Sigma‐1 receptor attenuates osteoclastogenesis by promoting ER‐associated degradation of SERCA2

Article25 May 2022Open Access Source DataTransparent process Sigma-1 receptor attenuates osteoclastogenesis by promoting ER-associated degradation of SERCA2 Xiaoan Wei Xiaoan Wei orcid.org/0000-0002-1132-8760 Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Writing - original draft, Project administration Search for more papers by this author Zeyu Zheng Zeyu Zheng Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Validation, Project administration Search for more papers by this author Zhenhua Feng Zhenhua Feng Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Validation, Project administration Search for more papers by this author Lin Zheng Lin Zheng Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Methodology, Project administration Search for more papers by this author Siyue Tao Siyue Tao Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Methodology, Project administration Search for more papers by this author Bingjie Zheng Bingjie Zheng Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Project administration Search for more papers by this author Bao Huang Bao Huang Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Project administration Search for more papers by this author Xuyang Zhang Xuyang Zhang Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Project administration Search for more papers by this author Junhui Liu Junhui Liu Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Writing - review & editing Search for more papers by this author Yilei Chen Yilei Chen Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Writing - review & editing Search for more papers by this author Wentian Zong Wentian Zong orcid.org/0000-0001-9511-0605 Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Resources Search for more papers by this author Zhi Shan Zhi Shan Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Writing - review & editing Search for more papers by this author Shunwu Fan Shunwu Fan Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Supervision, Funding acquisition Search for more papers by this author Jian Chen Corresponding Author Jian Chen [email protected] orcid.org/0000-0001-8568-9991 Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Data curation, Supervision, Funding acquisition, Writing - review & editing Search for more papers by this author Fengdong Zhao Corresponding Author Fengdong Zhao [email protected] orcid.org/0000-0002-2945-8707 Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - review & editing Search for more papers by this author Xiaoan Wei Xiaoan Wei orcid.org/0000-0002-1132-8760 Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Writing - original draft, Project administration Search for more papers by this author Zeyu Zheng Zeyu Zheng Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Validation, Project administration Search for more papers by this author Zhenhua Feng Zhenhua Feng Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Validation, Project administration Search for more papers by this author Lin Zheng Lin Zheng Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Methodology, Project administration Search for more papers by this author Siyue Tao Siyue Tao Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Methodology, Project administration Search for more papers by this author Bingjie Zheng Bingjie Zheng Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Project administration Search for more papers by this author Bao Huang Bao Huang Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Project administration Search for more papers by this author Xuyang Zhang Xuyang Zhang Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Project administration Search for more papers by this author Junhui Liu Junhui Liu Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Writing - review & editing Search for more papers by this author Yilei Chen Yilei Chen Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Writing - review & editing Search for more papers by this author Wentian Zong Wentian Zong orcid.org/0000-0001-9511-0605 Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Resources Search for more papers by this author Zhi Shan Zhi Shan Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Writing - review & editing Search for more papers by this author Shunwu Fan Shunwu