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CCAAT/enhancer binding protein gene promoter: binding of nuclear factors during differentiation of 3T3-L1 preadipocytes.

ABSTRACT: Differentiation of 3T3-L1 preadipocytes into adipocytes is accompanied by increased expression of the nuclear protein C/EBP (CCAAT/enhancer binding protein) and by transcriptional activation of a group of adipose-specific genes. We report here the isolation of the murine C/EBP gene and the characterization of its promoter. Consistent with its proposed role in coordinating transcription during preadipocyte differentiation, an increase in the rate of transcription of the C/EBP gene precedes that of several adipose-specific genes whose promoters are transactivated by C/EBP. DNase I cleavage-inhibition patterns (footprinting) of the C/EBP gene promoter by nuclear factors from differentiated and undifferentiated 3T3-L1 cells identified two sites of differential factor binding. One site in the C/EBP gene promoter between nucleotides -252 and -239 binds a nuclear factor(s) present in preadipocytes that is lost or modified upon differentiation. Another site, between nucleotides -203 and -176, exhibits different but overlapping footprints by nuclear factors present in differentiated and undifferentiated cells. Gel retardation analysis with oligonucleotides corresponding to these sites revealed protein-oligonucleotide complexes containing these differentially expressed nuclear factors. The factor present in differentiated cells that binds at this site was identified as C/EBP (possibly in heterodimeric form with a homologous leucine-zipper protein), suggesting that C/EBP may regulate expression of its own gene.

SUBMITTER: Christy RJ

PROVIDER: S-EPMC51279 | biostudies-other | 1991 Mar

REPOSITORIES: biostudies-other

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CCAAT/enhancer binding protein gene promoter: binding of nuclear factors during differentiation of 3T3-L1 preadipocytes.

Christy R J RJ Kaestner K H KH Geiman D E DE Lane M D MD

Proceedings of the National Academy of Sciences of the United States of America 19910301 6

Differentiation of 3T3-L1 preadipocytes into adipocytes is accompanied by increased expression of the nuclear protein C/EBP (CCAAT/enhancer binding protein) and by transcriptional activation of a group of adipose-specific genes. We report here the isolation of the murine C/EBP gene and the characterization of its promoter. Consistent with its proposed role in coordinating transcription during preadipocyte differentiation, an increase in the rate of transcription of the C/EBP gene precedes that o ...[more]

PMID: 2006196

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Project description:Objective:Ghrelin is a 28-amino-acid peptide that regulates energy hemostasis, glucose and lipid metabolism. We aimed to explore the effects of ghrelin on the proliferation and differentiation of 3T3-L1 and human primary preadipocytes. Methods:3-(4,5-Dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) spectrophotometry, Oil Red O staining, intracellular glycerol-3-phosphate dehydrogenase (G-3-PDH) assays and semiquantitative reverse transcription polymerase chain reaction were used to investigate the action of ghrelin. Results:Ghrelin (0.01-1000 ng/ml) significantly increased the numbers of 3T3-L1 cells, and the maximum stimulatory effect was observed with the 10 ng/ml ghrelin treatment for 24 h (p < 0.05). Ghrelin also promoted the proliferation of human primary preadipocytes from 24 h (p < 0.05) to 48 h (p < 0.05) at a concentration of 1000 ng/ml. Further investigation showed that IGF-1 levels were notably increased in ghrelin-treated 3T3-L1 and human preadipocytes, and IGF-1 antibody was capable to attenuate this stimulatory action of ghrelin (all p < 0.05). Additionally, ghrelin significantly suppressed the differentiation of 3T3-L1 and human primary preadipocytes; 10 ng/ml ghrelin notably downregulated G-3-PDH activities in 3T3-L1 cells on day 3 and in human cells from days 4 to 12 following differentiation (all p < 0.05), and the intracellular lipoprotein lipase mRNA levels were lower than that of the controls (p < 0.05). Further investigation showed that the mRNA levels of peroxisome proliferator-activated receptor ?2 (PPAR?2) and CCAAT/enhancer binding protein ? (C/EBPs) were also suppressed in ghrelin-treated human differentiating adipocytes. Conclusion:Ghrelin promotes the proliferation of 3T3-L1 and human primary preadipocytes by increasing the expression of IGF-1. Ghrelin inhibits murine and human adipocyte differentiation by downregulating PPAR?2 and C/EBP? levels, consequently leading to decreased lipid accumulation and lipogenic enzymes expression.

Dataset Information

CCAAT/enhancer binding protein gene promoter: binding of nuclear factors during differentiation of 3T3-L1 preadipocytes.

Publications

CCAAT/enhancer binding protein gene promoter: binding of nuclear factors during differentiation of 3T3-L1 preadipocytes.

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