Application of G alphai gene as osteoporosis treatment target

Abstract

The invention relates to the technical field of osteoporosis prevention and treatment. The invention provides an application of a G alpha i gene as an osteoporosis treatment target. According to the invention, the expression of the G alphai in osteoporosis patients and osteoclast / osteoclast progenitor cells is researched, and the influence of the G alphai on osteoclast formation and bone metabolism is evaluated. By explaining the specific mechanism of the G alphai for regulating bone metabolism and related signal pathways, the G alphai is confirmed to be a key medium for RANKL signal transduction. In addition, it is proved that a specific functional domain of G alpha i plays a key role in osteoclast differentiation. It is worthy of noting that when it is found that G alpha i is lacked, the anti-osteoporosis effect of the dinomonoclonal antibody is damaged, and it is prompted that the treatment effect of G alpha i on the dinomonoclonal antibody is indispensable. The research result emphasizes that the G alphai is used as a potential target spot for diagnosing and treating osteoporosis, and paves a way for developing a novel treatment strategy.

Description

Technical Field

[0001] The present invention relates to the technical field of osteoporosis prevention and treatment, and particularly relates to the application of the Gαi gene as a therapeutic target for osteoporosis. Background Art

[0002] Osteoporosis is a systemic skeletal disease that affects bone tissue, characterized by reduced bone mass, disrupted bone microstructure, and increased bone fragility, thereby increasing the risk of fractures. Although there have been advances in osteoporosis treatments such as denosumab and romosozumab, these therapies still have certain limitations. Therefore, further elucidating the pathophysiological mechanisms of osteoporosis and exploring new therapeutic targets are crucial for improving treatment efficacy and the quality of life of patients.

[0003] Recent studies have confirmed that bone tissue is an "osteoimmune" organ, which is not only a locomotor organ, a mineral storage depot, but also the main lymphoid organ that maintains hematopoietic stem cells. Bone and mineral homeostasis depend on the coordinated activities of osteoclasts, osteoblasts, and osteocytes, but pathological conditions can disrupt this balance through abnormal or persistent immune responses. In osteoporosis, monocytes differentiate into osteoclasts in response to increased demand, leading to bone loss. In postmenopausal osteoporosis, estrogen deficiency not only directly affects bone tissue but also alters the activity of immune cells, further exacerbating bone resorption. Given that osteoclasts belong to the monocyte / macrophage lineage, they interact closely with B lymphocytes and T lymphocytes, and their differentiation is regulated by receptor activator of nuclear factor-κB ligand (RANKL), which is also produced by lymphocytes and regulates lymphocyte production. These findings highlight the key role of the immune system in the pathogenesis of osteoporosis.

[0004] The Gαi protein (G protein αi subunit) is an important mediator of inhibitory signal transduction in the G protein-coupled receptor (GPCR) system. The three main Gαi subunits, Gαi1, Gαi2, and Gαi3, have 85-95% amino acid sequence homology and exhibit overlapping expression patterns. These subunits are widely expressed in the immune system, bone tissue, and endothelial cells. Notably, Gαi1 / 3 interacts with CD14 / Gab1 to regulate macrophage polarization in vitro and in vivo. Gαi signaling regulates multiple leukocyte functions, including adhesion and chemotaxis, and is involved in bone development. Although these studies suggest that Gαi is involved in immune function and bone metabolism, its specific role in osteoclast differentiation and osteoporosis remains unclear.

[0005] As a key molecule connecting bone and the immune system, RANKL signals activate RANK (receptor activator of nuclear factor-κB), promoting osteoclast maturation and regulating bone resorption. This pathway also affects lymphoid organ development and immune tolerance. Our previous studies have shown that Gαi1 and Gαi3 mediate key signal transduction events and regulate multiple cellular functions. Gαi1 and Gαi3 regulate macrophage polarization by forming a complex with CD14 and Gab1. Immunoprecipitation experiments showed that Gαi1 / 3 binds to interleukin-4 (IL-4)-stimulated IL-4 receptor α (IL-4Rα), promoting its trafficking to endosomes and activating the Gab1-Akt signaling cascade in macrophages. In addition, R-spondin3 enhances the interaction of Gαi1 / 3 with leucine-rich repeat-containing GPCRs and Gab1, promoting downstream activation of the Akt-mTOR pathway. Given that RANK is a key regulator of osteoclast differentiation and that RANKL-RANK signaling activates multiple downstream signaling pathways essential for osteoclast function, it is unclear whether Gαi is involved in osteoclastogenesis through RANKL-RANK signaling. Summary of the Invention