Fan Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Supervision, Funding acquisition Search for more papers by this author Jian Chen Corresponding Author Jian Chen [email protected] orcid.org/0000-0001-8568-9991 Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Data curation, Supervision, Funding acquisition, Writing - review & editing Search for more papers by this author Fengdong Zhao Corresponding Author Fengdong Zhao [email protected] orcid.org/0000-0002-2945-8707 Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - review & editing Search for more papers by this author Author Information Xiaoan Wei1,2,†, Zeyu Zheng1,2,†, Zhenhua Feng1,2,†, Lin Zheng1,2, Siyue Tao1,2, Bingjie Zheng1,2, Bao Huang1,2, Xuyang Zhang1,2, Junhui Liu1,2, Yilei Chen1,2, Wentian Zong1,2, Zhi Shan1,2, Shunwu Fan1,2, Jian Chen *,1,2 and Fengdong Zhao *,1,2 1Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China 2Key Laboratory of Musculoskeletal System Degeneration and Regeneration, Translational Research of Zhejiang Province, Hangzhou, China † These authors contributed equally to this work *Corresponding author. Tel: +86 057186006667; E-mail: [email protected] *Corresponding author. Tel: +86 057186006667; E-mail: [email protected] EMBO Mol Med (2022)14:e15373https://doi.org/10.15252/emmm.202115373 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Sigma-1 receptor (Sigmar1) is a specific chaperone located in the mitochondria-associated endoplasmic reticulum membrane (MAM) and plays a role in several physiological processes. However, the role of Sigmar1 in bone homeostasis remains unknown. Here, we show that mice lacking Sigmar1 exhibited severe osteoporosis in an ovariectomized model. In contrast, overexpression of Sigmar1 locally alleviated the osteoporosis phenotype. Treatment with Sigmar1 agonists impaired both human and mice osteoclast formation in vitro. Mechanistically, SERCA2 was identified to interact with Sigmar1 based on the immunoprecipitation-mass spectrum (IP-MS) and co-immunoprecipitation (co-IP) assays, and Q615 of SERCA2 was confirmed to be the critical residue for their binding. Furthermore, Sigmar1 promoted SERCA2 degradation through Hrd1/Sel1L-dependent ER-associated degradation (ERAD). Ubiquitination of SERCA2 at K460 and K541 was responsible for its proteasomal degradation. Consequently, inhibition of SERCA2 impeded Sigmar1 deficiency enhanced osteoclastogenesis. Moreover, we found that dimemorfan, an FDA-approved Sigmar1 agonist, effectively rescued bone mass in various established bone-loss models. In conclusion, Sigmar1 is a negative regulator of osteoclastogenesis, and activation of Sigmar1 by dimemorfan may be a potential treatment for osteoporosis in clinical practice. Synopsis Activation of Sigma-1 receptor by dimemorfan promoted SERCA2 degradation, causing reduction in osteoclast formation. Targeting Sigma-1 receptor and dimemorfan may be a novel potential therapeutic approach for osteoporosis. Loss of Sigma-1 receptor was found to cause severe osteoporosis phenotype compared with WT mice. Activation of Sigma-1 receptor both in vivo and in vitro rescued bone loss models and suppressed osteoclast formation. SERCA2 was interacted with Sigma-1 receptor and degraded in ERAD pathway, leading to impaired osteoclast's relative gene expression. Long term use of dimemorfan in clinical practice made it a promising drug towards osteoporosis treatment. The paper explained Problem Over activation of osteoclasts disturbs the balance between osteoclasts and osteoblasts, leading to bone-loss diseases. Sigma-1 receptor has been proved to take part in multiple physiological processes, and its role in regulating bone homeostasis remains unknown. Results Knockdown of Sigmar1 exacerbated osteoporosis phenotype in OVX mouse model, whereas overexpression of Sigmar1 exerted protective effects. Activation of Sigmar1 by dimemorfan inhibited osteoclast formation in vitro and attenuated bone loss in several pathological mouse models. In addition, Sigmar1 was found to interact with SERCA2 and mediated SERCA2 degradation by Hrd1/Sel1L-dependent ER-associated degradation. Impact Sigmar1 acts as a negative regulator in osteoclast formation in pathological conditions, providing new therapeutic target for bone-loss disease treatment. Introduction The skeleton is of prominent importance in maintaining the normal functions of humans, such as hematopoiesis, hormone