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• W2000392034 abstract "Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNFα impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/enhancer-binding protein α, in contrast to peroxisome proliferator-activated receptor γ, was markedly reduced. TNFα also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNFα maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and β-catenin levels. TNFα, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNFα prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation. Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNFα impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/enhancer-binding protein α, in contrast to peroxisome proliferator-activated receptor γ, was markedly reduced. TNFα also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNFα maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and β-catenin levels. TNFα, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNFα prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation. Adipose tissue is the major organ for storing and releasing surplus energy. Inability of the adipose cells to take up and store lipids, as seen in lipoatrophic and lipodystrophic conditions, is associated with the accumulation of ectopic triglycerides in the liver and skeletal muscles, insulin resistance, and diabetes (1Perseghin G. Petersen K. Shulman G.I. Int. J. Obes. Relat. Metab. Disord. 2003; 27 (Suppl. 3): S6-S11Crossref PubMed Scopus (190) Google Scholar). Transplantation of fat to animal models of lipoatrophy reverses these conditions (2Kim J.K. Gavrilova O. Chen Y. Reitman M.L. Shulman G.I. J. Biol. Chem. 2000; 275: 8456-8460Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). Interestingly, obesity and insulin resistance are also associated with ectopic lipid accumulation suggesting an insufficient uptake and storage of lipids in the adipose cells in these conditions, as well. The adipose tissue also plays an important role as an endocrine organ secreting different hormones and cytokines that can augment or impair whole-body insulin sensitivity (3Fukuhara A. Matsuda M. Nishizawa M. Segawa K. Tanaka M. Kishimoto K. Matsuki Y. Murakami M. Ichisaka T. Murakami H. Watanabe E. Takagi T. Akiyoshi M. Ohtsubo T. Kihara S. Yamashita S. Makishima M. Funahashi T. Yamanaka S. Hiramatsu R. Matsuzawa Y. Shimomura I. Science. 2005; 307: 426-430Crossref PubMed Scopus (1620) Google Scholar). Accumulation of body fat in adults is initially characterized by an increase in fat cell size followed by an increased cell number and, thus, recruitment of preadipocytes (4Kissebah A.H. Vydelingum N. Murray R. Evans D.J. Hartz A.J. Kalkhoff R.K. Adams P.W. J. Clin. Endocrinol. Metab. 1982; 54: 254-260Crossref PubMed Scopus (1556) Google Scholar, 5Krotkiewski M. Bjorntorp P. Sjostrom L. Smith U. J. Clin. Invest. 1983; 72: 1150-1162Crossref PubMed Scopus (1164) Google Scholar, 6Weyer C. Foley J.E. Bogardus C. Tataranni P.A. Pratley R.E. Diabetologia. 2000; 43: 1498-1506Crossref PubMed Scopus (558) Google Scholar). Very little is known about factors that regulate the commitment of pluripotent stem cells into the adipose lineage (7Camp H.S. Ren D. Leff T. Trends Mol. Med. 2002; 8: 442-447Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 8Otto T.C. Lane M.D. Cox M.M. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 229-242Crossref PubMed Scopus (409) Google Scholar). Once committed, the preadipocytes undergo an adipogenic program, which requires a coordinate activation of several pathways initiated by the down-regulation of the inhibitory preadipocyte factor-1 (Pref-1) 2The abbreviations used are: Pref-1, preadipocyte factor-1; C/EBP, CCAAT/enhancer binding protein; PPAR, peroxisome proliferator-activated receptor; Dvl, Dishevelled; PPRE, PPAR response element; GSK, glycogen synthase kinase; MCP-1, monocyte chemoattractant protein-1; Fz, Frizzled; IL-6, interleukin-6, TNFα, tumor necrosis factor α; PCNA, proliferation cell nuclear antigen; MAPK, mitogen-activate protein kinase; ELISA, enzyme-linked immunosorbent assay; sFRP2, secreted Frizzled-related protein 2. 