipogenesis through inhibition of PPARγ gene expression (9.Banerjee S.S. Feinberg M.W. Watanabe M. Gray S. Haspel R.L. Denkinger D.J. Kawahara R. Hauner H. Jain M.K. J. Biol. Chem. 2003; 278: 2581-2584Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). In addition, expression of KLF6 is transiently induced during adipogenesis in 3T3-L1 preadipocytes (10.Inuzuka H. Wakao H. Masuho Y. Muramatsu M.A. Tojo H. Nanbu-Wakao R. Biochim. Biophys. Acta. 1999; 1447: 199-207Crossref PubMed Scopus (19) Google Scholar). Furthermore, overexpression of KLF15 induces adipocyte maturation and GLUT4 expression (11.Gray S. Feinberg M.W. Hull S. Kuo C.T. Watanabe M. Sen-Banerjee S. DePina A. Haspel R. Jain M.K. J. Biol. Chem. 2002; 277: 34322-34328Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), although the physiological significance of KLF15 in the induction or maintenance of terminal differentiation has remained unclear. We now provide evidence that KLF15 promotes maintenance of the biochemical and morphological characteristics of mature adipocytes through direct induction of PPARγ expression in cooperation with C/EBPα.EXPERIMENTAL PROCEDURESAntibodies, Reagents, and Cells—Antibodies to C/EBPα, C/EBPβ, and C/EBPδ were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to cyclic AMP response element-binding protein (CREB) were from Cell Signaling Technologies (Beverly, MA). Antibodies to PPARγ (12.Hu E. Kin J.B. Sarraf P. Spiegelman B.M. Science. 1996; 274: 2100-2103Crossref PubMed Scopus (928) Google Scholar) and aP2 (13.Bernlohr D.A. Doering T.L. Kelly Jr., T.J. Lane M.D. Biochem. Biophys. Res. Commun. 1985; 132: 850-855Crossref PubMed Scopus (75) Google Scholar) were kindly provided by B. M. Spiegelman (Harvard Medical School, Boston, MA) and D. Bernlohr (University of Minnesota, Minneapolis, MN), respectively. Troglitazone was kindly provided by Sankyo Pharmaceutical Co. (Tokyo, Japan). Blasticidine S hydrochloride, hygromycin B, and puromycin dihydrochloride were obtained from Invitrogen (Carlsbad, CA), Wako (Osaka, Japan), and Sigma, respectively; hexadimethrine bromide was also from Sigma. 3T3-L1, NIH 3T3, and C2C12 cells were obtained from American Type Culture Collection (Manassas, VA), and Plat-E retroviral packaging cells (14.Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1338) Google Scholar) were kindly provided by T. Kitamura (University of Tokyo, Tokyo, Japan).Cell Culture and Staining—3T3-L1 preadipocytes were maintained as described previously (15.Sakaue H. Ogawa W. Matsumoto M. Kuroda S. Takata M. Sugimoto T. Spiegelman B.M. Kasuga M. J. Biol. Chem. 1998; 273: 28945-28952Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Their differentiation into adipocytes was induced by treatment of confluent cells first for 2 days with insulin (5 μg/ml), 0.25 μm dexamethasone, and 0.5 mm isobutylmethylxanthine (IBMX) in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and then for 2 days with insulin (5 μg/ml) alone in the same medium. The cells were then returned to the basal medium, which was replenished every other day.NIH 3T3 or C2C12 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and cultured to confluence. Adipogenesis in transfected cells was induced by treatment for 6 days with insulin (5 μg/ml), 0.25 μm dexamethasone, 0.5 mm IBMX, and 10 μm troglitazone in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells were then returned to the basal medium, which was replenished every other day.Mouse embryonic fibroblasts (MEFs) obtained from PPARγ knock-out mice or their wild-type littermates were immortalized by the 3T3 protocol (16.Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (1993) Google Scholar) and exposed to inducers of adipocyte differentiation as described above for NIH 3T3 cells. Cells were stained with oil red O as described (17.Sakaue H. Konishi M. Ogawa W. Asaki T. Mori T. Yamasaki M. Takata M. Ueno H. Kato S. Kasuga M. Itoh N. Genes Dev. 2002; 16: 908-912Crossref PubMed Scopus (112) Google Scholar).Oligonucleotide Microarray Analysis—Total RNA was isolated from 3T3-L1 preadipocytes or fully differentiated 3T3-L1 adipocytes (8 days after the induction of differentiation) and was processed with RNeasy columns (Qiagen, Hilden, Germany). Portions (10 μg) of the RNA were then used for