excretion, and mechanical support (Garrett & Emerson, 2009; Morrison & Scadden, 2014). The dynamic balance between bone formation and bone resorption is critical to maintain normal bone density and mineral homeostasis (Sobacchi et al, 2013), and the process is coupled both in time and space. Osteoclasts are the principal, if not the only cells, to resorb old bone. Excessive osteoclast activity contributes to osteoporosis, Paget’s disease, and rheumatoid arthritis (Singer & Leach, 2010; Walsh & Gravallese, 2010; Compston et al, 2019). Current drugs for osteoporosis are mainly aimed at preventing bone resorption; however, in clinical practice, these drugs are still facing many problems and challenges, such as unclear long-term efficacy, atypical fractures, and increased cardiovascular diseases risks (Khosla & Hofbauer, 2017; Compston et al, 2019; Reid, 2020). Therefore, understanding the underlying mechanism that regulates osteoclast differentiation and developing novel drugs for bone-loss disease treatment are necessary. Sigma-1 receptor (Sigmar1) is a nonopioid and evolutionarily isolated receptor with no homolog to any other known human protein (Hanner et al, 1996). Crystal analysis revealed that Sigmar1 was a single-pass transmembrane receptor and formed a trimer structure for ligand binding (Schmidt et al, 2016). Normally, Sigmar1 resides specifically at the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) and binds with GRP78 (78-kD glucose-related protein, also known as Bip) (Hayashi et al, 2009). Upon ligand stimulation or ER stress, Sigmar1 dissociates from GRP78 and translocates to the ER lumen to regulate calcium homeostasis and the unfolded protein reaction (UPR) to alleviate ER stress (Hayashi & Su, 2007; Su et al, 2016; Rosen et al, 2019). Thus, activation of Sigmar1 may be a potential therapeutic target for different diseases. Dimemorfan, an analog of dextromethorphan, is a selective Sigmar1 agonist and a nonopioid antitussive drug that has been safely used in the clinic in Japan for more than 40 years (Ida, 1997; Shen et al, 2017). Available studies related to Sigmar1 or dimemorfan mainly focused on neurodegenerative, neuromotor, and respiratory diseases (Luty et al, 2010; Al-Saif et al, 2011; Francardo et al, 2014; Lenze et al, 2020). However, the role of Sigmar1 and the potential application of dimemorfan in bone mineral homeostasis are unknown. Sacro/endoplasmic reticulum Ca2+-ATPases (SERCAs) are a family of proteins that are involved in calcium homeostasis in multiple cells (Periasamy et al, 2017). These proteins are encoded by a multigenic family consisting of SERCA1-3 (Atp2a1-3). SERCA2b, encoded by Atp2a2, is ubiquitously expressed in smooth muscle and nonmuscle tissues, including neurons (Kim et al, 2013). By catalyzing adenosine triphosphate (ATP), SERCA2 is the major calcium transport to reuptake calcium ions from the cytoplasm to the endoplasmic reticulum, and this process is essential for intracellular calcium oscillation (Dolmetsch et al, 1998; Negishi-Koga & Takayanagi, 2009). Knocking down SERCA2 remarkably suppressed calcium oscillation at both frequency and peak values, in contrast, overexpression of SERCA2 notably promoted calcium oscillation (Zhao et al, 2001; Morita & Kudo, 2010). Heterozygote SERCA2 (+/-) mice exhibited an osteopetrosis phenotype, and bone marrow-derived macrophages (BMMs) from these heterozygote mice displayed weak calcium oscillations and less osteoclast formation in the presence of receptor activator of nuclear factor-κB ligand (RANKL) than those from wild-type (WT) littermates (Yang et al, 2009). A recent study demonstrated that TMEM64 bound to SERCA2 and regulated SERCA2 activity, and knockout of TMEM64 resulted in reduced SERCA2 activity and decreased osteoclast formation (Kim et al, 2013). Here, we investigated the role of Sigmar1 in osteoclastogenesis and found that Sigmar1 global knockout (gKO) mice exhibited severe osteoporosis after ovariectomy surgery (OVX) compared with WT littermates. Activation of Sigmar1 by dimemorfan significantly inhibited osteoclast formation both in vivo and in vitro. Furthermore, SERCA2 was found to interact with Sigmar1, leading to its proteasomal degradation via the ERAD pathway. The results presented herein demonstrated that Sigmar1 had a profound effect on bone homeostasis and could be a potential therapeutic target for treating