In addition to its thoroughly investigated role in bone formation, the osteoblast master transcription factor RUNX2 also promotes osteoclastogenesis and bone resorption. Here we demonstrate that 17β-estradiol (E2), which is known to attenuate bone turnover in vivo and RUNX2 activity in vitro, strongly inhibits RUNX2-mediated osteoblast-driven osteoclastogenesis in co-cultures. Towards deciphering the underlying mechanism, we induced premature expression of RUNX2 in primary murine pre-osteoblasts, which resulted in robust differentiation of co-cultured splenocytes into mature osteoclasts. This was attributable to RUNX2-mediated increase in RANKL secretion, determined by ELISA, as well as to RUNX2-mediated increase in RANKL association with the osteoblast membrane, demonstrated using confocal fluorescence microscopy. The increased association with the osteoblast membrane was recapitulated by transiently expressed GFP-RANKL. E2 abolished the RUNX2-mediated increase in membrane-associated RANKL and GFP-RANKL, as well as the concomitant osteoclastogenesis. RUNX2-mediated RANKL cellular redistribution was attributable in part to a decrease in Opg expression, but E2 did not influence Opg expression either in the presence or absence of RUNX2. Diminution of RUNX2-mediated osteoclastogenesis by E2 occurred regardless of whether the pre-osteoclasts were derived from wild type or estrogen receptor alpha (ERα)-knockout mice, suggesting that activated ERα inhibited osteoblast-driven osteoclastogenesis by acting in osteoblasts, possibly targeting RUNX2. Furthermore, the selective ER modulators (SERMs) tamoxifen and raloxifene mimicked E2 in abrogating the stimulatory effect of osteoblastic RUNX2 on osteoclast differentiation in the co-culture assay. Thus, E2 antagonizes RUNX2-mediated RANKL trafficking and subsequent osteoclastogenesis. Targeting RUNX2 and/or downstream mechanisms that regulate RANKL trafficking may lead to the development of improved SERMs and possibly non-hormonal therapeutic approaches to high turnover bone disease.

The efficacy of glucocorticoids (GCs) in treating a wide range of autoimmune and inflammatory conditions is blemished by severe side effects, including osteoporosis. The chief mechanism leading to GC-induced osteoporosis is inhibition of bone formation, but the role of RUNX2, a master regulator of osteoblast differentiation and bone formation, has not been well studied. We assessed effects of the synthetic GC dexamethasone (dex) on transcription of RUNX2-stimulated genes during the differentiation of mesenchymal pluripotent cells into osteoblasts. Dex inhibited a RUNX2 reporter gene and attenuated locus-dependently RUNX2-driven expression of several endogenous target genes. The anti-RUNX2 activity of dex was not attributable to decreased RUNX2 expression, but rather to physical interaction between RUNX2 and the GC receptor (GR), demonstrated by co-immunoprecipitation assays and co-immunofluorescence imaging. Investigation of the RUNX2/GR interaction may lead to the development of bone-sparing GC treatment modalities for the management of autoimmune and inflammatory diseases.