2The abbreviations used are: Pref-1, preadipocyte factor-1; C/EBP, CCAAT/enhancer binding protein; PPAR, peroxisome proliferator-activated receptor; Dvl, Dishevelled; PPRE, PPAR response element; GSK, glycogen synthase kinase; MCP-1, monocyte chemoattractant protein-1; Fz, Frizzled; IL-6, interleukin-6, TNFα, tumor necrosis factor α; PCNA, proliferation cell nuclear antigen; MAPK, mitogen-activate protein kinase; ELISA, enzyme-linked immunosorbent assay; sFRP2, secreted Frizzled-related protein 2. and Wnt proteins (9Bennett C.N. Ross S.E. Longo K.A. Bajnok L. Hemati N. Johnson K.W. Harrison S.D. MacDougald O.A. J. Biol. Chem. 2002; 277: 30998-31004Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, 10Mei B. Zhao L. Chen L. Sul H.S. Biochem. J. 2002; 364: 137-144Crossref PubMed Scopus (109) Google Scholar, 11Moldes M. Zuo Y. Morrison R.F. Silva D. Park B.H. Liu J. Farmer S.R. Biochem. J. 2003; 376: 607-613Crossref PubMed Scopus (238) Google Scholar). Induction of the nuclear peroxisome proliferator-activated receptorγ2 (PPARγ2) also plays a crucial role in the early differentiation of the preadipocytes into lipid-accumulating cells (8Otto T.C. Lane M.D. Cox M.M. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 229-242Crossref PubMed Scopus (409) Google Scholar). Other key transcription factors are the family of CCAAT/enhancer-binding proteins: C/EBPα, C/EBPβ, and C/EBPδ. Several loss-of-function studies with cells either lacking PPARγ or C/EBPα have shown an absolute requirement of PPARγ for the induction of differentiation, whereas cells lacking C/EBPα, but expressing PPARγ, can still be induced to accumulate lipids. However, these cells are not the typical insulin-responsive and hormone-secreting adipose cells, because C/EBPα is required for the normal induction of many key adipogenic genes and proteins such as GLUT4, adiponectin, and IRS-1 (12Rosen E.D. Hsu C.H. Wang X. Sakai S. Freeman M.W. Gonzalez F.J. Spiegelman B.M. Genes Dev. 2002; 16: 22-26Crossref PubMed Scopus (1041) Google Scholar, 13Rosen E.D. Spiegelman B.M. J. Biol. Chem. 2001; 276: 37731-37734Abstract Full Text Full Text PDF PubMed Scopus (1063) Google Scholar). During preadipocyte differentiation, a carefully coordinated chain of events occurs where one of the earliest transcription factors to be expressed is C/EBPβ. However, due to its association with CHOP-10, C/EBPβ lacks early DNA-binding capacity (14Tang Q.Q. Lane M.D. Genes Dev. 1999; 13: 2231-2241Crossref PubMed Scopus (302) Google Scholar). Subsequently, C/EBPβ undergoes phosphorylation, achieves DNA-binding capacity, and induces both PPARγ2 and C/EBPα as well as the terminal clonal mitotic expansion (14Tang Q.Q. Lane M.D. Genes Dev. 1999; 13: 2231-2241Crossref PubMed Scopus (302) Google Scholar, 15Park B.H. Qiang L. Farmer S.R. Mol. Cell. Biol. 2004; 24: 8671-8680Crossref PubMed Scopus (157) Google Scholar, 16Tang Q.Q. Gronborg M. Huang H. Kim J.W. Otto T.C. Pandey A. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9766-9771Crossref PubMed Scopus (258) Google Scholar). Transcriptional activation of the C/EBPα gene occurs exceptionally late during differentiation, because repressors prevent premature expression of C/EBPα (17Jiang M.S. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12519-12523Crossref PubMed Scopus (38) Google Scholar, 18Tang Q.Q. Jiang M.S. Lane M.D. Mol. Cell. Biol. 1999; 19: 4855-4865Crossref PubMed Scopus (124) Google Scholar). Once expressed, C/EBPα activates its own gene through C/EBP binding sites in the promoter. This auto-activation is important for the terminal differentiation and maintained C/EBPα expression in the adipocytes (17Jiang M.S. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12519-12523Crossref PubMed Scopus (38) Google Scholar). C/EBPα is also anti-mitotic, and its expression ends clonal mitotic expansion. At the same time, genes related to the fully differentiated adipocyte are induced. Interleukin (IL)-6 and TNFα are major inflammatory cytokines that have been linked to the development of insulin resistance and Type 2 diabetes (19Pickup J.C. Mattock M.B. Chusney G.D. Burt D. Diabetologia. 1997; 40: 1286-1292Crossref PubMed Scopus (1034) Google Scholar). IL-6 is secreted by both adipose cells and inflammatory cells, and high circulating IL-6 levels are related to the future risk of developing Type 2 diabetes in humans (20Mohamed-Ali V. Goodrick S. Rawesh A. Katz D.R. Miles J.M. Yudkin J.S. Klein S. Coppack S.W. J. Clin. Endocrinol. Metab. 1997; 82: 4196-4200Crossref PubMed Google Scholar). In contrast, TNFα is only secreted by inflammatory cells, because the prohormone is not processed and cleaved in the adipocytes by the converting enzymes (21Xu H. Uysal K.T. Becherer J.D. Arner P. Hotamisligil G.S. Diabetes. 2002; 51: 1876-1883Crossref PubMed Scopus (151) Google Scholar). Recent studies have shown that the adipose tissue production of IL-6 in vivo is positively related to the size of the fat cells and that the interstitial concentrations are considerably higher than the circulating plasma levels (22Sopasakis V.R. Sandqvist M. Gustafson B. Hammarstedt A. Schmelz M. Yang X. Jansson P.A. Smith U. Obes. Res. 2004; 12: 454-460Crossref PubMed Scopus (189) Google Scholar). The additional recruitment of macrophages from the blood to the adipose tissue in obesity further creates an inflamed condition (23Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr., A.W. J. Clin. Invest. 2003; 112: 1796-1808Crossref PubMed Scopus (7207) Google Scholar, 24Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. J. Clin. Invest. 2003; 112: 1821-1830Crossref PubMed Scopus (5062) Google Scholar). The high interstitial concentrations make it likely that IL-6 is an important paracrine/autocrine regulator of the preadipocytes (22Sopasakis V.R. Sandqvist M. Gustafson B. Hammarstedt A. Schmelz M. Yang X. Jansson P.A. Smith U. Obes. Res. 2004; 12: 454-460Crossref PubMed Scopus (189) Google Scholar). We, therefore, examined the effects of IL-6 at concentrations found to be present in the adipose tissue in obesity (22Sopasakis V.R. Sandqvist M. Gustafson B. Hammarstedt A. Schmelz M. Yang X. Jansson P.A. Smith U. Obes. Res. 2004; 12: 454-460Crossref PubMed Scopus (189) Google Scholar), as well as TNFα, on the preadipocyte differentiation process using the well characterized 3T3-L1 cells. Materials—All chemicals were of high quality and obtained from commercial sources. Immunoblots were performed with the following antibodies; p18 (N-20), p107 (C-18), p130 (C-20), proliferation cell nuclear antigen (PCNA) (PC10), p21-activated kinase (N-20), C/EBPα (14AA), C/EBPβ (C-19), activator protein (AP2)α (C-18), Sp1 (E-3), p-αp21-activated kinase (Thr 423), PPARγ (H-100), Axin (H-98), dishevelled (Dvl)-1 (3F12) (all from Santa Cruz, Biotechnology, Inc., Santa Cruz, CA), MAPK (06-182) (Upstate Biotechnology, Lake Placid, NY), Perilipin (PROGP29), and LRP6 (AF1505) (R&D Systems, Inc.), Phospho-p44/42 MAPK (Cell Signaling, Beverly, MA and New England Biolabs Ltd., UK), and Bcl-2 and β-catenin (BD Transduction). Cell Culture Conditions—3T3-L1 preadipocytes were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (v/v), 2 mm glutamine, and antibiotics. After 2 days of confluence, the cells were treated with MDI (0.5 mm methylisobutylxanthine, 1 μm dexamethasone, and 865 nm insulin) for 48 h, followed by insulin alone for an additional 48 h. Cytokines (IL-6, mainly 20 ng/ml, and TNFα, mainly 1.5 ng/ml) and/or pioglitazone (1 μm) were added to the differentiation mixture and to cell culture media during the 8 (or longer) days of differentiation as indicated. To examine lipid accumulation, cells cultured in 6-well plates (Nunc, A/S, Roskilde, Denmark) were fixed with formalin and stained with Oil Red O. For photomicrographs, the