osteoporosis. Results Sigmar1 deletion has no influence on bone mass under steady conditions To investigate whether loss of Sigmar1 influenced bone mass in vivo, we examined 12-week-old male Sigmar1 gKO mice and their WT littermates. Compared with wild-type littermates, Sigmar1 gKO mice displayed normal body size, weight, and fertility. Using micro-CT, no increase or decrease in trabecular bone mass and cortical bone in the femur and the lumbar spine was observed between the two groups (Fig 1A–D). Similar results were shown in female Sigmar1 gKO mice versus WT littermates (Appendix Fig S1A–D). Tartrate-resistant acidic phosphatase (TRAP)-stained sections showed equal osteoclast formation in Sigmar1 gKO versus WT (Fig 1E–G). We performed a calcein labeling experiment and found the same bone formation rate in Sigmar1 gKO versus WT (Fig 1H and I). Immunofluorescence staining of SOST also indicates similar osteocyte number in both genotypes (Appendix Fig S1E and F). Furthermore, the serum procollagen I N-terminal propeptide (PINP) and C-terminal telopeptide of type I collagen (CTX-1), which represent bone formation and bone resorption, respectively, were also unchanged between the two mice in both sexes (Fig 1J and K and Appendix Fig S1G and H). In an in vitro bone formation assay, we isolated mesenchymal stem cells (MSCs) from the two gene types and induced them using an osteogenic cell culture medium. ALP and Alizarin Red staining showed similar extracellular calcium deposits between gKO and WT MSCs, indicating that deletion of Sigmar1 had little effect on the osteogenic process (Fig 1L). These results demonstrated that Sigmar1 knockout had no influence on bone mass under steady conditions. Figure 1. Sigmar1 deletion has no influence on bone mass under steady conditions A. Microcomputed tomography (micro-CT) images of the proximal femur from 12-week-old male WT and Sigmar1 gKO mice. Scale bars, 1 mm. B. Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp), trabecular thickness (Tb. Th), cortical region BV/TV (Ct. BV/TV), and cortical thickness (Ct. Th, mm) (n = 5 biological replicates). C. Coronal images of the fifth lumbar spine. Scale bars, 1 mm. D. Quantification of trabecular bone parameters of lumbar spine (n = 5 biological replicates). E. TRAP staining of tibias from male WT and Sigmar1 gKO mice. Scale bars, 50 μm. F, G. Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates). H, I. Representative images and quantitative analysis of calcein double labeling. Scale bars, 20 μm (n = 6 biological replicates). J, K. Serum PINP (procollagen I N-terminal propeptide) and CTX-I (C-terminal telopeptide of type I collagen) concentrations measured by ELISA in male Sigmar1 gKO mice and their WT littermates (n = 6 biological replicates). L. MSCs from male WT or Sigmar1 gKO mice underwent osteogenic differentiation for 7 or 21 days and staining for alkaline phosphatase or alizarin red, respectively. Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. The unpaired two-tailed Student’s t-test (B, D, F, G, I, J, and K) was used for statistical analysis. Download figure Download PowerPoint Loss of Sigmar1 exacerbates OVX-induced bone loss and promotes osteoclastogenesis in vitro To further unveil the role of Sigmar1 in bone homeostasis, we performed ovariectomy surgery (OVX) to investigate events under pathological conditions. Briefly, Sigmar1 gKO and WT littermates were ovariectomized at 12 weeks of age, and 6 weeks later, these mice were harvested for radiographic and histologic analyses. Significant uterus mass loss confirmed the successful establishment of OVX surgery (Fig EV1A). Micro-CT revealed that Sigmar1 gKO-OVX mice exhibited a severe osteoporosis phenotype with lower bone volume/tissue volume (BV/TV), trabecular number (Tb. N), and elevated trabecular separation (Tb. Sp) (Fig 2A and B). Radiographic analysis of the lumbar spine also indicated severe bone loss in Sigmar1 gKO mice after OVX surgery (Fig EV1B and C). Next, we performed TRAP staining for osteoclasts and immunohistochemistry (IHC) staining of osteocalcin (Ocn) for osteoblasts in the femur sections and found that Sigmar1 knockout in OVX mice resulted in more osteoclast number, whereas the osteoblast number decreased after OVX surgery and showed no difference between WT and Simgar1 gKO mice (Figs 2C–E and EV1D and E). Click here to expand this figure. Figure EV1. Sigmar1 deletion