Osteoblast-specific down-regulation of NLRP3 inflammasome by aptamer-functionalized liposome nanoparticles improves bone quality in postmenopausal osteoporosis rats

AUTHORS

Lijun Xu, Jie Zhu, Lingjun Rong, Huinan Yang, Bin Wang, Shuai Lu, Lingxiao Zhang, Fuyi Li, Shihua Yang, Zhifang Wang, Chong Li, Xiao Hu, Ruoyun Liu, Lili Zheng, Hongjian Liu, Haohao Zhang, Yanling Liu, Di Zhao, Shuiying Zhao, Lun Zhang, Yingbo Jia, Shiyu Liang, Zhikang Guo, Xixiu Xi, Ruitian Liu, Lixia Zhang

ABSTRACT

Rationale: NLRP3 inflammasome is critical in the development and progression of many metabolic diseases driven by chronic inflammation, but its effect on the pathology of postmenopausal osteoporosis (PMOP) remains poorly understood.

Methods: We here firstly examined the levels of NLRP3 inflammasome in PMOP patients by ELISA. Then we investigated the possible mechanisms underlying the effect of NLRP3 inflammasome on PMOP by RNA sequencing of osteoblasts treated with NLRP3 siRNA and qPCR. Lastly, we accessed the effect of decreased NLRP3 levels on ovariectomized (OVX) rats. To specifically deliver NLRP3 siRNA to osteoblasts, we constructed NLRP3 siRNA wrapping osteoblast-specific aptamer (CH6)-functionalized lipid nanoparticles (termed as CH6-LNPs-siNLRP3).

Results: We found that the levels of NLRP3 inflammasome were significantly increased in patients with PMOP, and were negatively correlated with estradiol levels. NLRP3 knock-down influenced signal pathways including immune system process, interferon signal pathway. Notably, of the top ten up-regulated genes in NLRP3-reduced osteoblasts, nine genes (except Mx2) were enriched in immune system process, and five genes were related to interferon signal pathway. The in vitro results showed that CH6-LNPs-siNLRP3 was relatively uniform with a dimeter of 96.64 ± 16.83 nm and zeta potential of 38.37 ± 1.86 mV. CH6-LNPs-siNLRP3 did not show obvious cytotoxicity and selectively delivered siRNA to bone tissue. Moreover, CH6-LNPs-siNLRP3 stimulated osteoblast differentiation by activating ALP and enhancing osteoblast matrix mineralization. When administrated to OVX rats, CH6-LNPs-siNLRP3 promoted bone formation and bone mass, improved bone microarchitecture and mechanical properties by decreasing the levels of NLRP3, IL-1β and IL-18 and increasing the levels of OCN and Runx2.

Conclusion: NLRP3 inflammasome may be a new biomarker for PMOP diagnosis and plays a key role in the pathology of PMOP. CH6-LNPs-siNLRP3 has potential application for the treatment of PMOP.

Long noncoding RNA Malat1 protects against osteoporosis and bone metastasis

AUTHORS

Yang Zhao, Jingyuan Ning, Hongqi Teng, Yalan Deng, Marisela Sheldon, Lei Shi, Consuelo Martinez, Jie Zhang, Annie Tian, Yutong Sun, Shinichi Nakagawa, Fan Yao, Hai Wang & Li Ma

ABSTRACT

MALAT1, one of the few highly conserved nuclear long noncoding RNAs (lncRNAs), is abundantly expressed in normal tissues. Previously, targeted inactivation and genetic rescue experiments identified MALAT1 as a suppressor of breast cancer lung metastasis. On the other hand, Malat1-knockout mice are viable and develop normally. On a quest to discover the fundamental roles of MALAT1 in physiological and pathological processes, we find that this lncRNA is downregulated during osteoclastogenesis in humans and mice. Remarkably, Malat1 deficiency in mice promotes osteoporosis and bone metastasis of melanoma and mammary tumor cells, which can be rescued by genetic add-back of Malat1. Mechanistically, Malat1 binds to Tead3 protein, a macrophage-osteoclast–specific Tead family member, blocking Tead3 from binding and activating Nfatc1, a master regulator of osteoclastogenesis, which results in the inhibition of Nfatc1-mediated gene transcription and osteoclast differentiation. Notably, single-cell transcriptome analysis of clinical bone samples reveals that reduced MALAT1 expression in pre-osteoclasts and osteoclasts is associated with osteoporosis and metastatic bone lesions. Altogether, these findings identify Malat1 as a lncRNA that protects against osteoporosis and bone metastasis.