cells were counterstained with Mayer's hematoxylin. Optical density for Oil Red O was determined by washing the stained cells with dH2O, and, after removal of all water, 1 ml of isopropanol was added for 10 min, and the optical density was measured in a plate reader at 510 nm. DNA synthesis was assessed by monitoring incorporation of 5-bromo-2′-deoxyuridine using immunocytochemistry with the Cell Proliferation Kit (Amersham Biosciences). Cells were plated on 1-well chamber slides (Nunc, A/S) and allowed to reach confluence before treatment with MDI with or without the cytokine. After induction of differentiation (1, 2, 4, and 6 days) the cells were incubated for 1 h with 5-bromo-2′-deoxyuridine prior to detection, and the number of positive cells per 1000 cells was identified. PPRE, C/EBP, and NFY-A Binding Assays—DNA-binding capacity in nuclear extracts of PPARγ to PPRE (TransAM PPARγ ELISA), C/EBPα and C/EBPβ to C/EBP binding sites (TransAM C/EBPβ/α ELISA), and NFY-A binding to NFY-A binding sites (TransAM NFY-A ELISA, all from ActiveMotif, Rixensart, Belgium), were measured as described by the manufacturer. Whole Cell Extracts and Western Blots—3T3-L1 cells, cultured in 25-cm2 flasks, were rinsed with ice-cold phosphate-buffered saline and lysed in lysis buffer containing 25 mm Tris-HCl, pH 7.4, 0.5 mm EGTA, 25 mm NaCl, 1% Nonidet P-40, 1 mm Na3VO4, 10 mm NaF, 0.2 mm leupeptin, 1 mm benzamidine, and 0.1 mm 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride. The insoluble material was sedimented by centrifugation at 20,000 × g for 10 min at 4 °C, and the supernatants were collected and frozen at -80 °C. Protein content was determined using a BCA Protein Assay kit (Pierce). 25-50 μg of whole cell extracts were used for electrophoresis through either 7.5% or 12% SDS-polyacrylamide gels (PAGEr, Cambrex, Biosciences Inc.) and transferred to BA85 nitrocellulose membranes (Schleicher & Schuell, BioScience Gmbh, Germany). The membranes were blocked for 1 h with 5% dry milk in Tris-saline (T/S) buffer (10 mm Tris-base, 0.154 m NaCl, and 1% Tween 20). After washing in T/S buffer, the membranes were incubated with primary antibodies for 1 h. For detection, the membranes were incubated with secondary antibody for 1 h, and the bands were visualized with ECL reagent (Amersham Biosciences). Nuclear Extracts—Nuclear extracts were prepared as described previously (25Timchenko N.A. Harris T.E. Wilde M. Bilyeu T.A. Burgess-Beusse B.L. Finegold M.J. Darlington G.J. Mol. Cell. Biol. 1997; 17: 7353-7361Crossref PubMed Google Scholar). Briefly, 3T3-L1 cells from one 75-cm2 flask were washed, scraped in phosphate-buffered saline buffer, and centrifuged for 10 min at 2,000 × g. The pelleted cells were dissolved in buffer A (25 mm Tris-HCl (pH 7.5), 50 mm KCl, 2 mm MgCl2, 1 mm EDTA, and 5 mm dithiothreitol), and homogenized in a tight homogenizer. The homogenate was pelleted by centrifugation at 3,300 × g for 10 min and washed twice with buffer. High salt extraction of nuclear proteins was performed by incubation of nuclei with buffer B (25 mm Tris-HCl (pH 7.5), 0.42 m NaCl, 1.5 mm MgCl2, 1 mm dithiothreitol, 0.5 mm EDTA, 25% sucrose) for 30 min on ice. The nuclei were pelleted at 20,000 × g for 30 min, and the supernatant was frozen at -80 °C prior to analyses. Protein content was determined using the Bradford protein assay kit (Bio-Rad). Real-time PCR—RNA was isolated from the cultured cells with RNeasy (Qiagen Gmbh, Hilden Germany). Gene expression was analyzed with the ABI PRISM 7900 sequence detection system (TaqMan, Applied Biosystems, Foster City, CA). Gene-specific primers and probes were designed using Primer Express software (Applied Biosystems), and the sequences used are available on request. The real-time PCR reaction was essentially performed as recommended using 100 nm probe, 200 nm of both forward and reverse primers, and 10 ng of total RNA in a final volume of 20 μl. The standard curve method or relative