synthesis of biotin-labeled cRNA, which in turn was used to probe Genechip Mouse MU74 microarrays (Affymetrix, Santa Clara, CA). After washing and staining, the arrays were scanned with a Hewlett Packard confocal laser scanner and visualized with Affymetrix Genechip 3.1 software. Finally, the results were analyzed with Genechip Analysis Suite software version 4.0 (Affymetrix), and the -fold differences in hybridization intensity between samples from undifferentiated 3T3-L1 preadipocytes and fully differentiated 3T3-L1 adipocytes were determined.Expression Plasmids—A mammalian expression vector for KLF15 (pcDNA3.1/KLF15) was constructed by inserting the products of reverse transcription (RT) and the PCR obtained from rat adipose tissue RNA into pcDNA3.1 (Invitrogen). To construct an expression vector for a deletion mutant of KLF15 that lacks the NH2-terminal 318 amino acids (Δ318), we amplified a DNA fragment encoding Δ318 by PCR with the sense primer 5′-CTGCCATGCACAAATGCACTTTC-3′ and the antisense primer 5′-CTTCAGTTGATGGCGCGTAC-3′ and then inserted the PCR product into the PT7-blue T vector (Takara, Tokyo, Japan). The sequence of the inserted fragment was verified and then subcloned into pcDNA3.1. Mammalian expression vectors for C/EBPα or C/EBPβ were constructed by subcloning the corresponding mouse cDNAs (kindly provided by S. Akira, Osaka University, Osaka, Japan) into pcDNA3.1.Retroviral Vectors and Infection—To produce retroviral vectors for green fluorescent protein (GFP), C/EBPα, C/EBPβ, C/EBPδ, KLF6, KLF9, KLF15, or Δ318, we subcloned cDNAs for GFP, mouse C/ebpα, mouse C/ebpβ, mouse C/ebpδ (kindly provided by S. Akira), mouse KLF6 (RT-PCR product of mouse adipose tissue RNA), mouse KLF9 (RT-PCR product of mouse adipose tissue RNA), rat KLF15, or Δ318 into pWZL containing blasticidine or hygromycin resistance genes (kindly provided by G. P. Nolan, Stanford University, Stanford, CA). To construct a retroviral vector for PPARγ2, we inserted a cDNA for mouse PPARγ2 (RT-PCR product of mouse adipose tissue RNA) into pMXs containing a puromycin resistance gene (kindly provided by T. Kitamura).NIH 3T3 cells stably expressing KLF15 (NIH/KLF15 cells) were generated with the retroviral vector pWZL/KLF15 as described (18.Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2283) Google Scholar), with the following modifications: Plat-E packaging cells were transfected with the retroviral vector with the use of FuGENE 6 (Roche Applied Science, Indianapolis, IN). After 24 h, the cells were incubated for an additional 24 h in fresh medium to obtain retrovirus-containing supernatants. NIH 3T3 cells (50 to 60% confluence) were then incubated with these supernatants in the presence of hexadimethrine bromide (4 μg/ml) for 12 h. Cells expressing KLF15 were selected on the basis of their resistance to blasticidine S (10 μg/ml) or hygromycin B (500 μg/ml).NIH 3T3 cells stably expressing other ectopic proteins with the exception of PPARγ2; 3T3-L1 cells stably expressing GFP (3T3-L1/GFP cells), KLF15, or Δ318; and C2C12 cells stably expressing GFP (C2C12/ GFP cells) or KLF15 were generated as described above for NIH/KLF15 cells. NIH 3T3 cells stably expressing PPARγ2 (NIH/PPARγ2 cells) were similarly generated with pMXs/PPARγ2 and were selected on the basis of their resistance to puromycin (2 μg/ml).Adenovirus Vectors and Infection—An adenoviral vector encoding rat KLF15 (AxCA-Klf15) was generated with the use of an Adenovirus Expression Kit (Takara, Tokyo, Japan) as described previously (15.Sakaue H. Ogawa W. Matsumoto M. Kuroda S. Takata M. Sugimoto T. Spiegelman B.M. Kasuga M. J. Biol. Chem. 1998; 273: 28945-28952Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). An adenoviral vector encoding β-galactosidase (AxCA-lacZ) was kindly provided by I. Saito (University of Tokyo, Tokyo, Japan). 