Piperlongumine, a Piper longum-derived amide alkaloid, protects mice from ovariectomy-induced osteoporosis by inhibiting osteoclastogenesis via suppression of p38 and JNK signaling†

AUTHORS

Na Luo, Lei Zhang, Chunmei Xiu, Xi Luo, Siyuan Hu, Kaizhong Ji, Qingbai Li, Jianquan Chen

ABSTRACT

Postmenopausal osteoporosis (PMOP) is a metabolic bone disease that results from overproduction and hyperactivation of osteoclasts caused by insufficient estrogen in women after menopause. Current therapeutic strategies are mainly focused on treating PMOP patients who have already developed severe bone loss or even osteoporotic fractures. Obviously, a better strategy is to prevent PMOP from occurring in the first place. However, such reagents are largely lacking. Piperlongumine (PLM), an amide alkaloid extracted from long pepper Piper longum, exhibits the anti-osteoclastogenic effect in normal bone marrow macrophages (BMMs) and the protective effect against osteolysis induced by titanium particles in mice. This study examined the preventive effect of PLM on PMOP and explored the potential mechanism of this effect using both ovariectomized mice and their primary cells. The result showed that PLM (5 and 10 mg kg−1) administered daily for 6 weeks ameliorated ovariectomy-induced bone loss and osteoclast formation in mice. Further cell experiments showed that PLM directly suppressed osteoclast formation, F-actin ring formation, and osteoclastic resorption pit formation in BMMs derived from osteoporotic mice, but did not obviously affect osteogenic differentiation of bone marrow stromal cells (BMSCs) from these mice. Western blot analysis revealed that PLM attenuated maximal activation of p38 and JNK pathways by RANKL stimulation without affecting acute activation of NF-κB, AKT, and ERK signaling. Furthermore, PLM inhibited expression of key osteoclastogenic transcription factors NFATc1/c-Fos and their target genes (Dcstamp, Atp6v0d2, Acp5, and Oscar). Taken together, our findings suggest that PLM inhibits osteoclast formation and function by suppressing RANKL-induced activation of the p38/JNK-cFos/NFATc1 signaling cascade, thereby preventing ovariectomy-induced osteoporosis in mice. Thus, PLM can potentially be used as an anti-resorption drug or dietary supplement for the prevention of PMOP.

Progranulin deficiency associates with postmenopausal osteoporosis via increasing ubiquitination of estrogen receptor α☆,

AUTHORS

Guangfei Li, Aifei Wang, Wei Tang, Wenyu Fu, Qingyun Tian, Jinlong Jian, Michal Lata, Aubryanna Hettinghouse, Yuanjing Ding, Jianlu Wei, Xiangli Zhao, Mingyong Wang, Qirong Dong, Chuanju Liu, Youjia Xu

ABSTRACT

Estrogen deficiency is considered the most important cause of postmenopausal osteoporosis. However, the underlying mechanism is still not completely understood. In this study, progranulin (PGRN) was isolated as a key regulator of bone mineral density in postmenopausal women through high throughput proteomics screening. In addition, PGRN-deficient mice exhibited significantly lower bone mass than their littermates in an ovariectomy-induced osteoporosis model. Furthermore, estrogen-mediated inhibition of osteoclastogenesis and bone resorption as well as its protection against ovariectomy-induced bone loss largely depended on PGRN. Mechanistic studies revealed the existence of a positive feedback regulatory loop between PGRN and estrogen signaling. In addition, loss of PGRN led to the reduction of estrogen receptor α, the important estrogen receptor involved in estrogen regulation of osteoporosis, through enhancing its degradation via K48-linked ubiquitination. These findings not only provide a previously unrecognized interplay between PGRN and estrogen signaling in regulating osteoclastogenesis and osteoporosis but may also present a new therapeutic approach for the prevention and treatment of postmenopausal osteoporosis by targeting PGRN/estrogen receptor α.