quantification of mRNA levels was plotted as -fold change, generally compared with day 0 (initiation of adipogenesis) when additions were made. 18 S ribosomal RNA was used as endogenous control (Applied Biosystems). Analyses were performed in duplicates, and all experiments were repeated at least three times. Statistical Analyses—Conventional statistical methods were used to calculate means ± S.E. Student's t test was used to compare differential gene expression and transcription factor binding between untreated and cytokine-treated samples. A value of p < 0.05 was considered statistically significant. IL-6, Like TNFα, Reduces Lipid Accumulation and Supports a Proliferative State—In the presence of IL-6, there was a marked inhibition in the early lipid accumulation (4 days) (Fig. 1, A and C) and a clear reduction (20-30%, p < 0.05) in the Oil Red O accumulation was also seen after 8 days (Fig. 1, B and D) as well as after 11 days (data not shown). In addition, many of the preadipocytes maintained a fibroblast-like appearance in the presence of IL-6 (Fig. 1, E and F). Also these fibroblast-like cells accumulated lipids, albeit to a much lesser extent. In the presence of IL-6, the cells were significantly larger (19.4%), although they contained less lipids, were less adherent to the surface, and exhibited a higher migration rate that resulted in cluster formations (see supplemental Fig. S1, A and B), which were obvious 4-6 days after induction of differentiation. Addition of the PPARγ ligand, pioglitazone, made the cells round up and accumulate more lipids but the cluster formation remained (see supplemental Fig. S1, C and D). These morphological changes induced by IL-6 suggest that the cells remain in a proliferative state. This was further confirmed by 5-bromo-2′-deoxyuridine labeling, which was elevated also at differentiation day 6 (not shown). The IL-6-stimulated proliferation was also associated with an increased expression of the anti-apoptotic protein, Bcl-2, both in the presence and absence of pioglitazone (see supplemental Fig. S2). Together, these data show that the presence of IL-6 both delayed and reduced the cellular lipid accumulation and, in addition, altered the cell morphology and size as well as the migration pattern during the adipocyte differentiation process. We also examined the effect of TNFα on the differentiation pattern. This cytokine was more potent than IL-6 and virtually no lipid accumulation (<5% of the cells) was seen in the presence of as low a concentration as 1.5 ng/ml (Fig. S1, G and J). Thus, early preadipocytes are highly sensitive to the inhibitory effect of cytokines on differentiation. IL-6 and TNFα Impair the Normal Differentiation Pattern—We then examined the effects of IL-6 on the expression of genes related to the differentiation and development of a normal adipocyte phenotype. Most genes related to adipose cell differentiation and function, such as adiponectin, resistin, perilipin, and Foxc2 were reduced, whereas aP2, which is mainly regulated by PPARγ, was not reduced (Fig. 2, A-E). Also a number of other important genes induced during differentiation and related to insulin action, lipid synthesis, and uptake, such as glucose transfer 4 (GLUT4), insulin receptor substrate (IRS)-1, c-CBL-associated protein (CAP), fatty acid synthase, and lipoprotein lipase (LPL) were markedly reduced in the presence of IL-6 (Fig. 2, F-J). In contrast, IL-6 increased the expression of IL-6 itself as well as MCP-1 and plasminogen activator inhibitor (PAI)-1 (Fig. 3, A-C). In general, the addition of the PPARγ-ligand abrogated the IL-6 effect and normalized CAP and perilipin; this was also verified at the protein level (see supplemental Fig. S3).FIGURE 3IL-6 and TNFα induces inflammatory markers. A, induction of IL-6. 