Conditional ectopic expression of C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis.
Activation of adipogenesis in 3T3 preadipocytes by exposure to the adipogenic inducers dexamethasone, methylisobutylxanthine, insulin, and fetal bovine serum is accompanied by a transient burst of C/EBP beta protein expression that precedes the induction of the fat gene program. In this study we have investigated the role of C/EBP beta in initiating the adipogenic program by overexpressing C/EBP beta in multipotential NIH-3T3 fibroblasts. Conditional ectopic expression of C/EBP beta was accomplished by using an artificial transcriptional regulatory system based on the Escherichia coli tetracycline repressor to generate a stable cell line, beta 2, that expresses C/EBP beta mRNA and protein in a tightly controlled tetracycline dose-dependent manner. Induction of C/EBP beta DNA-binding activity in NIH-3T3 beta 2 cells exposed to dexamethasone in the presence of insulin and fetal bovine serum activates the expression of an adipocyte-specific nuclear hormone receptor, PPAR gamma, that stimulates the conversion of these fibroblasts into committed preadipocytes. Either ectopic expression of C/EBP beta or treatment with dexamethasone alone is incapable of inducing PPAR gamma expression, but when present together, they have a synergistic effect on the adipogenic program. Exposure of these stimulated cells to a PPAR activator 5,8,11,14-eicosatetraynoic acid (ETYA) results in the accumulation of fat droplets and expression of the adipocyte-enriched genes aP2 and glycerol phosphate dehydrogenase (GPD). The number of beta 2 cells that can differentiate into adipocytes is related to the concentration of tetracycline and, therefore, the amount of the exogenous C/EBP beta protein expressed. C/EBP beta can induce PPAR gamma mRNA in the absence of ETYA; however, expression of aP2 mRNA and maximum fat deposition is dependent on the PPAR activator. Our results suggest that enhanced expression of C/EBP beta converts multipotential mesenchymal precursor cells into preadipocytes that respond to adipogenic inducers, including dexamethasone and PPAR activators to differentiate into adipocytes.
Wu
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Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118, USA.
Xie
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Bucher
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Farmer
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D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D016475:3T3 Cells; D015117:5,8,11,14-Eicosatetraynoic Acid / Q000494:pharmacology; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002454:Cell Differentiation / Q000235:genetics; D002458:Cell Fractionation; D004247:DNA / Q000378:metabolism; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D015342:DNA Probes; D004268:DNA-Binding Proteins; D007328:Insulin; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D015117:5,8,11,14-Eicosatetraynoic Acid; D003907:Dexamethasone; D004247:DNA; D013752:Tetracycline; D015056:1-Methyl-3-isobutylxanthine