results in severe osteoporosis in the OVX model and promotes osteoclastogenesis in vitro A. Uterus weight from different groups (n = 6 biological replicates). B. Coronal images of the fifth lumbar spine. Scale bars, 1 mm. C. Quantification of trabecular bone parameters of lumbar spine (n = 5 biological replicates). D, E. Immunohistochemistry staining of osteocalcin (Ocn) in femur sections (D) and quantification of Ocn-positive osteoblast on trabecular bone surface (E) (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. The one-way ANOVA with the Tukey’s multiple comparison test (A, C, and E) was used for statistical analysis. Source data are available online for this figure. Download figure Download PowerPoint Figure 2. Sigmar1 deletion results in severe osteoporosis in the OVX model and promotes osteoclastogenesis in vitro A. Micro-CT images of the proximal femur from female WT or Sigmar1 gKO mice that received sham or ovariectomy surgery for 6 weeks. Scale bars, 1 mm. B. Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp), and trabecular thickness (Tb. Th) (n = 5 biological replicates). C. TRAP staining of femur sections from the four groups. Scale bars, 200 μm. D, E. Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 5 biological replicates). F. TRAP staining to detect osteoclastogenesis of BMMs from WT or Sigmar1 gKO mice. Scale bars, 200 μm. G, H. Quantification of the size and nuclei numbers of TRAP-positive multinuclear cells (n = 6 biological replicates for G and n = 3 biological replicates for H). I, J. Representative images and quantification of the relative pit resorption area of hydroxyapatite-coated plates. WT or Sigmar1 gKO BMMs were seeded on hydroxyapatite-coated plates and treated with 50 ng/ml RANKL (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. The one-way ANOVA with the Tukey’s multiple comparison test (B, D, and E) and unpaired two-tailed Student’s t-test (G, H, and J) were used for statistical analysis. Download figure Download PowerPoint To further investigate the influence of knockout of Sigmar1 on bone mass, we carried out a bone marrow transfer mice model. 6-week-old WT mice were subjected to sublethal irradiation and WT or Sigmar1 gKO bone marrow cells were transferred to these mice through tail vein injection (Fig EV2A). 6 weeks after the transfer, we collected mice blood for genotype identification (Fig EV2B), those successfully transferred with WT or Sigmar1 gKO mice were ovariectomized, and all these mice were collected 6 weeks after the OVX surgery for further analysis. Micro-CT scanning of the femurs showed that mice transferred with Sigmar1 gKO bone marrow exhibited severe osteoporosis after the OVX (Fig EV2C and D). Histological staining also confirmed that Sigmar1 gKO bone marrow transferred mice displayed less trabecular bone and more osteoclast number (Fig EV2E–H). Click here to expand this figure. Figure EV2. Bone marrow transfer of Sigmar1 gKO cells exacerbates OVX-induced osteoporosis A. The schematic illustrates the protocol for bone marrow transfer experiment and OVX surgery. B. PCR bands for identifying success transfer of WT and Sigmar1 gKO bone marrow cells. Sigmar1 gKO cells had negative wt bands (upper), and positive mut bands (lower) and WT cells had the opposite results. C. Micro-CT images of the proximal femur from female ovariectomized mice that transferred with WT or Sigmar1 gKO bone marrow cells previously. Scale bars, 1 mm. D. Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp), and trabecular thickness (Tb. Th) (n = 8 biological replicates). E. H&E staining of femur sections. Scale bars, 200 μm. F. TRAP staining of femur sections. Scale bars, 200 μm. G, H. Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 8 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student’s t-test (D and G and H) was used for statistical analysis. Download figure Download PowerPoint To elucidate the impact of Sigmar1 on osteoclastogenic cells in vitro, we isolated bone marrow-derived macrophages (BMMs) from both Sigmar1 gKO and WT mice for osteoclast induction. We found that Sigmar1 knockout significantly promoted osteoclast formation (Fig 2F), which showed an increasing number and size of TRAP-positive cells (Fig 2G and H). Bone resorption analysis indicated that Sigmar1 knockout osteoclast had higher bone resorption capacity (Fig 2I and J). Further RT-qPCR analysis revealed that mar