Octyl itaconate inhibits osteoclastogenesis by suppressing Hrd1 and activating Nrf2 signaling - PubMed (original) (raw)
Octyl itaconate inhibits osteoclastogenesis by suppressing Hrd1 and activating Nrf2 signaling
Xuewu Sun et al. FASEB J. 2019 Nov.
Abstract
The endogenous metabolite itaconate has emerged as a regulator of macrophage function that limits inflammation. However, its effect on cell differentiation and osteoclast-related diseases is unclear. Here, for the first time, we explored the effect of itaconate and its cell-permeable itaconate derivative, 4-octyl itaconate (OI) on osteoclast differentiation in vitro and in vivo. Firstly, we demonstrated that itaconate concentration was lower in estrogen-deficient mice. OI released itaconate and induced the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) in bone marrow-derived macrophages during osteoclastogenesis. Furthermore, OI significantly suppressed the early, middle, and late stages of osteoclastogenesis induced by receptor activator of NF-κB ligand in vitro, as confirmed by tartrate-resistant acid phosphatase staining. Moreover, it significantly inhibited fibrous actin ring formation and bone resorption in vitro. Mechanistically, we observed that OI enhanced Nrf2 expression by suppressing its association with ubiquitin via inhibition of the E3 ubiquitin ligase (Hrd1). OI also inhibited LPS-induced the reactive oxygen species and inflammatory responses via Hrd1. An estrogen deficiency (via ovariectomy)-induced osteoporosis model was also established. Here, on micro-computed tomography and histologic analysis showed that OI effectively suppressed ovariectomy-induced bone loss. In summary, OI, an itaconate derivative, can inhibit osteoclastogenesis in vitro and in vivo, indicating that OI could be a potential drug to treat osteoclast-related diseases; our results also link itaconate to the development of osteoporosis.-Sun, X., Zhang, B., Pan, X., Huang, H., Xie, Z., Ma, Y., Hu, B., Wang, J., Chen, Z., Shi, P. Octyl itaconate inhibits osteoclastogenesis by suppressing Hrd1 and activating Nrf2 signaling.
Keywords: metabolite; osteoclast; osteoporosis.
Conflict of interest statement
This work was funded by grants from the Nature Science Foundation of China (81874015; 81873985) and the National Natural Science Fund of Zhejiang Province (LY16H060004). The authors declare no conflicts of interest.
Figures
Figure 1
Itaconate concentrations are reduced in estrogen-deficient mice. A) A mouse model of estrogen deficiency and bone mass loss was first established. Micro-CT and quantitative analysis of BMD were carried out to explore bone loss associated with estrogen deficiency; n = 5. B) BMMs were isolated from the estrogen deficiency (OVX) and sham-surgery (sham) groups. The concentration of itaconate was analyzed by LC-MS assay. C) The concentration of itaconate was analyzed in BMMs during osteoclastogenesis by LC-MS assay. D) CCK-8 assays were performed to test the cytotoxicity of exogenous itaconate, OI, in BMMs. E) RANKL-induced osteoclastogenesis was carried out in BMMs stimulated with OI (40 μM). The concentration of itaconate was analyzed at the indicated time points. F) BMMs were stimulated with different concentrations of OI for 24 h. The expression of Nrf2 was analyzed by Western blotting. Quantitative analysis of Western blot results was also carried out. mBMM, mouse-derived bone marrow macrophage. All results in bar graphs are expressed as means ±
sem
. *P < 0.05, #P < 0.01.
Figure 2
OI inhibits osteoclastogenesis in vitro. A) TRAP staining was performed to detect RANKL-induced osteoclastogenesis. BMMs were cultured for 7 d to generate mature osteoclasts. d 1–7: OI was added for the entire period of osteoclastogenesis. d 3–7: OI was added to the medium from d 3 until d 7. d 5–7: OI was added to the medium from d 5 to 7. B) TRAP-positive cell numbers and TRAP-positive cell size relative to control (Ctrl) levels were quantitatively analyzed using ImageJ. C) The expression of osteoclast-related genes, including c-FOS, Acp5, Ctsk, and Dc-stamp, was examined by real-time PCR at the indicated time point. D) The expression of Nfatc1 was analyzed at different culture times by Western blotting. E) Actin ring formation assays were performed to determine the effect of OI on the generation of mature osteoclasts. BMMs were first cultured for 5 d to generate osteoclasts and then cultured with OI for another 48 h. The actin ring was detected using phalloidin under a fluorescence microscope after treatment with OI. F) The effect of OI on bone resorption was analyzed using a scanning electron microscope as described in Materials and Methods. BMMs were cultured for 7 d to generate mature osteoclasts, and OI was added to the medium on d 7 to test its effect on osteoclast function. The bone resorption area was analyzed using ImageJ. The concentration of OI was 10 μM unless otherwise noted. Scale bars, 200 μm. All results in bar graphs are expressed as means ±
sem
. *P < 0.05, #P < 0.01, **P < 0.005.
