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. 2023 Aug 29;26(10):107760.
doi: 10.1016/j.isci.2023.107760. eCollection 2023 Oct 20.

CGK733 alleviates ovariectomy-induced bone loss through blocking RANKL-mediated Ca2+ oscillations and NF-κB/MAPK signaling pathways

Minglian Xu  1   2 , Dezhi Song  1   2 , Xiaoxiao Xie  1   2 , Yiwu Qin  1   2 , Jian Huang  1   2 , Chaofeng Wang  1   2 , Junchun Chen  1   2 , Yuangang Su  1   2 , Jiake Xu  3   4 , Jinmin Zhao  1   2 , Qian Liu  1   2
Affiliations

CGK733 alleviates ovariectomy-induced bone loss through blocking RANKL-mediated Ca2+ oscillations and NF-κB/MAPK signaling pathways

Minglian Xu et al. iScience. .

Abstract

Osteoporosis is a prevalent systemic metabolic disease in modern society, in which patients often suffer from bone loss due to over-activation of osteoclasts. Currently, amelioration of bone loss through modulation of osteoclast activity is a major therapeutic strategy. Ataxia telangiectasia mutated (ATM) inhibitor CGK733 (CG) was reported to have a sensitizing impact in treating malignancies. However, its effect on osteoporosis remains unclear. In this study, we investigated the effects of CG on osteoclast differentiation and function, as well as the therapeutic effects of CG on osteoporosis. Our study found that CG inhibits osteoclast differentiation and function. We further found that CG inhibits the activation of NFATc1 and ultimately osteoclast formation by inhibiting RANKL-mediated Ca2+ oscillation and the NF-κB/MAPK signaling pathway. Next, we constructed an ovariectomized mouse model and demonstrated that CG improved bone loss in ovariectomized mice. Therefore, CG may be a potential drug for the prevention and treatment of osteoporosis.

Keywords: Biological sciences; Molecular biology; Molecular physiology.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

