Rosiglitazone suppresses RANKL-induced NFATc1 autoamplification by disrupting the physical interaction between NFATc1 and PPARγ

Abstract

Receptor activator of nuclear factor-κB ligand (RANKL) is required for initiation of osteoclastogenesis, with the signaling pathway including the NF-kB, c-Fos, and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) transcription factors. Because NFATc1 expression is autoamplified, we investigated the molecular mechanism by which peroxisome proliferator-activated receptor gamma (PPARγ) activation by the thiazolidinedione drug rosiglitazone decreases NFATc1 expression during RANKL stimulation. Western blotting demonstrated that rosiglitazone attenuated the increase in NFATc1 protein level induced by RANKL without affecting that of PPARγ. Immunofluorescence data indicated that rosiglitazone tended to suppress RANKL-induced NFATc1 nuclear translocation, partly by reducing calcineurin activity, as reflected by the observed decrease in nuclear NFATc1 abundance. On coimmunoprecipitation, the intensity of the physical interaction between NFATc1 and PPARγ was unexpectedly higher in the RANKL-stimulated group than in the control, but rosiglitazone reduced this to basal levels. Furthermore, RANKL failed to elevate mRNA expression of NFATc1 after PPARγ knockdown. ChIP assay indicated that rosiglitazone significantly reduced the binding of NFATc1 to its own promoter despite RANKL stimulation. These findings suggest that PPARγ activation by rosiglitazone blocks NFATc1 from binding to its own promoter, thereby reducing RANKL-induced NFATc1 autoamplification.

Keywords: NFATc1; PPARγ; osteoclastogenesis; physical interaction; rosiglitazone.

Figure 1

Rosiglitazone decreases RANKL‐induced osteoclastogenesis and NFATc1 expression. Cells were cultured in 96‐well plates and treated with vehicle or different doses of rosiglitazone for 5 days in the presence of RANKL (50 μg·mL⁻¹). (A) A representative image of TRAP‐stained osteoclast differentiation is shown at × 100 magnification. Scale bar, 100 μm. (B) The number of TRAP‐positive cells with more than three nuclei was counted. Data were drawn from three wells of a single experiment. *P < 0.05 vs. control. Rosi, rosiglitazone. (C) Cytotoxicity of rosiglitazone was determined using MTT assay. In this experiment, cells were treated with different doses of rosiglitazone for 24 h and 5 days in the absence of RANKL. (D) Cells were treated with vehicle or 5 μm of rosiglitazone for 24 h in the presence or absence of RANKL. Cell lysates were prepared, and detection of NFATc1 and PPARγ was determined by western blotting. Shown is a representative immunoblot from three experiments. Densitometric analyses of the level of RANKL (E) and PPARγ (F) were shown relative to the control (n = 3). *P < 0.05 vs. control. ^# P < 0.05 vs. RANKL. Rosi, rosiglitazone.

Figure 2

Rosiglitazone blocks NFATc1 nuclear import induced by RANKL. Cells were cultured for 24 h with the addition of 10 μm rosiglitazone in the presence of RANKL (50 μg·mL⁻¹). (A) Ca⁺⁺/calmodulin‐dependent calcineurin activity of cell lysates was measured in duplicate from three experiments. *P < 0.05 vs. control. ^# P < 0.05 vs. RANKL. (B) Protein levels of calcineurin isoforms in cell lysates were shown using anti‐calcineurin Aα (catalytic subunit A isoform α) and anti‐calcineurin B2 (regulatory subunit B type 2). (C) Immunofluorescence image for NFATc1 localization obtained using anti‐NFATc1 (green). Arrows indicate NFATc1 nuclear translocation. Scale bar, 20 μm. (D) Cytosolic and nuclear fractions were prepared for localization of NFATc1 and PPARγ. Equal amounts (10 μg) were loaded in each fraction. The image is representative of three experiments. (E) Based on (D), relative NFATc1 levels in the nuclear fraction were calculated through densitometric analyses (n = 3). *P < 0.05 vs. control.

Figure 3

Rosiglitazone disrupts the physical interaction between PPARγ and NFATc1. After cells were cultured in the same fashion as described in Fig. 2, whole cell lysates were prepared and (A) immunoprecipitated with polyclonal anti‐PPARγ antibody and the NFATc1‐PPARγ complex was detected with monoclonal anti‐NFATc1 antibody or (B) immunoprecipitated with monoclonal anti‐NFATc1 antibody and the NFATc1‐PPARγ complex was detected with polyclonal anti‐PPARγ antibody. Two replicates are shown, but input control was not loaded in the second replicate of either (A) or (B).

Figure 4

PPARγ knockdown inhibits mRNA expression of RANKL‐induced NFATc1 and inhibits RANKL‐induced NFATc1 binding to its own promoter. Cells were transfected with control siRNA or PPARγ siRNA, followed by incubation with rosiglitazone (10 μm) for 24 h in the presence or absence of RANKL. (A) PPARγ knockdown was confirmed by western blotting. (B) End‐point RT‐PCR was carried out under control siRNA and PPARγ siRNA to evaluate mRNA expression of the NFATc1‐dependent genes NFATc1 and TRAP and the NFATc1‐independent genes RANK and β‐actin. The image is representative of two experiments. (C) After in situ ChIP assay, quantitative PCR was performed and levels of NFATc1 mRNA bound to the promoter were calculated. ChIP data are pooled from six measurements of two experiments. Statistical analysis was performed via two‐way ANOVA. *P < 0.05 vs. control siRNA. **P < 0.05 vs. RANKL. ^# P < 0.05 vs. matched control siRNA.