Figure 3
OI attenuates ovariectomy-induced bone loss in vivo. A) Representative 3-dimensional micro-CT reconstruction images of the 4 groups [sham-surgery group (sham); OVX + PBS group; OVX + OI group; OVX + ZOL group] at 6 wk postsurgery. B) Micro-CT analyses of BV/TV, Tb.Th, and BS/BV. Data were confirmed by 3 independent experiments. All results in bar graphs are expressed as means ±
sem
. NS, no significance. *P < 0.05, #P < 0.01.
Figure 4
Histologic analysis shows that OI significantly attenuates ovariectomy-induced bone loss. A) H&E staining was carried out to test bone loss in the femurs, and TRAP staining was used to detect osteoclasts in each group as described in Materials and Methods. Scale bars, 500 μm. B) Quantitative analysis was performed to determine the mean number of TRAP-positive cells from 5 random fields and TRAP-positive cell numbers per bone surface (OC.S/BS). C) Serum levels of C-telopeptide 1 (CTx-1) in mice from each group were determined by ELISA. D) The expression of Ho-1, NAD(P)H quinone dehydrogenase 1 (Nqo-1), and glutamate-cysteine ligase catalytic subunit (Gclc) was analyzed by real-time PCR. mRNA was isolated from whole mouse femurs of the indicated groups. E) Serum levels of IL-1β, TNF-α, and IL-6 in mice from each group were determined by ELISA. All results in bar graphs are expressed as means ±
sem
. NS, no significance. *P < 0.05, #P < 0.01.
Figure 5
OI induces the expression of Nrf2 by suppressing Hrd1 activity. A) BMMs were pretreated with CHX for 6 h. Then, they were stimulated with OI for another 12 h. The expression of Nrf2 in the cytoplasm was then analyzed by Western blotting after nuclear and cytoplasmic protein extraction. Quantitative analysis of the expression of Nrf2 is also shown. B) Degradation rate of Nrf2 over time. BMMs were treated with CHX for the indicated times. The expression of Nrf2 in the cytoplasm was measured by Western blotting. C) RANKL-induced osteoclastogenesis was performed with OI stimulation. The gene expression of Hrd1 at the indicated times was measured by real-time PCR. D) Protein levels of Hrd1 were also analyzed at the indicated times during osteoclastogenesis and stimulation with OI. E) Adenovirus-mediated overexpression of Hrd1 was proven by Western blotting. F) BMMs were transfected with AdCtrl and AdHrd1 for 24 h with OI or DMSO stimulation. The expression of Nrf2 and Ho-1 was then analyzed by Western blotting. G) BMMs were transfected with AdCtrl and AdHrd1 for 24 h with OI stimulation. Immunoprecipitation was performed as described in Materials and Methods. Ctrl, control; IB, immunoblot; IP, immunoprecipitation; Ub, ubiquitin. All results in bar graphs are expressed as means ±
sem
. *P < 0.05, #P < 0.01.
Figure 6
Hrd1 is involved in the OI-mediated suppression of osteoclastogenesis. A) BMMs were transfected with AdCtrl and AdHrd1, and RANKL-induced osteoclastogenesis was carried out with OI stimulation for 7 d. TRAP staining was performed to detect osteoclasts, and quantitative analysis (TRAP-positive cell numbers per well and relative TRAP-positive cell areas) was used to confirm the results of TRAP staining. BMMs were transfected with AdCtrl and AdHrd1, and RANKL-induced osteoclastogenesis was carried out with OI stimulation for 4 d. Scale bar, 150 μm. B) The expression of osteoclast-related genes (Dc-stamp, Acp5, Ctsk, and c-Fos) was measured by real-time PCR. C) The expression of Nfatc1 was analyzed by Western blotting. D) BMMs were transfected with AdCtrl and AdHrd1 and stimulated with OI. Bone resorption analysis was carried out to test the effect of Hrd1 on OI-mediated osteoclast function. Scale bar, 200 μm. All results in bar graphs are expressed as means ±
sem
. NS, no significance. *P < 0.05, #P < 0.01.
Figure 7
Hrd1 is involved in the OI-mediated inhibition of ROS production and inflammatory responses. A) BMMs were transfected with AdCtrl and AdHrd1 and then pretreated with OI for 6 h. LPS (10 ng/ml)–induced ROS production was measured as described in Materials and Methods. BMMs were transfected with AdCtrl and AdHrd1 and then pretreated with OI for 6 h. B) After stimulation with LPS (10 ng/ml) for 12 h, the expression of pro–IL-1β in BMMs was analyzed by Western blotting. C) The concentration of IL-1β and IL-18 in the culture medium was measured by ELISA. Fl2, fluorescence 2; M1, marker 1; MFI, mean fluorescence intensity. All results in bar graphs are expressed as means ±
sem
. *P < 0.05, #P < 0.01.
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