Characterization of rat CD14 promoter and its regulation by transcription factors AP1 and Sp family proteins in hepatocytes.
CD14, a 55kDa glycoprotein, serves as a lipopolysaccharide (LPS) recognition molecule. CD14 is a monocyte differentiation antigen expressed by myeloid-derived cells, or other cells such as hepatocytes, as either a membrane-bound protein or a soluble serum protein. Increasing evidence indicates that soluble CD14 in plasma is an acute-phase protein derived, among other sources, from liver cells. Although information is available on the cellular expression of CD14, little is known about the cis- and trans-acting factors that regulate basal CD14 transcription in liver cells. We show here that liver cells have a relatively high basal CD14 transcription rate as determined by nuclear run-on assay. We cloned and sequenced an 883bp 5'-flanking region of the rat CD14 gene and demonstrated functional promoter activity in liver cells. Sequence analysis revealed that, like in the human and mouse CD14 genes, multiple Sp1 and AP1 binding elements exist in rat CD14. Site-directed mutagenesis and transient transfection assays demonstrated that an Sp1 element located at -836 and an AP1 element located at -270 are required for basal promoter activity in liver cells. Electrophoretic mobility shift assays indicate that both Sp1 and Sp3 nuclear factors interact with the -836 Sp1 element, while the AP1-related proteins Fra-2 and JunD bind to the AP1 motif. These data provide novel insights into the regulation of basal CD14 expression in liver cells.
Liu
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Department of Surgery, University of Pittsburgh School of Medicine, PA 15261, USA. shubing@pitt.edu
Shapiro
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RA
Nie
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Zhu
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Vodovotz
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Billiar
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D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D018950:Lipopolysaccharide Receptors / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000502:physiology*; D013047:Specific Pathogen-Free Organisms; D018808:Transcription Factor AP-1 / Q000502:physiology*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D018950:Lipopolysaccharide Receptors; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D004247:DNA; D008156:Luciferases