Glucocorticoids antagonize RUNX2 during osteoblast differentiation in cultures of ST2 pluripotent mesenchymal cells

Abstract

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.

Keywords: GLUCOCORTICOID-INDUCED OSTEOPOROSIS; OSTEOBLAST DIFFERENTIATION; TRANSCRIPTION.

Fig. 1

Dex inhibits RUNX2-driven formation of ALP-positive osteoblast-like nodules. A: Representative images from Day-2 (top) and Day-5 (bottom) ST2/Rx2^dox cultures treated with dox and/or dex as indicated and stained for ALP activity. B: ALP-positive nodules in ST2/Rx2^dox cultures treated as in A were enumerated using the ImageJ64 software. Data are Mean±SD (n = 3) after adjustment to the number of ALP-positive nodules in the dex-free cultures, defined as 100%. Continuous and dashed lines represent Days 2 and 5, respectively and thin lines represent the number of ALP-positive nodules that formed in the absence of dox or dex. C: ST2/Rx2^dox cells were plated and treated as in A and their proliferation was assessed by performing MTT assays on Days 0, 2, and 5 (Mean±SD; n = 3). The error bars in C are smaller than the symbols.

Fig. 2

GR physically interacts with and inhibits RUNX2. A: ST2/Rx2^dox cells were transiently transfected with the RUNX2 reporter p6XOSE2-luc and luciferase activity was measured after 48 h of treatment with dox (filled circles) or vehicle (open circles) along with dex at the indicated concentrations (mean±SD, n = 3). B: ST2/Rx2^dox cells were treated for 48 h with dox (filled circles) or vehicle (open circles) along with dex at the indicated concentrations, and RUNX2 mRNA levels were measured by RT-qPCR (mean±SD, n = 3). C: ST2/Rx2^dox cells were treated with dox in the presence of dex or vehicle. RUNX2 complexes were immunoprecipitated using FLAG antibodies, or non-specific IgG antibodies were used as control. Presence of GR and RUNX2 in the precipitates or in 15% of the input (in) was assessed by Western analysis with anti-GR and anti-FLAG antibodies, respectively.

Fig. 3

RUNX2 interacts with GR in living cells. ST2/Rx2^dox cells grown on cover slips were treated with dox and/or 1 μM dex as indicated and subjected to confocal microscopy to image FLAG-RUNX2 (red), GR (green) and collocalization of both (yellow). Cells were stained with DAPI to visualize their nuclei.

Fig. 4

Dex inhibits RUNX2-stimulated genes to various degrees. ST2/Rx2^dox cells were treated for 48 h with dox and/or dex as depicted, and the mRNA levels of the indicated genes were measured by RT-qPCR (mean±SD, n = 3).