3T3-L1 adipocytes were infected with adenovirus vectors as essentially described previously (15.Sakaue H. Ogawa W. Matsumoto M. Kuroda S. Takata M. Sugimoto T. Spiegelman B.M. Kasuga M. J. Biol. Chem. 1998; 273: 28945-28952Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar).Northern and Immunoblot Analyses—Total RNA (15 μg) extracted from cells was subjected to Northern blot analysis with a full-length mouse aP2 cDNA, a fragment of mouse PPARγ cDNA, full-length mouse C/EBPα cDNA, full-length mouse C/EBPβ cDNA, full-length mouse C/EBPδ cDNA, full-length mouse KLF6 cDNA, full-length mouse KLF9 cDNA, full-length mouse KLF15 cDNA (RT-PCR product of mouse adipose tissue RNA), or a fragment of human GLUT4 cDNA as probes. Cell lysates (∼100 μg of protein) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis as described (15.Sakaue H. Ogawa W. Matsumoto M. Kuroda S. Takata M. Sugimoto T. Spiegelman B.M. Kasuga M. J. Biol. Chem. 1998; 273: 28945-28952Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar).Electroporation—3T3-L1 preadipocytes were transfected with plasmids by electroporation as described previously (19.Sakaue H. Ogawa W. Nakamura T. Mori T. Nakamura K. Kasuga M. J. Biol. Chem. 2004; 279: 39951-39957Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). For transfection of 3T3-L1 adipocytes, the cells were detached from culture dishes 5 days after exposure to inducers of differentiation by treatment with 2.5% trypsin and collagenase (0.5 mg/ml); they were then washed with phosphate-buffered saline before resuspension with plasmid DNA in the solution provided with Nucleofector Kit V (Amaxa, Cologne, Germany). The plasmid DNA was then introduced into the cells by electroporation with the use of a Nucleofector instrument (program U-28). After electroporation, the cells were resuspended in culture medium and replated on culture dishes.Luciferase Reporter Assays—For GLUT4 gene promoter assays, NIH 3T3 cells plated in 24-well dishes (50 to 60% confluence) were transiently transfected with 500 ng of the reporter plasmid pGL3-basic (Promega, Madison, WI) containing the human GLUT4 gene promoter (nucleotides -894 to +84 relative to the transcriptional start site), 500 ng of the indicated expression plasmids, and 100 ng of pCMV encoding β-galactosidase (pCMV/β-gal) with the use of the Lipofectamine reagent (Invitrogen). After incubation for 48 to 72 h, the cells were lysed in 100 μl of 1 times passive lysis buffer (Promega), and portions of the lysate were subjected to assays for firefly luciferase (Promega) and β-galactosidase (Clontech, Palo Alto, CA) activities. Promoter activity was determined as the ratio of luciferase to β-galactosidase activities. For PPARγ2 gene promoter assays, 3T3-L1 adipocytes (∼3 × 106) were transfected with 500 ng of pGL3-basic containing the mouse PPARγ2 gene promoter (nucleotides -626 to +60), 500 ng of the indicated expression plasmids, and 100 ng of pCMV/β-gal by electroporation and were subsequently assayed for firefly luciferase and β-galactosidase activities as described for the GLUT4 gene promoter assay.RNA Interference—The target sequences of the KLF15 gene selected for RNA interference (RNAi) comprised nucleotides 300 to 319 and nucleotides 1112 to 1131 relative to the transcriptional start site. To construct mammalian expression vectors for each of the corresponding small interfering RNAs (siRNAs), designated si300 and si1112, respectively, we amplified by PCR a DNA fragment including the mouse U6 (mU6) promoter and encoding the sense sequence of the siRNA, a short hairpin sequence, the antisense sequence of the siRNA, and a poly(A) terminator. PCR was performed for 35 cycles of 95 °C for 30 s, 45 °C for 30 s, and 72 °C for 30 s with specific primers, the pMXs/mU6 plasmid (kindly provided by K. I. Nakayama, Kyushu University, Fukuoka, Japan) as the template, and Ex-taq polymerase (Takara). The PCR primers were 5′-AGCGCCCGACCGAAAGGAG-3′ (sense) and 5′-ACTAAGGTCGACAAAAAGCCTTCTGTTCCTGATACATCTCTTGAATGTAGCAGGAACAGAAGGCAAACAAGGCTTTTCTCCAAG-3′ (antisense) for si300 and 5′-AGCGCCCGACCGAAAGGAG-3′ (sense) and 5′-ACTAAGGTCGACAAAAAGATGTACACCAAGAGCAGCTCTCTTGAAGCTGCTCTTGGTGTACATCAAACAAGGCTTTTCTCCAAG-3′ (antisense) for si1112. The PCR products were subcloned into PT7-blue T and their sequences were verified with the CUGA sequencing system (Nippongene, Tokyo, Japan). They were then subcloned into the NotI and SalI sites of pcDNA3.1, from which the cytomegalovirus promoter was subsequently deleted.RESULTSUp-regulation of KLF15 Gene Expression during Adipocyte Differentiation in 3T3-L1 Cells—We first profiled genes whose level of expression changed in association with the differentiation of 3T3-L1 preadipocytes induced by IBMX, dexamethasone, and