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. Author manuscript; available in PMC: 2014 Feb 23.
Published in final edited form as: Arthritis Rheumatol. 2014 Jan;66(1):107–120. doi: 10.1002/art.38195

Figure 4.

Figure 4

The β-catenin signaling pathway down-regulates Rankl expression in a GR-dependent manner. A–D, ATDC5 cells were cultured with Wnt-3a (50 μg/ml), LiCl (10 mM), or vehicle (A and B), transfected with β-catenin or control small interfering RNA (siRNA) (C), or treated with dexamethasone (Dex) (10−7M) or vehicle (D) for 12, 24, and 48 hours. The fold change in Rankl mRNA expression (A and C), Opg mRNA expression (B), and Rankl promoter activity (D) was significantly different between the groups after 24 or 48 hours of culture. E, Treatment of ATCD5 cells with dexamethasone enhanced Rankl mRNA expression at all time points. F, Transfection of ATDC5 cells with a mutant Rankl promoter construct (mutation of the GR binding site in the Rankl promoter) inhibited basal and dexamethasone-induced Rankl promoter activity, as compared to transfection with a wild-type (WT) reporter construct. G and H, Wnt-3a and LiCl inhibited GR mRNA expression in WT reporter–transfected cells (G) and inhibited Rankl promoter activity in WT reporter–transfected cells, but not in mutant (Mut) reporter–transfected cells (H). I and J, Transfection of cells with a Rankl-luc reporter construct and β-catenin siRNA up-regulated Rankl promoter activity (I), whereas treatment of these cells with Wnt-3a inhibited Rankl promoter activity (J). No effects were observed on empty vector–transfected cells. K, Interaction of β-catenin with GR was detected by chromatin immunoprecipitation (ChIP) assay. L, ChIP assay was performed using sonicated chromatin extracted from ATDC5 cells treated with either anti-GR antibody or normal rabbit IgG. Purified DNA was analyzed by standard polymerase chain reaction, using mouse Rankl–specific primer set 1 (−682/−562), which amplifies the fragment spanning the GR binding sequence (−647/−633). Primer set 2 was used as a negative control, amplifying a fragment downstream of the transcription start site (+2463/+2592). GR specifically binds to the proximal promoter region of the Rankl promoter, and treatment of cells with Wnt-3a inhibited GR binding to the Rankl promoter. Results are the mean ± SD of 3 mice per group. * = P < 0.05 versus controls, by Student’s unpaired t-test. WB = Western blotting.