Runx-2 is not essential for the vitamin D-regulated expression of RANKL and osteoprotegerin in osteoblastic cells

doi:10.1016/j.bbrc.2004.09.101

Biochemical and Biophysical Research Communications

Volume 324, Issue 2, 12 November 2004, Pages 655-660

https://doi.org/10.1016/j.bbrc.2004.09.101 Get rights and content

Abstract

The differentiation and activity of osteoclasts are positively and negatively controlled by receptor activator of nuclear factor-κB ligand (RANKL), which is expressed on the surface of osteoblasts and stromal cells, and its decoy receptor osteoprotegerin (OPG), which is secreted by osteoblasts and stromal cells, respectively. The expression of the genes for RANKL and OPG is regulated by 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. Runt-related gene-2 (Runx-2) is essential for osteoblast differentiation and there are several reports that Runx-2 is involved in osteoclast formation. Therefore, to clarify the role of Runx-2 in osteoclastogenesis, we designed a series of experiments using C2 cells and C6 cells, which are derived from calvariae of runx2-deficient mice. Treatment of C2 cells and C6 cells with 1α,25(OH)₂D₃ for 2–4 days increased and decreased the levels of expression of the mRNAs for RANKL and OPG, respectively, and the effects were dose-dependent. However, by day 8, the level of RANKL mRNA had fallen and that of OPG mRNA had risen. Furthermore, C6 cells induced the differentiation of mouse spleen cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated cells (osteoclast-like cells) in the presence of 10⁻⁷ M 1α,25(OH)₂D₃. Such formation of osteoclast-like cells was inhibited by exogenous OPG in a dose-dependent manner. Thus, our findings indicate that Runx-2 is not essential for the expression of RANKL and OPG, and the formation of osteoclast-like cells.

Section snippets

Materials and methods

Materials. α-Modified minimum essential medium (α-MEM), fetal bovine serum, and penicillin/streptomycin antibiotic mixture were purchased from Life Technologies (Grand Island, NY, USA). 1α,25(OH)₂D₃ and ISOGEN were purchased from Wako Pure Chemicals (Osaka, Japan). Recombinant human OPG was obtained from PeproTech EC (London, UK).

Reverse transcriptase-polymerase chain reaction. We used reverse transcriptase-polymerase chain reaction (RT-PCR) to detect mRNAs for Runx-2, RANKL, and OPG in runx-2

Results

Both C2 cells and C6 cells were established from calvariae of runx-2-deficient mice. Although these cells exhibit very low level of alkaline phosphatase activity and no osteocalcin synthesis when they were cultured in the control medium, the treatment with BMP-2 increased alkaline phosphatase activity and induced osteocalcin synthesis in these cells, indicating that C2 and C6 cells are capable of differentiating into osteoblastic cells in the presence of BMP-2 (unpublished data). Analysis by

Discussion

The formation of osteoclasts from hematopoietic cells is regulated by various factors, such as RANKL, OPG, and M-CSF, that are produced by cells of the osteoblastic lineage. Recently, we demonstrated that premyoblastic C2C12 cells differentiate into osteoblast-like cells upon treatment with bone morphogenetic protein-2 (BMP-2) and synchronously induce the formation of osteoclasts from spleen cells in vitro [29]. These observations suggest that osteoblastic differentiation might be associated

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by grants from the Japan Science Society and the Smoking Research Foundation.

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