[0006] The object of the present invention is to provide an application of the Gαi gene as a therapeutic target for osteoporosis, establishing Gαi1 / 3 as a key regulator of osteoclastogenesis and suggesting it as a promising target for the treatment of osteoporosis.

[0007] To achieve the above object of the invention, the present invention provides the following technical solutions: The present invention provides an application of the Gαi gene as a therapeutic target for osteoporosis.

[0008] Preferably, the types of the Gαi gene are Gαi1 and / or Gαi3.

[0009] The present invention also provides an application of the Gαi protein as a diagnostic marker for osteoporosis.

[0010] Preferably, the types of the Gαi protein are Gαi1 and / or Gαi3.

[0011] The present invention also provides an application of the Gαi protein in regulating osteoclast function and bone metabolism.

[0012] Preferably, the regulation of osteoclast function is the regulation of osteoclast differentiation, and the regulation of bone metabolism is the regulation of bone resorption; The types of the Gαi protein are Gαi1 protein and / or Gαi3 protein.

[0013] The present invention also provides a method for regulating osteoclast differentiation and bone resorption, by knocking out or inhibiting the expression of Gαi1 and / or Gαi3 genes, or inhibiting the activity of Gαi1 and / or Gαi3 proteins, blocking the formation of the RANK-TRAF6 complex, thereby reducing osteoclast differentiation and bone resorption.

[0014] The present invention also provides a Gαi1 / 3 protein inhibitor for preventing or treating osteoporosis, and the Gαi1 / 3 protein inhibitor targets the Asp173 residue of the Gαi1 / 3 protein.

[0015] The present invention also provides the use of an inhibitor of Gαi protein in the preparation of a drug for preventing or treating osteoporosis.

[0016] The present invention provides the use of a Gαi gene as a therapeutic target for osteoporosis. The present invention studied the expression of Gαi in osteoporosis patients and osteoclasts / osteoclast progenitor cells, and evaluated its effect on osteoclast formation and bone metabolism. By clarifying the specific mechanism of Gαi regulating bone metabolism and related signaling pathways, we confirmed that Gαi is a key mediator of RANKL signal transduction. In addition, we demonstrated that specific functional domains of Gαi play a key role in osteoclast differentiation. Notably, we found that the anti-osteoporosis effect of denosumab is impaired in the absence of Gαi, suggesting that Gαi is indispensable for its therapeutic effect. Our research results highlight Gαi as a potential target for the diagnosis and treatment of osteoporosis, paving the way for the development of novel therapeutic strategies. Brief Description of the Drawings

[0017] Figure 1 For Gαi participating in the development of osteoporosis, osteoclast formation and signal transduction; Figure 2 For conditional knockout of Gαi1 / 3 in myeloid cells can reduce OVX-induced bone loss; Figure 3 For double knockout of Gαi1 and Gαi3 inhibits rankl-induced osteoclast differentiation of BMMs; Figure 4 For overexpression of Gαi1 / 3 promotes rankl-induced osteoclastogenesis and signal transduction; Figure 5 For double knockout of Gαi1 and Gαi3 inhibits the activation of downstream signals of rankl-induced BMMs; Figure 6 For Gαi1 / 3 mediates the binding of RANK-TRAF6 through the 173 Asp residue, regulating the RANKL signaling pathway and osteoclastogenesis; Figure 7In BMMs with Gαi1 and Gαi3 mutations, rankl-induced signal transduction and osteoclast differentiation are inhibited; Figure 8 Gαi1 and Gαi3 mediate the binding of RANK and TRAF6 through NeE, promote downstream signal transduction, and affect osteoclast differentiation. Detailed implementation manners

[0018] The present invention provides the application of Gαi genes as therapeutic targets for osteoporosis.

