Hand Proteins Control the Onset of Osteoblast Differentiation

Objectives: Members of the basic helix-loop-helix (bHLH) family of transcription factors regulate the specification and differentiation of numerous cell types during embryonic development. Here we report that bHLH Hand proteins, Hand2/dHand and Hand1/eHand, regulate osteoblast differentiation and membranous bone formation by interacting with and inhibiting the activity of Runx2, a transcription factor necessary for osteoblast differentiation. Methods: Hand2^BA/BA mice, which lack the Hand2 branchial arch enhancer, Hand1^NCKO mice, which lack Hand1 expression specifically in neural crest cells, Hand2^BA/BA;Hand1^NCKO mice, and transgenic mice (Hand2-LacZ) in which the LacZ gene is expressed under the control of a 11 kb mouse Hand2 promoter, were used for this study. The bone phenotype was analyzed from cartilage and bone staining. The expression of osteoblast marker genes and mineralization were compared in mutant and wild-type embryos by section in situ hybridization, whole mount in situ hybridization, alkaline phosphatase staining, and von Kossa staining. Function of Hand proteins was analyzed by western blotting, immunocytochemistry, promoter assays, immunoprecipitation, pull-down assay, and chromatin immunoprecipitation (ChIP) using COS and ROS17/2.8 cells. Results: Hand2^BA/BA, and Hand2^BA/BA;Hand1^NCKO mice display premature ossification of developing bones due to ectopic and accelerated osteoblast differentiation. Hand2 and Runx2 are co-expressed in osteoblasts of the first branchial arch. The regulatory function of osteoblast differentiation by Hand2 is mediated by its N-terminal domain, which interacts with the Runx2 DNA binding domain to inhibit its function. Inhibitory function by Hand2 requires neither dimerization with other bHLH proteins nor DNA binding, suggesting that Hand proteins act via novel transcriptional mechanisms mediated by protein-protein interactions. Conclusion: Hand proteins act as corepressors of Runx2 and regulators of osteoblast differentiation and skeletogenesis in branchial arch development. This research is supported by the JSPS Postdoctoral Fellowships for Research Abroad 2005 (to N.F.) and the March of Dimes Birth Defects Foundation (to H.Y.).