Activation of the insulin-like growth factor-binding protein-5 promoter inosteoblasts 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-depend ent and -independent actions. PGE(2) rapidly increases IGFBP-5 expression b y osteoblasts through cAMP-dependent processes. A minimal DNA sequence requ ired for basal and PGE(2)-stimulated IGFBP-5 promoter activity spans -69 to -35 bp. This region adjoins a functional TATA box and contains E box, CCAA T 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 e lement reduced basal promoter activity by 50% and eliminated the 2-fold sti mulatory effect of PGE(2). In contrast, mutations in the C/EBP- or NF-l-rel ated elements also reduced basal promoter activity without fully eliminatin g the PGE(2) effect. Overexpression of C/EBP delta stimulated basal IGFBP-5 ; promoter activity, and this effect was eliminated by mutating the C/EBP-b inding site. However, mutation of the AP-2-binding site or overexpression o f AP-2 did not correlate with basal or PGE(2)-induced promoter activation. By electrophoretic mobility shift assay, prominent gel shift complexes occu rred with osteoblast nuclear extracts and P-32-labeled probes spanning the E box-, C/EBP-, and NF-l-related motifs. These gel shift complexes were dep leted by specific binding site mutations and were enhanced by PGE(2). Incre ased binding by extracts from PGE(2)-treated cultures was blocked by cycloh eximide treatment. These results identify several elements as integral bind ing sequences for both basal and PGE(2)-stimulated IGFBP-5 promoter activit y. They further reveal that multiple sequences within this cluster form a b asic transcription unit where nuclear factors can accumulate in a protein s ynthesis-dependent way and enhance IGFBP-5 expression by osteoblasts in res ponse to PGE(2).