REGULATION OF INSULIN-LIKE GROWTH-FACTOR-I TRANSCRIPTION BY CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE (CAMP) IN FETAL-RAT BONE-CELLS THROUGH AN ELEMENT WITHIN EXON-1 - PROTEIN-KINASE A-DEPENDENT CONTROL WITHOUT A CONSENSUS AMP RESPONSE ELEMENT

Insulin-like growth factor I(IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblas ts (Ob) is stimulated by agents that increase the intracellular cAMP c oncentration, including prostaglandin E(2) (PGE(2)). Previous studies with Ob cultures demonstrated that PGE, enhanced IGF-I transcription t hrough selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now all owed localization of a potential cis-acting promoter element(s) respon sible for cAMP-stimulated gene expression to the 5'-untranslated regio n (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observatio ns: 1) a transfection construct containing only 122 nucleotides (nt) o f promoter 1 and 328 nt of the 5'-UTR retained full PGE(2)-stimulated reporter expression; 2) maximal PGE(2)-driven reporter expression requ ired the presence of nt 196 to 328 of exon 1 when tested within the co ntext of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-lucifer ase-reporter constructs with a plasmid encoding the alpha-isoform of t he catalytic subunit of murine cAMP-dependent protein kinase (PKA) pro duced results comparable to those seen with PGE(2) treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE(2)-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts . Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I ex on 1. These results directly implicate PKA in the control of IGF-I gen e transcription by PGE(2) and identify a segment of IGF-I exon 1 as be ing essential for this hormonal regulation.