Signaling via fibroblast growth factor receptors(FGFRs) -1 and -2 coordinates connective tissue remodeling during wound repair and contributes to the responses of osteoblasts to metastatic bone cancers. FGFs active at least 2 of 5 known mitogen activated protein kinase (MAPK) cascades; increase phospholipid turnover; and elevate intracellular calcium. The signal transduction pathways and protein - DNA interactions that confer transcriptional regulation in response to FGFR activation are virtually uncharacterized. Characterizing FGFR - dependent transcription in osteoblasts will provide insights useful for designing novel therapeutic strategies for treating metabolic and metastatic bone disease. We propose to identify protein-DNA interactions and kinase cascades conferring FGFR1/FGFR2-regulated gene expression in osteoblasts, using osteocalcin (OC) and interstitial collagenase (MMP) FGF response elements (FREs) as models for study. We mapped these FREs by assessing transcriptional activity of of promoter fragments (luciferase reporter) in MC3T3-E1 calvarial osteoblasts and in primary calvarial osteoblast cultures. The FREs in the OC and MMP1 promoters confer transcriptional upregulation in response to ligand - activated FGFR1 and a constituitively-active FGFR2 oncoprotein. Activation of both elements is inhibited by expression of dominant negative Ras or MAPK ohosphatase -2, yet is unaffected by inhibition of the classic p44/p42 MAPK cacade. The OCFRE binding protein complex ( OC-FREB ) has been purified to apparent homegeneity from an osteoblastic cell line, and contains approximately 80 kDa and approximately 70 kDa proteins as constituents. The MMPFRE maps to DNA- protein interactions at Ets and AP1 DNA cognates. FGFR activation of the human MMP1 promoter is mediated by (i) and FGF-regulated, Fra1 - containing AP1 complex present in osteoblasts; and (ii) de-repression and activation of the human MMP1 promoter at the Ets DNA cognate. We ve identified that PE1 is an Ets repressor expressed in MC3T3-E1 calvarial osteoblasts that regulates the interstitial collagenase promoter activity.
Specific aims are:
Aim 1 : To identify, characterize, and clone OC-FREB in order to study its role and regulation with respect to FGFR1/FGFR2 signaling in osteoblasts.
Aim 2 : To determine the mechanisms whereby Fra1 expression, phosphorylation, and protein-protein interactions regulate interstitial collagenase induction by FGF.
Aim 3 : To define the role, regulation, and signaling and interstitial collagenase gene expression during murine skeletal development.
