Activation of the insulin-like growth factor-binding protein-5 promoter in osteoblasts by cooperative E box, CCAAT enhancer-binding protein, and nuclear factor-1 deoxyribonucleic acid-binding sequences.
Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) has IGF-dependent and -independent actions. PGE2 rapidly increases IGFBP-5 expression by osteoblasts through cAMP-dependent processes. A minimal DNA sequence required for basal and PGE2-stimulated IGFBP-5 promoter activity spans -69 to -35 bp. This region adjoins a functional TATA box and contains E box, CCAAT enhancer-binding protein (C/EBP), nuclear factor-1 (NF-1), and activator protein-2 (AP-2) transcription factor related binding motifs. In this study we compared minimal promoter sequences of -74 to +120 bp, without or with mutations in each potential regulatory element, by reporter gene expression and electrophoretic mobility shift assays. Mutation of the E box-related element reduced basal promoter activity by 50% and eliminated the 2-fold stimulatory effect of PGE2. In contrast, mutations in the C/EBP- or NF-1-related elements also reduced basal promoter activity without fully eliminating the PGE2 effect. Overexpression of C/EBPdelta stimulated basal IGFBP-5 promoter activity, and this effect was eliminated by mutating the C/EBP-binding site. However, mutation of the AP-2-binding site or overexpression of AP-2 did not correlate with basal or PGE2-induced promoter activation. By electrophoretic mobility shift assay, prominent gel shift complexes occurred with osteoblast nuclear extracts and 32P-labeled probes spanning the E box-, C/EBP-, and NF-1-related motifs. These gel shift complexes were depleted by specific binding site mutations and were enhanced by PGE2. Increased binding by extracts from PGE2-treated cultures was blocked by cycloheximide treatment. These results identify several elements as integral binding sequences for both basal and PGE2-stimulated IGFBP-5 promoter activity. They further reveal that multiple sequences within this cluster form a basic transcription unit where nuclear factors can accumulate in a protein synthesis-dependent way and enhance IGFBP-5 expression by osteoblasts in response to PGE2.
Ji
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C
Yale University School of Medicine, Section of Plastic Surgery, New Haven, Connecticut 06520-8041, USA.
Chen
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Y
Centrella
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McCarthy
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TL
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics*; D015232:Dinoprostone / Q000494:pharmacology; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000235:genetics*; D010006:Osteoblasts / Q000187:drug effects / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014157:Transcription Factors; D015854:Up-Regulation; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D018975:Insulin-Like Growth Factor Binding Protein 5; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D004247:DNA; D015232:Dinoprostone