[0019] In the present invention, the types of the Gαi genes are preferably Gαi1 and / or Gαi3.

[0020] The present invention also provides the application of Gαi proteins as diagnostic markers for osteoporosis.

[0021] In the present invention, the types of the Gαi proteins are preferably Gαi1 and / or Gαi3.

[0022] The present invention also provides the application of Gαi proteins in regulating osteoclast function and bone metabolism.

[0023] In the present invention, the regulation of osteoclast function is preferably the regulation of osteoclast differentiation, and the regulation of bone metabolism is the regulation of bone resorption; The types of the Gαi proteins are Gαi1 protein and / or Gαi3 protein.

[0024] The present invention also provides a method for regulating osteoclast differentiation and bone resorption, by knocking out or inhibiting the expression of Gαi1 and / or Gαi3 genes, or inhibiting the activity of Gαi1 and / or Gαi3 proteins, blocking the formation of the RANK-TRAF6 complex, thereby reducing osteoclast differentiation and bone resorption.

[0025] The present invention also provides a Gαi1 / 3 protein inhibitor for preventing or treating osteoporosis, and the Gαi1 / 3 protein inhibitor targets the Asp173 residue of the Gαi1 / 3 protein.

[0026] The present invention also provides the application of inhibitors of Gαi proteins in the preparation of drugs for preventing or treating osteoporosis.

[0027] The following combines examples to detail the technical solutions provided by the present invention, but they cannot be understood as limiting the protection scope of the present invention.

[0028] Example 1 Gαi1 / 3 is involved in osteoporosis and osteoclast formation

[0029] Ovariectomy (OVX) is a commonly used experimental model for studying osteoporosis. To detect the cellular localization of Gαi subunits in bone marrow, we performed single-cell sequencing on mouse bone marrow cells. A total of 14,123 bone marrow cells were analyzed, and through dimensionality reduction, clustering, and annotation, 9 different cell clusters were identified ( Figure 1 A). Among them, we identified 2,368 osteoclast precursors, osteoclasts, and osteoblasts. Notably, Gαi3 was highly expressed in osteoclast precursor cells and osteoclasts ( Figure 1 B-C).

[0030] Violin plots showed that the expression of Gαi3 increased in the bone marrow of OVX mice, and the expression level in osteoclasts was higher than that in osteoclast precursors ( Figure 1 D-E). In addition, bone marrow samples from osteoporosis patients were collected, and Western blot analysis showed increased expression of Gαi1 and Gαi3, while the expression of Gαi2 remained unchanged ( Figure 1 F). These findings suggest that Gαi subunits may be involved in osteoporosis and osteoclastogenesis.

[0031] We performed enrichment analysis on co-expressed genes (CEGs) significantly correlated with Gαi3, and the results showed that the CEGs significantly correlated with Gαi3 were mainly involved in biological processes such as "osteoclast differentiation", "organelle fission", and "nuclear chromosome segregation" ( Figure 1 G). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of Gαi3 CEGs revealed the top 20 pathways ( Figure 1 H), including pathways closely related to osteoclast formation such as "PI3K-Akt signaling pathway", "p65 signaling pathway", and "DNA replication" ( Figure 1 H). These results indicate that Gαi1 / 3 is involved in osteoporosis, osteoclast formation, and related signal transduction.

[0032] Example 2 Silencing of Gαi1 / 3 in BMDMs prevents OVX-induced osteoporosis

[0033] Previous single-cell sequencing and clinical data indicated that Gαi1 / 3 might be involved in the development of osteoporosis. To test this hypothesis, we generated Gαi1 / 3 conditional knockout mice (Gαi1 / 3oc-) in osteoclast precursor bone marrow-derived macrophages (BMMs). According to the previously described protocol, AAV9-CMV-FLEX-Cas9-U6-sgGαi1 (5 μL) and AAV9-CMV-FLEX-Cas9-U6-sgGαi3 were injected into the femoral marrow cavity of LysM-Cre C57BL / 6J mice, and Gαi1 / 3oc- mice were generated three weeks later. As a control, LysM-Cre mice were injected with AAV9-CMV-FLEX-Cas9-U6-sgC. After injection, Gαi1 and Gαi3 were effectively deleted in BMMs and their differentiated osteoclasts, and Western blot confirmed that the protein levels of Gαi1 and Gαi3 in BMMs of Gαi1 / 3oc- mice were significantly reduced, while the expression of Gαi2 remained unchanged ( Figure 2 A).