insulin. Total RNA isolated from 3T3-L1 preadipocytes and adipocytes was analyzed with mouse oligonucleotide microarrays, revealing that the differentiation of these cells was accompanied by an ∼80-fold increase in the abundance of KLF15 mRNA (Fig. 1A). The amounts of KLF9, KLF10, and KLF13 mRNAs were increased to much smaller extents, whereas those of KLF6 mRNA and of several other KLF gene transcripts were decreased, in association with 3T3-L1 cell differentiation. Consistent with these results, Northern blot analysis showed that the abundance of KLF15 and KLF9 mRNAs increased during the differentiation of 3T3-L1 cells, whereas that of KLF6 mRNA decreased (Fig. 1B). KLF15 mRNA was first detected 4 days after the induction of differentiation, 2 days later than the initial increase in the amounts of PPARγ and aP2 mRNAs, and its abundance was increased maximally at 6 and 8 days. The time course of the up-regulation of KLF15 mRNA during adipocyte differentiation was similar to that apparent for C/EBPα and GLUT4 mRNAs. The amounts of C/EBPβ and C/EBPδ mRNAs were already maximal at 2 days and had returned to basal levels by 4 to 6 days after the induction of differentiation.Inhibition of KLF15 Function by a Dominant Negative Mutant in 3T3-L1 Cells—To determine whether KLF15 is required for adipocyte differentiation in 3T3-L1 cells, we constructed an expression vector for a putative dominant negative mutant (Δ318) that lacks the NH2-terminal 318 amino acids of KLF15, which contain the transactivation domain, leaving the DNA binding domain intact. To verify that the Δ318 mutant was able to inhibit KLF15 function, we examined the effect of its expression on the activity of the GLUT4 gene promoter, a target of KLF15 action (11.Gray S. Feinberg M.W. Hull S. Kuo C.T. Watanabe M. Sen-Banerjee S. DePina A. Haspel R. Jain M.K. J. Biol. Chem. 2002; 277: 34322-34328Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). We transiently cotransfected NIH 3T3 cells with a GLUT4 promoter-luciferase gene reporter construct together with an expression plasmid either for full-length KLF15 or for the Δ318 mutant. The activity of the GLUT4 gene promoter was increased by ∼50% by expression of wild-type KLF15 and was reduced by ∼50% by expression of Δ318 (Fig. 2A). In addition, Δ318 prevented the increase in GLUT4 gene promoter activity induced by the full-length protein, confirming that Δ318 acts in a dominant negative manner.Fig. 2Effect of a dominant negative mutant of KLF15 on adipocytic differentiation of 3T3-L1 preadipocytes.A, NIH 3T3 cells were transfected with a GLUT4 promoter-luciferase gene reporter plasmid (or pGL3-basic as a control), a β-galactosidase expression plasmid, and expression vectors for KLF15, the Δ318 mutant, or both KLF15 and Δ318 (or the pcDNA3.1 empty vector). The cells were subsequently assayed for luciferase and β-galactosidase activities, and the former was normalized by the latter. Data represent normalized luciferase activity expressed relative to the value for cells transfected with pGL3-basic and pcDNA3.1; data are mean ± S.D. of triplicates from a representative experiment. B, 3T3-L1 preadipocytes stably expressing GFP, KLF15, or Δ318, as indicated, were subjected to Northern blot analysis of KLF15 mRNA. Control 3T3-L1 cells before or 8 days after exposure to inducers of differentiation were similarly analyzed. C, 3T3-L1 preadipocytes stably expressing GFP, KLF15, Δ318, or both Δ318 and PPARγ2 were stained with oil red O 8 days after exposure to inducers of differentiation. Macroscopic (upper panel) and microscopic (×100, lower panel) views are shown. D, 3T3-L1 preadipocytes stably expressing GFP, KLF15, Δ318, or both Δ318 and PPARγ2 were subjected to immunoblot analysis with antibodies to PPARγ, C/EBPα, aP2, or CREB (control) 8 days after exposure to inducers of differentiation. E and F, 3T3-L1 adipocytes 8 days after exposure to inducers of differentiation were infected with adenovirus vector AxCA-lacZ or AxCA-Klf15 at the indicted multiplicity of infection. 