None
Graphical abstract
Figure 1
Figure 1
CG suppresses RANKL-induced osteoclastogenesis in vitro (A) The chemical structure of CGK733 (CG). (B) The cell viability of BMMs treated with CG was detected by CCK-8 assay (n = 3). (C) Flow cytometry was applied to examine the apoptosis of BMMs treated with 3 μM and 6 μM CG for 48 h. (D) TRAP staining images of BMMs cultured with or without RANKL in the presence of indicated concentrations of CG from 0 to 6 μM (scale bar = 400 μm). (E) TRAP+ osteoclasts were quantified in each well (nuclei ≥3). (F) Stage representative images of TRAP+ osteoclasts treated with CG (scale bar = 400 μm). (G) Quantitative analysis of TRAP+ osteoclasts (nuclei ≥3). (H) Representative images of podosome belts in the presence or absence of CG (scale bar = 1000 μm). (I) The podosome belt area was quantified by ImageJ software. All data are presented as mean ± SD. Statistical significance was assessed via one-way ANOVA method. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 2
Figure 2
CG inhibits the expression of osteoclast-specific genes and RANKL-induced NFATc1 (A) qPCR was used to detect the expression levels of Nfatc1, c-Fos, Acp5, Dc-stamp, Mmp9 and Ctsk relative to β-actin in BMMs stimulated with RANKL for 5 days in the presence of CG (n = 3). All data are presented as mean ± SD. Statistical significance was assessed via one-way ANOVA method. (B) Representative Western blot images of the expression levels of NFATc1, CTSK, ATP6V0D2 and c-Fos. BMMs were cultured with or without CG treatment in the presence of RANKL for 1, 3, and 5 days, and the expression of related proteins was detected by western blot. (C–F) Quantification of the ratios of band intensity of NFATc1, CTSK, ATP6V0D2, and c-Fos relative to β-actin (n = 3). All data are presented as mean ± SD. Statistical significance was assessed via two-way ANOVA method. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 3
Figure 3
CG blocks RANKL-induced activation of the NF-κB/MAPK signaling pathway (A) The representative immunofluorescence images of p65 nuclear translocation following RANKL stimulation for 30 min without or with CG (6 μM). (scale bar = 100 μm, the inset enlarged images scale bar = 25 μm). (B and C) Quantification of nuclear translocation of P65 and nuclear/cytoplasmic Fluorescence ratio (n = 3). All data are presented as mean ± SD. Statistical significance was assessed via one-way ANOVA method. (D) Representative western blot images of the effects of CG on P65 phosphorylation and IκBα degradation induced by RANKL. BMMs starved for 3 h with or without CG pretreatment were stimulated with RANKL (50 ng/mL) at series time points (0, 5, 10, 20, 30, 60 min). The expression of total and phosphorylated proteins was detected by western blot. (E and F) Quantification of the ratios of band intensity of p-P65 relative to P65 and IκBα relative to β-actin. (n = 3). (G) Representative Western blot images of the effects of CG on MAPK pathway. The effect of CG on RANKL-induced activation of P38, ERK, and JNK was detected by Western blot. (H–J) The ratios of phosphorylated and non-phosphorylated forms of P38, ERK and JNK were quantified (n = 3). All data are presented as mean ± SD. Statistical significance was assessed via two-way ANOVA method. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 4
Figure 4
CG attenuates Ca2+ oscillations and bone resorption activity of osteoclasts in vitro (A) Representative images of bone resorption areas formed by osteoclasts that were treated with CG were obtained by scanning electron microscopy (scale bar = 200 μm) and representative images of TRAP-stained cells (scale bar = 2000 μm, the inset enlarged images scale bar = 400 μm). (B and C) The area of bone resorption pits and TRAP-positive cells per well was quantified using ImageJ software (n = 3). (D) Line chart of Ca2+ oscillatory fluorescence intensity from negative control, positive control, and CG (6 μM) treated groups. (E) Quantitative analysis of changes in Ca2+ oscillation intensity in each group (n = 10 cells per group). The values of intensity change were calculated by the maximum peak intensity minus the minimum intensity and then comparing it to the average value. All data are presented as mean ± SD. Statistical significance was assessed via one-way ANOVA method. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 5
Figure 5
CG treatment prevents bone loss induced by ovariectomized in vivo (A) Diagram of the process and grouping of animal experiments was created by https://www.biorender.com/. ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry. (B) Weight curve of mice following OVX surgery at various time periods (n = 6 mice per group). (C) CTX-1 levels in serum were measured using an ELISA kit (n = 6 mice per group). (D) Representative of Micro CT images showed that the bone loss was prevented by CG administration (n = 6 mice per group). (E–H) Quantitative analysis of parameters relating to bone microstructure, including volume/tissue volume (BV/TV), the trabecular number (Tb. N), the trabecular separation (Tb. Sp), and the trabecular thickness (Tb. Th) (n = 6 mice per group). All data are presented as mean ± SD. Statistical significance was assessed via one-way ANOVA method. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 6
Figure 6
CG ameliorates osteoclast activity in a mouse model of osteoporosis induced by ovariectomized (A) Representative images of histological analysis of the left femur stained with H&E. (B) Representative images of histological analysis of the left femur stained with TRAP. (C) CTSK-positive cells were marked as brownish-yellow (scale bar = 300 μm, the inset enlarged images scale bar = 120 μm). (D) BV/TV was quantitatively analyzed in tissue sections (n = 6 mice per group). (E) TRAP-positive osteoclast surface per trabecular surface (OC. S/BS) was quantitatively analyzed (n = 6 mice per group). (F) The level of CTSK was quantitatively measured in the left femur (n = 6 mice per group). All data are presented as mean ± SD. Statistical significance was assessed via one-way ANOVA method. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 7
Figure 7
A proposed scheme depicting the inhibition of CG on RANKL-induced NFATc1 activation during osteoclast differentiation Our findings demonstrate that CG suppresses NFATc1 activation and downregulates osteoclast-specific genes such as Mmp9, Dc-stamp, Ctsk, and Acp5 via inhibiting the calcium signaling pathway and the RANKL-induced NF-B/MAPK signaling pathway. NFATc1, nuclear factor of activated T cells 1; Mmp9, matrix metallopeptidase 9; Ctsk, cathepsin K; Dc-stamp, dendritic cell-specific transmembrane protein; Acp5, tartrate-resistant acid phosphatase; RANKL, receptor activator of nuclear factor-κB (NF-κB) ligand; NF-κB, nuclear factor-κB; MAPKs, mitogen-activated protein kinases.
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