VSIG4 inhibits RANKL-induced osteoclastogenesis by enhancing Nrf2-dependent antioxidant response against reactive oxygen species production

AUTHORS

Jiansen Miao, Yiting Tu, Junchen Jiang, Rufeng Ren, Qihang Wu, Haibo Liang, Tengjie Wang, Binghao Lin, Jingtao Wu, Youjin Pan, Xiangyang Wang, Haiming Jin

ABSTRACT

Osteoporosis is a prevalent systemic skeletal disorder, particularly affecting postmenopausal women, primarily due to excessive production and activation of osteoclasts. However, the current anti-osteoporotic drugs utilized in clinical practice may lead to certain side effects. Therefore, it is necessary to further unravel the potential mechanisms regulating the osteoclast differentiation and to identify novel targets for osteoporosis treatment. This study revealed the most significant decline in VSIG4 expression among the VSIG family members. VSIG4 overexpression significantly inhibited RANKL-induced osteoclastogenesis and bone resorption function. Mechanistically, both western blot and immunofluorescence assay results demonstrated that VSIG4 overexpression attenuated the expression of osteoclast marker genes and dampened the activation of MAPK and NF-κB signaling pathways. Furthermore, VSIG4 overexpression could inhibit the generation of reactive oxygen species (ROS) and stimulate the expression of Nrf2 along with its downstream antioxidant enzymes via interaction with Keap1. Notably, a potent Nrf2 inhibitor, ML385, could reverse the inhibitory effect of VSIG4 on osteoclast differentiation. In line with these findings, VSIG4 overexpression also mitigated bone loss induced by OVX and attenuated the activation of osteoclasts in vivo. In conclusion, our results suggest that VSIG4 holds promise as a novel target for addressing postmenopausal osteoporosis. This is achieved by suppressing osteoclast formation via enhancing Nrf2-dependent antioxidant response against reactive oxygen species production.

Inhibition of PPP1R15A alleviates osteoporosis via suppressing RANKL-induced osteoclastogenesis

AUTHORS

Zong-bao Ding, Yan Chen, Yu-rong Zheng, Yi-yuan Wang, Wen-de Deng, Jie-huang Zheng, Qin Yang, Zi-ye Chen, Li-hong Li, Hui Jiang & Xiao-juan Li

ABSTRACT

Osteoporosis results from overactivation of osteoclasts. There are currently few drug options for treatment of this disease. Since the successful development of allosteric inhibitors, phosphatases have become attractive therapeutic targets. Protein phosphatase 1, regulatory subunit 15 A (PPP1R15A), is a stress-responsive protein, which promotes the UPR (unfolded protein response) and restores protein homeostasis. In this study we investigated the role of PPP1R15A in osteoporosis and osteoclastogenesis. Ovariectomy (OVX)-induced osteoporosis mouse model was established, osteoporosis was evaluated in the left femurs using micro-CT. RANKL-stimulated osteoclastogenesis was used as in vitro models. We showed that PPP1R15A expression was markedly increased in BMMs derived from OVX mice and during RANKL-induced osteoclastogenesis in vitro. Knockdown of PPP1R15A or application of Sephin1 (a PPP1R15A allosteric inhibitor in a phase II clinical trial) significantly inhibited osteoclastogenesis in vitro. Sephin1 (0.78, 3.125 and 12.5 μM) dose-dependently mitigated the changes in NF-κB, MAPK, and c-FOS and the subsequent nuclear factor of activated T cells 1 (NFATc1) translocation in RANKL-stimulated BMMs. Both Sephin1 and PPP1R15A knockdown increased the phosphorylated form of eukaryotic initiation factor 2α (eIF2α); knockdown of eIF2α reduced the inhibitory effects of Sephin1 on NFATc1-luc transcription and osteoclast formation. Furthermore, Sephin1 or PPP1R15A knockdown suppressed osteoclastogenesis in CD14+ monocytes from osteoporosis patients. In OVX mice, injection of Sephin1 (4, 8 mg/kg, i.p.) every two days for 6 weeks significantly inhibited bone loss, and restored bone destruction and decreased TRAP-positive cells. This study has identified PPP1R15A as a novel target for osteoclast differentiation, and genetic inhibition or allosteric inhibitors of PPP1R15A, such as Sephin1, can be used to treat osteoporosis.