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Identification of hepatocyte nuclear factor-3 binding sites in the Clara cell secretory protein gene.

ABSTRACT: To determine the mechanisms of cell-specific gene expression in the developing pulmonary epithelium the Clara cell secretory protein (CCSP) gene promoter was analysed by DNAase I footprinting. A prominent site of protein-DNA interaction was detected from nucleotides -132 to -76 using nuclear extract from mouse lung and human H441 cells. Mobility shift analysis revealed that an oligonucleotide corresponding to this region interacted with multiple proteins from lung and H441 cell nuclear extracts. Analysis of the nucleotide sequence of this region identified two potential binding sites for hepatocyte nuclear factor 3 (HNF-3), and consistent with this finding binding to this CCSP oligonucleotide was specifically competed for by an oligonucleotide corresponding to the HNF-3-binding site from the mouse transthyretin gene. Mobility shift of the CCSP oligonucleotide was supershifted using antisera specific to HNF-3 alpha and HNF-3 beta, and HNF-3 alpha and HNF-3 beta translated in vitro were found to bind specifically to this same oligonucleotide. Co-transfection of HNF-3 alpha- and HNF-3 beta-expression plasmids increased cell-specific reporter gene activity in H441 cells transfected with a CCSP-CAT gene chimeric construct containing this -132 to -76 region. Taken together, these results suggest a role for HNF-3 in mediating cell-specific CCSP gene expression within the bronchiolar epithelium. These findings support the hypothesis that members of the HNF-3 'forkhead' family of transcription factors determine gene expression and cell fate in multiple cell lineages derived from the primitive gut endoderm.

SUBMITTER: Bingle CD

PROVIDER: S-EPMC1134843 | biostudies-other | 1993 Oct

REPOSITORIES: biostudies-other

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Project description:The sensitization of hepatocytes to cell death from tumor necrosis factor ? (TNF?) underlies many forms of hepatic injury, including that from toxins. Critical for hepatocyte resistance to TNF? toxicity is activation of nuclear factor ?B (NF-?B) signaling, which prevents TNF?-induced death by the up-regulation of protective proteins. To further define the mechanisms of hepatocyte sensitization to TNF? killing, immunoblot analysis comparing livers from mice treated with lipopolysaccharide (LPS) alone or LPS together with the hepatotoxin galactosamine (GalN) was performed to identify TNF?-induced protective proteins blocked by GalN. Levels of CCAAT/enhancer-binding protein ? (C/EBP?) were increased after LPS treatment but not GalN/LPS treatment. In a nontransformed rat hepatocyte cell line, TNF?-induced increases in C/EBP? protein levels were dependent on NF-?B-mediated inhibition of proteasomal degradation. Pharmacological inhibition of c-Jun N-terminal kinase (JNK) did not affect C/EBP? degradation, indicating that the process was JNK-independent. C/EBP? functioned to prevent cell death as adenoviral C/EBP? overexpression blocked TNF?-induced apoptosis in cells sensitized to TNF? toxicity by NF-?B inhibition. C/EBP? inhibited TNF?-induced caspase 8 activation and downstream mitochondrial cytochrome c release and caspase 3 and caspase 7 activation. Studies in primary hepatocytes from c/ebp?(-/-) mice confirmed that loss of C/EBP? increased death from TNF?. c/ebp?(-/-) mice were also sensitized to liver injury from a nontoxic dose of LPS or TNF?. The absence of jnk2 failed to reverse the GalN-induced block in C/EBP? induction by LPS, again demonstrating that C/EBP? degradation was JNK-independent.C/EBP? is up-regulated by TNF? and mediates hepatocyte resistance to TNF? toxicity by inhibiting caspase-dependent apoptosis. In the absence of NF-?B signaling, proteasomal degradation of C/EBP? is increased by a JNK-independent mechanism and promotes death from TNF?.

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Identification of hepatocyte nuclear factor-3 binding sites in the Clara cell secretory protein gene.

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