Osteoblast precursors must secrete an extracellular matrix before they will differentiate, thus explaining the requirement for ascorbic acid in bone formation. The osteoblast: ECM interaction conveys fundamental information to bone cells allowing them to sense and respond to their extracellular microenvironment. Our long-term goal is to understand how ECM signals control osteoblast-specific gene expression. Major advances during the previous support period led to the development of the following three hypotheses which will be addressed in this project: 1. Osteoblast-specific gene expression requires integrin activation of the MAPK pathway and subsequent phosphorylation of specific amino acid residues of Cbfal, an osteoblast-specific transcription factor. These phosphorylation sites are essential for ECM responsiveness. 2. ECM/MAPK-dependent activation of Cbfal affects its ability to interact with accessory nuclear factors and activate transcription. 3. ECM: integrin signaling and BMP signals mediated by Smad proteins synergistically interact to activate bone specific gene expression. These hypotheses will be addressed through achievement of the following specific aims: 1. To identify specific amino acid residues in Cbfal that are phosphorylated by MAPK and in intact cells and assess the significance of these phosphorylation sites in the responsiveness of Cbfal to MAPK and matrix signals in cultured cells and in vivo. 2. To define the involvement of the MAPK pathway in ECM-dependent activation of Cbfal. 3. To identify critical interactions between Cbfal and accessory nuclear proteins and relate ECM/MAPK-dependent activation of transcription to changes in accessory factor binding and intranuclear Cbfal localization. 4. To determine the basis for the synergy between the ECM and bone morphogenetic proteins (BMP5) in stimulating osteoblast differentiation. These studies will greatly advance our understanding of how genes are regulated in bone. They will elucidate a novel mechanism to explain how ECM signals control the activity of a critical osteoblast transcription factor, and show how matrix signals interact with signals generated by BMPs to regulate bone formation.
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