3T3-L1 preadipocytes were differentiated for 8 days with or without 20 ng/ml IL-6 or 1.5 ng/ml TNFα. RNA was extracted, and mRNA levels were determined with real-time PCR. The data were first normalized to 18 S rRNA. Then, all mRNA levels were normalized to expression level in the control sample (without cytokines = 1). B, both IL-6 (left bar) and, in particular, TNFα (right bar) dramatically increase expression of MCP-1 mRNA. Differentiation and RNA extraction performed as above. C, IL-6 induces expression of PAI-1 (left axis). Differentiation and RNA extraction were performed as above. Data are presented as the mean ± S.E. (n = 4-5). *, p < 0.05; ***, p < 0.002, compared with untreated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also examined the effect of TNFα which, in addition to the ablated lipid accumulation, also completely prevented the normal differentiation (discussed later). However, the expression of chemo- and cytokines like IL-6 and MCP-1 was dramatically increased (Fig. 3, A-C) and considerably more than for IL-6, whereas specific macrophage markers like F4/80 and MAC-1 were not induced (data not shown). TNFα, in contrast to IL-6, did not show an increase but actually decreased PAI-1 expression (Fig. 3C). The Early Induction of Adipocyte Differentiation Is Not Altered by IL-6—Because IL-6 (like TNFα) exerts such profound effects on the growth and differentiation of the preadipocytes, we set out to systematically explore the molecular mechanisms involved. Down-regulation of Pref-1 is required for preadipocytes to undergo adipose conversion. However, Pref-1 mRNA was decreased within hours after induction of differentiation whether or not IL-6 was present (Fig. 4A). Wnt signaling and secretion also play a key role for maintaining the cells in an undifferentiated state (11Moldes M. Zuo Y. Morrison R.F. Silva D. Park B.H. Liu J. Farmer S.R. Biochem. J. 2003; 376: 607-613Crossref PubMed Scopus (238) Google Scholar). However, also Wnt10b mRNA, previously shown to inhibit adipocyte differentiation (26Ross S.E. Hemati N. Longo K.A. Bennett C.N. Lucas P.C. Erickson R.L. MacDougald O.A. Science. 2000; 289: 950-953Crossref PubMed Scopus (1492) Google Scholar), declined rapidly upon induction of differentiation whether or not IL-6 was present (Fig. 4B). We also measured Wnt1 and Wnt3a, but these ligands were not expressed under any conditions used (data not shown). Activation of the retinoblastoma family proteins, p130 and p107, is directly correlated with the entry into the clonal mitotic expansion phase. Following induction of differentiation, the expression of these proteins is switched with an increase in p107 and a decrease in p130 (27Liu K. Guan Y. MacNicol M.C. MacNicol A.M. McGehee Jr., R.E. Mol. Cell. Endocrinol. 2002; 194: 51-61Crossref PubMed Scopus (16) Google Scholar, 28Timchenko N.A. Wilde M. Iakova P. Albrecht J.H. Darlington G.J. Nucleic Acids Res. 1999; 27: 3621-3630Crossref PubMed Scopus (50) Google Scholar). Again, IL-6 did not interfere with the normal p130/p107 switch (Fig. 4C). Thus, IL-6 did not interfere with these critical initial steps required for the induction of differentiation. Clonal Mitotic Expansion—C/EBPβ and C/EBPδ are expressed immediately after induction of differentiation. Initially, C/EBPβ is inactivated by binding to CHOP-10, a dominant-negative member of the C/EBP family. However, CHOP-10 decreases rapidly and C/EBPβ is released (29Clarke S.L. Robinson C.E. Gimble J.M. Biochem. Biophys. Res. Commun. 1997; 240: 99-103Crossref PubMed Scopus (186) Google Scholar, 30Tang Q.Q. