Runx2-mediated activation of the Bax gene increases osteosarcoma cell sensitivity to apoptosis.

The Runx family of transcription factors regulate cell growth and differentiation, and control the expression of target genes involved in cell fate decisions. We examined the role of the bone-related member of this family, Runx2, in regulating apoptosis via modulation of the Bcl2 family of genes in the osteosarcoma cell line Saos2. Our data demonstrate that Runx2 directly binds to two Runx-specific regulatory elements on the human bax promoter thereby inducing Bax expression. Furthermore, bone morphogenetic protein-induced or vector-mediated expression of Runx2 resulted in upregulation of Bax expression, and subsequent increased sensitivity of Saos2 cells to apoptosis. Finally, the observed upregulation of Bax expression and increased apoptosis were Runx2 dependent as Runx2 loss of function abrogated these effects. Our study provides the first evidence for Bax as a direct target of Runx2, suggesting that Runx2 may act as a proapoptotic factor in osteosarcoma cells.

A rapid multiparameter approach to study factors that regulate osteoclastogenesis: demonstration of the combinatorial dominant effects of TNF-alpha and TGF-ss in RANKL-mediated osteoclastogenesis.

Macrophages differentiate into osteoclasts in response to the critical cytokine RANKL. However, the efficiency of RANKL-mediated osteoclastogenesis can be profoundly influenced by various cytokines. While studies describing the isolated effects of particular cytokines on osteoclastogenesis have been performed, combinatorial effects of cytokines have not been addressed routinely due to the absence of an efficient assay system. To study the effects of cytokine combinations on osteoclast formation, we performed in vitro assays using either the RAW293 cell line or primary murine splenic macrophages as osteoclast precursors. Using a multiparameter cytokine plating method, we analyzed osteoclastogenesis in response to multiple combinations of the following inflammation-related cytokines: RANKL, IFN-gamma, TNF-alpha, IL-1beta, IL-6, IL-10. We further investigated the role of T-cell-related cytokine combinations on osteoclastogenesis by measuring osteoclast area in response to RANKL with IFN-gamma, IL-2, IL-4, IL-6, TGF-ss, and TNF-alpha. Treatments with RANKL, TNF-alpha, and TGF-ss induced maximal osteoclast formation, suggesting a role for these cytokines in the most aggressive forms of inflammatory bone loss. TNF-alpha alone, however, was unable to induce osteoclast formation in the absence of RANKL despite co-administration of other proinflammatory cytokines. IFN-gamma was a potent inhibitor under all conditions, implicating T cells and NK cells in osteoclast inhibition. These studies demonstrate a rapid screening approach for identifying the potential collective effects of multiple factors on osteoclastic bone resorption.