PROMOTER-DEPENDENT AND PROMOTOR-INDEPENDENT ACTIVATION OF INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-5 GENE-EXPRESSION BY PROSTAGLANDIN E(2) IN PRIMARY RAT OSTEOBLASTS

Insulin-like growth factor (IGF) action is mediated by high affinity c ell surface IGF receptors and modulated by a family of secreted IGF bi nding proteins (IGFBPs), IGFBP-5, the most conserved of six IGFBPs cha racterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells, In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E(2) (PGE(2)), a local factor that mediates certain e ffects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain, In this s tudy, we show that transcriptional and post-transcriptional events ini tiated by PGE(2) collaborate to enhance IGFBP-5 gene expression in pri mary fetal rat osteoblast cultures, PGE(2) treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h o f incubation, Analysis of nascent IGFBP-5 mRNA suggested that PGE(2) h ad only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes c onfirmed that PGE, enhanced promoter activity by similar to 2-fold. Si milar stimulatory effects were seen with forskolin. A DNA fragment wit h only 51 base pairs of the 5'-flanking sequence retained hormonal res ponsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal LGFBP-5 p romoter, Incubation of osteoblasts with the mRNA transcriptional inhib itor 5,6-dichforo-1-beta-D-ribofilranosylbenzimidazole demonstrated th at PGE(2) enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t( 1/2) from 9 to 18 h. The effects of PGE(2) on steady-state IGFBP-5 tra nscripts were abrogated by preincubating cells with cycloheximide, ind icating that the effects of PGE(2) on both gene transcription and mRNA stability required ongoing protein synthesis, Therefore, both promote r-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE(2) in osteoblasts.