Transcriptional repression of TFF1 in gastric epithelial cells by CCAAT/enhancer binding protein-beta

TFF1 is a member of a unique family of gastrointestinal peptides. Loss of TFF1 expression has been observed in the majority of human gastric cancers and the biological significance of this loss has been demonstrated in a Tff1 knockout mouse model. However, few TFF1 gene mutations or allelic loss have also been documented. To understand the molecular mechanism repressing the TFF1 gene expression, the 5'-flanking region of the human TFF1 gene was characterized. We found a repressor region (-241 to -84), which is active in MKN45 and IMGE5 cells expressing endogenous TFF1 gene. A consensus binding site for C/EBPbeta was identified and EMSA analysis demonstrated specific binding of CEBPbeta. Mutation of this C/EBPbeta element potentiated the transactivation of TFF1 by 50% and 145% for MKN45 and IMGE5 cells respectively. Furthermore, co-transfection of C/EBPbeta isoforms specifically decreased TFF1 promoter activity. These findings suggest that C/EBPbeta is involved in the down-regulating of TFF1 gene expression and this mode of repression may account at least in part for the loss of TFF1 gene expression in transformed human and mice gastric epithelial cells.     

CCAAT/enhancer binding protein-beta negatively regulates the expression of glycerol-3-phosphate dehydrogenase 1 in pig PK-15 cells

Soluble glycerol-3-phosphate dehydrogenase 1 (GPD1, EC 1.1.1.8) plays important roles in the synthesis of triacylglycerol and in the glycerol-3-phosphate shutter. Though GPD1 is expressed in most adult tissues, little is known about the regulation of its expression. In this study, we analyzed the characters, organization and core region of the promoter of pig GPD1 gene by in silico analysis and activity detection of deletion mutants. We also identified and testified the negative regulation effect of C/EBP β on pig GPD1 gene by Chromatin immunoprecipitation (ChIP) assay and over-expression experiments in cultured pig kidney cells. Compared to that of human, pig GPD1 gene promoter has three conserved regions and one deletion region. In silico analysis indicated that pig GPD1 promoter was TATA-less with at least 3 CpG islands of over 200 bp in length and over 60% in GC content. The activity detection of deletion mutants suggested that the essential elements required for the optimal promoter activity scatter in the promoter region, while the core promoter region was from -422 bp to -1 bp. Chromatin immunoprecipitation (ChIP) assay results indicated that C/EBP β had plenty of binding sites in pig GPD1 promoter with the common cis-element (5’- TKNNGCAAK -3’). The over-expression examination of C/EBP β showed that the expression of GPD1 was negatively regulated by C/EBP β in pig kidney cells. Overall, our study revealed that the pig GPD1 promoter is a TATA-less promoter, and in promoter region, the binding sites of C/EBP β share common motif of (5’-TKNNGCAAK -3’). We also showed that pig GPD1 gene is regulated negatively by C/EBP β in cultured kidney cells.     

Expression and function of CCAAT/enhancer binding proteinbeta (C/EBPbeta) LAP and LIP isoforms in mouse mammary gland, tumors and cultured mammary epithelial cells

CCAAT/Enhancer binding proteins (C/EBPs) play important roles in the regulation of cell growth and differentiation. This study investigated the expression and function of C/EBPbeta isoforms in the mouse mammary gland, mammary tumors, and a nontransformed mouse mammary epithelial cell line (HC11). C/EBPbeta mRNA levels are 2-5-fold higher in mouse mammary tumors derived from MMTV/c-neu transgenic mice compared with lactating and involuting mouse mammary gland. The "full-length" 38 kd C/EBPbeta LAP ("Liver-enriched Activator Protein") isoform is the predominant C/EBPbeta protein isoform in mammary tumor whole cell lysates, however, the truncated 20 kd C/EBPbeta LIP ("Liver-enriched Inhibitory Protein") isoform is also present at detectable levels (mean LAP:LIP ratio 5.3:1). The mammary tumor C/EBPbeta LAP:LIP ratio decreases 70% (from 5.3:1 to 1.6:1) when lysate preparation is switched from a rapid whole cell lysis protocol to a multistep nuclear/cytoplasmic fractionation protocol. In contrast to mammary tumors, only the C/EBPbeta LAP isoform is detectable in the mammary gland whole cell and nuclear lysates; the truncated "LIP" isoform is undetectable regardless of isolation protocol. Ectopic over expression of C/EBPbeta LIP or C/EBPbeta LAP did not alter HC11 growth rates. However, C/EBPbeta LIP over expressing HC11 cells (LAP:LIP ratio of approximately 1:1) exhibited a consistent 2-4 h delay in G(0)/S phase transition. C/EBPbeta LIP overexpressing HC11 cells did not express beta-casein mRNA (mammary epithelial cell differentiation marker) in response to lactogenic hormones. This defect in beta-casein expression was not corrected by carrying out the differentiation protocol in the presence of an artificial extracellular matrix. These results demonstrate that the "full-length" C/EBPbeta LAP isoform is the predominant C/EBPbeta protein isoform expressed in mouse mammary gland in vivo and mouse mammary epithelial cell cultures in vitro. C/EBPbeta LIP detected in mammary tumor lysates may result from in vivo production or ex vivo isolation-induced proteolysis of C/EBPbeta LAP. Ectopic overexpression of C/EBPbeta LIP (LAP:LIP ratio of approximately 1:1) inhibits mammary epithelial cell differentiation (beta-casein expression).     

CCAAT/enhancer-binding protein-beta participates in insulin-responsive expression of the factor VII gene

Expression of the human coagulation factor VII (FVII) gene by hepatoma cells was modulated in concert with levels of glucose and insulin in the culture medium. In low glucose medium without insulin, amounts of both FVII mRNA and secreted FVII protein were coordinately increased; in the presence of glucose with insulin, both were decreased. Analysis of the FVII promoter showed that these effects could be reproduced in a reporter-gene system, and a small promoter element immediately upstream of the translation start site of the gene, which mediated these effects, was identified. Mutation of this element largely abrogated the glucose/insulin-responsive change in expression of the reporter gene. Several members of the CCAAT/enhancer-binding protein family were found to be capable of binding the identified sequence element but not the mutated element. The expression of a FVII minigene directed by a segment of the native FVII promoter responded to co-expressed activating and inhibiting forms of CCAAT/enhancer-binding protein beta.