IDENTIFICATION OF THE CAMP RESPONSE ELEMENT THAT CONTROLS TRANSCRIPTIONAL ACTIVATION OF THE INSULIN-LIKE GROWTH-FACTOR-I GENE BY PROSTAGLANDIN E(2) IN OSTEOBLASTS

Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replica tion and differentiation. We previously showed that prostaglandin E(2) (PGE(2)) and other agents that increase cAMP activated IGF-I gene tra nscription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction wa s mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have charact erized inducible DNA-protein interactions involving this site. Transie nt transfections of IGF-I P1 reporter genes into primary rat osteoblas ts showed that the 328-base pair untranslated region of exon 1 was req uired for a full 5.3-fold response to PGE(2); mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction b y 65%. PGE(2) stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extrac ts from untreated osteoblast cultures, was detected within 2 h of PGE( 2) treatment, and was maximal. by 4 h. This DNA-protein interaction wa s not observed in cytoplasmic extracts from PGE(2)-treated cultures, i ndicating nuclear localization of the protein kinase A-activated facto r(s). Activation of this factor was not blocked by cycloheximide (Chx) , and Chx did not impair stimulation of IGF-I gene expression by PGE(2 ). In contrast, binding to a consensus cAMP response element (CRE; 5'- TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE(2 ) or Chx. Competition gel mobility shift analysis using mutated DNA pr obes identified 5'-CGCAATCG-3' as the minimal sequence needed for indu cible binding. All modified IGF-I P1 promoter-reporter genes with muta tions within this CRE sequence also showed a diminished functional res ponse to PGE(2). These results identify the CRE within the 5'-untransl ated region of IGF-I exon 1 that is required for hormonal activation o f IGF-I gene transcription by cAMP in osteoblasts.