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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 Sequences¹

Yale University School of Medicine, Section of Plastic Surgery, New Haven Connecticut 06520

Address all correspondence and requests for reprints to: Thomas L. McCarthy, Ph.D., Section of Plastic Surgery, 333 Cedar Street, P.O. Box 208041, New Haven, Connecticut 06520-8041. E-mail: thomas.mccarthy{at}yale.edu

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/EBP 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.

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