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12446-12450Crossref PubMed Scopus (138) Google Scholar). IL-6, like TNFα (data not shown), did not alter the normal down-regulation of CHOP-10, and the mRNA levels remained low until the cells had entered the stage of terminal differentiation (Fig. 4D). Binding activity of C/EBPβ and -δ coincides with the entry of the preadipocytes into the S phase of the cell cycle and the onset of clonal mitotic expansion. C/EBPδ gene expression peaked after ∼24 h, and was similar whether or not IL-6 was present (Fig. 4E), while C/EBPβ mRNA was slightly, but significantly (p < 0.05), increased in the presence of IL-6 but showed a similar time course as the control cells (Fig. 4E). Neither C/EBPβ activating (LAP) or inhibitory (LIP) proteins in nuclear extracts (Fig. 4F), nor phosphorylation (data not shown), were altered by the presence of IL-6. To further verify that C/EBPβ was not changed by IL-6 we examined the binding to C/EBP-binding sites. However, specific binding by nuclear extracts prepared for up to 72 h after induction of differentiation was also not altered by the presence of IL-6 (data not shown). Similarly, TNFα did not inhibit the early induction of either C/EBPβ or C/EBPδ (data not shown). PPARγ, C/EBPβ, and C/EBPδ Induction Is Normal, whereas C/EBPα Expression Is Reduced by IL-6—An absolute requirement for adipogenesis and lipid accumulation is expression of the PPARγ isoforms (12Rosen E.D. Hsu C.H. Wang X. Sakai S. Freeman M.W. Gonzalez F.J. Spiegelman B.M. Genes Dev. 2002; 16: 22-26Crossref PubMed Scopus (1041) Google Scholar, 13Rosen E.D. Spiegelman B.M. J. Biol. Chem. 2001; 276: 37731-37734Abstract Full Text Full Text PDF PubMed Scopus (1063) Google Scholar), PPARγ1 and PPARγ2; the latter only being expressed in the adipose cells. Both C/EBPβ and C/EBPδ are activators of the PPARγ gene, but, once expressed, there is feedback activation between C/EBPα and PPARγ through C/EBP regulatory elements in the PPARγ promoter (29Clarke S.L. Robinson C.E. Gimble J.M. Biochem. Biophys. Res. Commun. 1997; 240: 99-103Crossref PubMed Scopus (186) Google Scholar). We analyzed the binding of PPARγ in nuclear extracts to PPRE during the initial 72 h of differentiation (Fig. 5A). This was not altered by the presence of IL-6, also supported by the finding that induction for both PPARγ1 and -2 proteins was normal (Fig. 5B). After a delay of ∼30 h after the induction of differentiation, C/EBPα is transactivated by C/EBPβ and C/EBPδ through C/EBP regulatory elements in the C/EBPα promoter (31Lane M.D. Tang Q.Q. Jiang M.S. Biochem. Biophys. Res. Commun. 1999; 266: 677-683Crossref PubMed Scopus (238) Google Scholar). PPARγ also induces transactivation of C/EBPα through PPRE in the C/EBPα promoter. C/EBPα gene expression peaked at 96 h (Fig. 5C), and this late activation corresponds to induction of terminal differentiation and the anti-mitotic effect of C/EBPα (31Lane M.D. Tang Q.Q. Jiang M.S. Biochem. Biophys. Res. Commun. 1999; 266: 677-683Crossref PubMed Scopus (238) Google Scholar, 32Morrison R.F. Farmer S.R. J. Biol. Chem. 1999; 274: 17088-17097Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 33Tang Q.Q. Otto T.C. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 44-49Crossref PubMed Scopus (613) Google Scholar). The induction of C/EBPα mRNA expression, 24-48 h and later, was markedly reduced by the presence of IL-6 (Fig. 5C). Similarly, C/EBPα protein expression in whole cell lysates (data not shown), as well as in nuclear extracts (Fig. 5B), was reduced by ∼50-70%. Addition of pioglitazone did not prevent the inhibitory effect of IL-6 on C/EBPα gene activation (Fig. 5C), whereas the protein expression was clearly increased (Fig. 5B). The binding of C/EBPα in nuclear extracts to the C/EBP binding sites was markedly decreased 48 h and later during the differentiation process (Fig. 5D). Protein dilution experiments (data not shown) indicated that nuclear extracts from IL-6-exposed cells contained one or more factors that altered the normal binding of C/EBPα, but the nature of this remains to be identified. These data show that the early process leading to the induction of differentiation and activation of C/EBPβ, C/EBPδ, and PPARγ2 are not altered by IL-6, whereas the b" @default.
• W2000392034 created "2016-06-24" @default.
• W2000392034 creator A5028506321 @default.
• W2000392034 creator A5028599620 @default.
• W2000392034 date "2006-04-01" @default.
• W2000392034 modified "2023-10-16" @default.
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