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We recently identified a bipartite element in the rat osteocalcin (OC) promoter that confers synergistic induction by fibroblast growth factor receptor 2 (FGF2) and cAMP. A GCAGTCA motif (OCFRE) at -146 to -138 in the OC promoter is necessary for synergy and participates in a FGF2-regulated DNA-protein interaction. We have isolated the FGF-regulated component of this transcriptional response for detailed study. Two or three copies of the OC promoter fragment -154 to -113 with the intact OCFRE confer 10- or 30-fold FGF2-inductive responses, respectively, on the unresponsive basal promoter 92 OCLUC (luciferase reporter) in MC3T3-E1 cells; a single copy is insufficient. As in the native context, induction depends upon an intact OCFRE motif (mutant GCATTTA motifs unresponsive). FGF receptor 1 can mediate activation; expression of this receptor in L6 cells (no endogenous FGF receptors) permits FGF2 induction of (OCFRE)2 92 OCLUC. FGF2 induction of (OCFRE)2 92 OCLUC in MC3T3-E1 cells is not recapitulated by platelet-derived growth factor-BB, epidermal growth factor, insulin-like growth factor I, or transforming growth factor-beta (&lt; 10% the activity of FGF2). OCFRE activation is not inhibited by kinase inhibitors H-89, wortmannin, staurosporine, KN-62, or H-7. However, the phosphoprotein phosphatase inhibitors okadaic acid (OKA), calyculin A, and vanadate decrease FGF induction of (OCFRE)2 92 OCLUC or (OCFRE)3 92 OCLUC, without inhibiting induction of the interstitial collagenase promoter. OKA and calyculin A do not decrease OCFRE DNA-protein interactions, suggesting that important protein-protein interactions are phosphatase regulated. These data provide evidence that; 1) FGF receptors elaborate transcriptional activation signals that functionally differ from those of other receptor tyrosine kinases; 2) an OKA-sensitive phosphatase participates in FGF receptor-dependent activation of the OCFRE; and 3) two transcriptional activation signals are initiated by FGF receptor activation in MC3T3-E1 cells, reflected in the divergent sensitivities of OCFRE and interstitial collagenase promoter induction to OKA and vanadate.

The rat osteocalcin fibroblast growth factor (FGF)-responsive element: an okadaic acid-sensitive, FGF-selective transcriptional response motif.