48 h after adenovirus infection (MOI), the cells were subjected to Northern blot analysis of KLF15 mRNA (E) or subjected to immunoblot analysis with antibodies to PPARγ, C/EBPα, or CREB (control) (F). Data in B–F are representative of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next constructed retroviral vectors encoding Δ318, wild-type KLF15, and GFP for expression of these proteins in 3T3-L1 preadipocytes (Fig. 2B). Expression of Δ318 in these cells greatly inhibited both the lipid accumulation (Fig. 2C) and the induction of PPARγ, C/EBPα, and aP2 (Fig. 2D) normally apparent 8 days after exposure of the cells to inducers of differentiation. Expression of GFP or of wild-type KLF15 did not affect these aspects of adipocyte differentiation. The amount of exogenous KLF15 mRNA by a retrovirus vector was one-half of that of endogenous KLF15 in 3T3-L1 adipocytes (Fig. 2B). We have therefore investigated the effect of overexpression of KLF15 by an adenovirus vector on the induction of PPARγ and C/EBPα. Although the abundance of KLF15 mRNA in 3T3-L1 adipocytes infected with an adenovirus vector for this protein at an multiplicity of infection of 30 plaque-forming units (pfu)/cell was eight times to that of endogenous KLF15 mRNA in 3T3-L1 adipocytes (Fig. 2E), this overexpression of KLF15 did not affect the amount of PPARγ and C/EBPα protein (Fig. 2F) in 3T3-L1 adipocytes. To examine whether this inhibitory effect of Δ318 on adipogenesis was prevented by coexpression of PPARγ2, we infected 3T3-L1 cells both with a Δ318-encoding retroviral vector containing a blasticidine resistance gene and with a PPARγ2-encoding retroviral vector containing a puromycin resistance gene. The infected cells were then subjected to selection in the presence of both blasticidine S and puromycin. The inhibitory effect of Δ318 on adipogenesis, as monitored by both lipid accumulation (Fig. 2C) and the expression of C/EBPα and aP2 (Fig. 2D), was indeed prevented by coexpression of PPARγ2, indicating that it was attributable to the prevention of PPARγ induction.Lack of Effect of Δ318 on the Expression of C/EBPβ in 3T3-L1 Cells—The increase in the amount of KLF15 mRNA during adipocytic differentiation of 3T3-L1 cells occurred slightly later than did that in the amount of PPARγ mRNA (Fig. 1B). PPARγ mRNA was thus detected 2 days after exposure of cells to inducers of differentiation, whereas KLF15 mRNA was not apparent at this time. The transient increase in the abundance of the mRNAs for C/ebpβ and C/ebpδ, both of which directly induce the expression of PPARγ (20.Yeh W.C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (806) Google Scholar, 21.Wu Z. Bucher N.L. Farmer S.R. Mol. Cell. Biol. 1996; 16: 4128-4136Crossref PubMed Google Scholar), was maximal 2 days after induction of differentiation. Furthermore, MEFs that lack C/EBPβ and C/EBPδ are unable to differentiate into adipocytes (22.Tanaka T. Yoshida N. Kishimoto T. Akira S. EMBO J. 1997; 16: 7432-7443Crossref PubMed Scopus (634) Google Scholar). These two proteins thus likely induce the expression of PPARγ before the induction of C/EBPα or KLF15 during adipogenesis in 3T3-L1 cells. We next investigated whether the expression of the dominant negative mutant of KLF15 (Δ318) affected the induction of C/EBPβ or PPARγ during the 4 days of exposure of 3T3-L1 cells to inducers of differentiation, a period when the levels of C/EBPβ and C/EBPδ are increased. Immunoblot analysis revealed that expression of Δ318 in 3T3-L1 cells inhibited the expression of PPARγ on day 4 (Fig. 3) as it did on day 8 (Fig. 2D); it did not affect PPARγ abundance on day 2 (Fig. 3), however, a time before induction of KLF15 gene expression is apparent. The up-regulation of C/EBPβ expression, which was maximal 1 and 2 days after exposure to the inducers of differentiation, was not inhibited by Δ318 (Fig. 3). Moreover, overexpression of wild-type KLF15 slightly increased the abundance of PPARγ at 1 or 2 days after the induction of differentiation (Fig. 3), consistent with the previous observation that KLF15 promoted adipocyte differentiation (9.Banerjee S.S. Feinberg M.W. Watanabe M. Gray S. Haspel R.L. Denkinger D.J. Kawahara R. Hauner H. Jain M.K. J. Biol. Chem. 2003; 278: 2581-2584Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar).Fig. 3Effect of a dominant negative mutant of KLF15 on the expression of C/EBPβ or PPARγ during adipocyte differentiation in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes stably e
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