Calcineurin regulates phosphorylation status of transcription factor osterix

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Abstract

Osterix is an osteoblast-specific transcriptional factor that is essential for osteoblast differentiation and bone formation. Calcineurin regulates bone formation through modulating osteoblast differentiation. However, post-translational modification of osterix such as phosphorylation and interactions between osterix and calcineurin remains unclear. In the present study, we demonstrated that calcineurin interacted with osterix determined by immunoprecipitation assay and Western analysis. Immunocytochemical study also revealed that osterix and calcineurin were co-localized in nucleus. Deletion of calcineurin binding motif on osterix molecule disrupted osterix–calcineurin interaction. Phosphorylation status of osterix was augmented by treatment with phosphatase inhibitors, FK506 and calyculin A. In contrast, treatment of recombinant calcineurin reduced phosphorylation status of osterix. Our present study suggests that calcineurin has an important role in the function of osterix through its modification of phosphorylation.

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2. Materials and methods

Plasmid construction and stable cell lines. Mouse osterix full length cDNA was cloned from total cellular RNA of mouse osteoblastic MC3T3-E1 cells by RT-PCR as previously reported [12]. Osterix cDNA was subcloned into pcDNA3.1-Flag (modified pcDNA3.1, Invitrogen, Carlsbad, CA, USA) and pEGFP-C3 (BD Biosciences, Palo Alto, CA, USA) expression vectors, and confirmed by sequencing. To generate Flag-OsxΔVP (carrying a deletion of valine and proline at position 109 and 110, respectively), we carried

Calcineurin associates with osterix

To investigate whether calcineurin could interact with osterix, proteins prepared from HEK-Flag-Osx and HEK-Flag cells were immunoprecipitated with Flag affinity gel, and subjected to Western blot analysis. Anti-calcineurin antibody interacted with Flag-osterix complex eluted from Flag affinity gel (Fig. 1A, upper panel, Flag-Osx). The molecular weight of a positive band was estimated as 61 kDa, corresponding to that of the calcineurin. Anti-calcineurin antibody also recognized the protein in

4. Discussion

To identify a new factor that could participate in osterix regulation, we used the cell lysates from HEK293 cells transfected with Flag or Flag-osterix plasmid and performed immunoprecipitation assay. We demonstrated that calcineurin associates with osterix in a direct manner, as determined by immunoprecipitation and Western analysis. The binding of calcineurin and osterix was further supported by their localization in nucleoplasm of HEK293 cells, determined by immunocytochemical study. Osterix

Acknowledgments

We thank Mrs. E. Sasaki for her skilful technical assistance. This study was supported in part by grants from the Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan.

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