THE RAT OSTEOCALCIN FIBROBLAST GROWTH-FACTOR (FGF)-RESPONSIVE ELEMENT- AN OKADAIC ACID-SENSITIVE, FGF-SELECTIVE TRANSCRIPTIONAL RESPONSE MOTIF

We recently identified a bipartite element in the rat osteocalcin (OC) promoter that confers synergistic induction by fibroblast growth fact or 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 FGF 2-regulated DNA-protein interaction. We have isolated the FGF-regulate d component of this transcriptional response for detailed study. Two o r three copies of the OC promoter fragment -154 to -113 with the intac t OCFRE confer 10- or 30-fold FGF2-inductive responses, respectively, on the unresponsive basal promoter 92 OCLUC (luciferase reporter) in M C3T3-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 th is receptor in L6 cells (no endogenous FGF receptors) permits FGFP ind uction of (OCFRE)(2) 92 OCLUC. FGF2 induction of (OCFRE), 92 OCLUC in MC3T3-E1 cells is not recapitulated by platelet-derived growth factor- BE, epidermal growth factor, insulin-like growth factor I, or transfor ming growth factor-beta (<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 oka daic acid (OKA), calyculin A, and vanadate decrease FGF induction of ( OCFRE)(2) 92 OCLUC or (OCFRE)(3) 92 OCLUC, without inhibiting inductio n of the interstitial collagenase promoter. OKA and calyculin A do not decrease OCFRE DNA-protein interactions, suggesting that important pr otein-protein interactions are phosphatase regulated. These data provi de evidence that: 1) FGF receptors elaborate transcriptional activatio n signals that functionally differ from those of other receptor tyrosi ne kinases; 2) an OKA-sensitive phosphatase participates in FGF recept or-dependent activation of the OCFRE; and 3) two transcriptional activ ation signals are initiated by FGF receptor activation in MC3T3-E1 cel ls, reflected in the divergent sensitivities of OCFRE and interstitial collagenase promoter induction to OKA and vanadate.