Fibroblast growth factor 2 and cyclic AMP synergistically regulate bone sialoprotein gene expression

Bone sialoprotein (BSP) is a noncollagenous protein of the mineralized bone extracellular matrix. We here report that FGF2 and cAMP act synergistically to stimulate BSP gene expression. Treatment of ROS 17/2.8 cells with either 10 ng/ml FGF2 or 1 μM FSK for 6 h resulted in 5.4- and 8.2-fold increases, respectively, in the levels of BSP mRNA. However, in the presence of both FGF2 and forskolin (FGF/FSK), BSP mRNA levels were increased synergistically by 20.4-fold. Using a luciferase reporter construct, encompassing BSP promoter nucleotides −116 to +60, transcription was also increased synergistically by 15.0-fold with FGF/FSK, compared to stimulations of 2.6- and 5.3-fold, respectively, for FGF2 and FSK alone. Transcriptional stimulation by FGF/FSK abrogated in constructs included 2 bp mutations in the inverted CCAAT, CRE, FRE and Pit-1 elements. Whereas the FRE–protein complex was increased by FGF2 and FGF/FSK, the Pit-1–protein complex was decreased by FSK and FGF/FSK. Notably, transcriptional activity induced by FGF/FSK was blocked by protein kinase A, tyrosine kinase and MEK inhibitors. These studies indicate that the combinatorial effects of FGF and FSK act through PKA, tyrosine kinase and MAP-kinase-dependent pathways, which target the inverted CCAAT, CRE, FRE and Pit-1 elements in the BSP gene to synergistically increase BSP expression.