SYNERGISTIC INDUCTION OF OSTEOCALCIN GENE-EXPRESSION - IDENTIFICATIONOF A BIPARTITE ELEMENT CONFERRING FIBROBLAST GROWTH-FACTOR-2 AND CYCLIC-AMP RESPONSIVENESS IN THE RAT OSTEOCALCIN PROMOTER

Fibroblast growth factors (FGFs) are important regulators of calvarial osteoblast growth and differentiation. We have studied the regulation of the osteoblast-specific gene osteocalcin (OC) by FGF2 in phenotypi cally immature MC3T3-E1 calvarial osteoblastic cells. FGF2 markedly in duces OC mRNA accumulation in MC3T3-E1 cells in She presence of forsko lin (FSK). Similarly, OC promoter activity (luciferase reporter) is up -regulated 6-10-fold by FGF2/FSK or by FGF2/8-bromo cyclic AMP. Half-m aximal induction of OC promoter activity occurs at 1 nM FGF2, By 5' de letion analysis and dinucleotide point mutations, we map one component of this FGF2/FSK response to a GCAGTCA motif in the region -144 to -1 38 relative to the OC transcription initiation site. The OC promoter r egion -154 to -90 confers FGF2/FSK responsiveness on the Rous sarcoma virus minimal promoter. By 3' and internal deletion analyses, the regi on between -90 to -99 is also found to be necessary for FGF2/FSK syner gy (encodes a PuGGTCA motif previously identified as a component of FS K induction). A DNA binding activity that recognizes the region -148 t o -125 of the rat OC promoter is induced in crude nuclear extracts fro m MC3T3-E1 cells treated with FGF2 or FGF2/FSK. This binding activity is sequence-specific and does not recognize the TCAGTCA DNA cognate of AP1. Members of the ATF, Fos, and Jun family are not immunologically detected in this inducible DNA binding activity. However, transient co -expression of ATF3 but not ATF2 selectively attenuates the FGF2 compo nent of induction. Thus, a novel FGF2-regulated DNA-protein interactio n in the OC promoter participates in the transcriptional control of OC expression by FGF and cyclic AMP in MC3T3-E1 calvarial osteoblasts.