[0034] OVX was performed on Gαi1 / 3oc- and control mice (Gαi1 / 3oc+) to establish an osteoporosis mouse model. After 6 weeks, bone mass in the femoral metaphysis was analyzed using Micro-CT. 3D reconstruction images showed that bone loss was induced after ovariectomy, but this process was delayed after knockdown of Gαi1 / 3 ( Figure 2 B). In addition, compared with Gαi1 / 3oc+ OVX mice, the bone morphology-related parameters BV / TV, Tb.Th, and Tb.N increased, while Tb.Sp decreased in Gαi1 / 3oc- OVX mice ( Figure 2 C-G). Histological examination of the femur showed an increase in the number of TRAP-positive osteoclasts on the bone surface in OVX mice, while this increase was inhibited in Gαi1 / 3oc+ mice ( Figure 2 H-I). Immunofluorescence staining of NFATc1 in OVX bone tissue showed that the expression level was lower in Gαi1 / 3oc- mice than in Gαi1 / 3oc+ mice ( Figure 2 J, K). H&E staining showed that BMM-specific knockdown of Gαi1 / 3 did not affect the number of osteoblasts ( Figure 2 L,M).

[0035] To further investigate the role of Gαi1 / 3 in osteoclastogenesis in vivo, we generated myeloid cell-specific Gαi1 / 3-deficient mice (LysMCre; Gαi1 / 3fl / fl) by crossing LysMCre mice with Gαi1 / 3fl / fl mice and compared them with Gαi1 / 3fl / fl (control) mice. After ovariectomy, bone mass loss was not observed in LysMCre; Gαi1 / 3fl / fl mice. Consistent with the above results, the BMD, BV / TV, Tb. Th, and Tb.N values in LysMCre; Gαi1 / 3fl / fl mice increased, while Tb.Sp decreased. Bone morphometric analysis of the femur showed that the number of trabecular osteoclasts in LysMCre; Gαi1 / 3fl / fl mice was significantly lower than that in Gαi1 / 3fl / fl mice.

[0036] Example 3 RANKL-induced osteoclast development and function are abolished in BMMs derived from Gαi1 / 3-DKO mice

[0037] RANKL is an important regulator of osteoclast development and function. The role of Gαi1 / 3 protein in RANKL-induced osteoclastogenesis was investigated using bone marrow macrophages (BMMs) from wild-type (WT) and Gαi1 / 3 double-knockout (Gαi1 / 3-DKO) mice. The protein expression of Gαi1 and Gαi3 was significantly reduced in BMMs derived from Gαi1 / 3-DKO mice, while the expression of Gαi2 remained unchanged compared with BMMs from wild-type mice ( Figure 5 A).

[0038] Next, BMMs were differentiated into osteoclasts by adding RANKL, regardless of the presence of Gαi1 and Gαi3. TRAP staining was performed 6 days after induction of osteoclast differentiation. RANKL induced the differentiation of BMMs into osteoclasts, but the number of osteoclasts differentiated from BMMs derived from Gαi1 / 3-DKO mice was significantly reduced compared with WT mice ( Figure 3 A, B). By phalloidin staining, the area of the actin ring structure in Gαi1 / 3-DKO osteoclasts was significantly reduced ( Figure 3 C, D). The osteoclast function was evaluated by bone resorption assays. The bone resorption capacity of osteoclasts derived from Gαi1 / 3-DKO BMMs was reduced compared with WT BMMs, as shown by a decrease in the number of resorption pits and traces on the bone surface ( Figure 3 E, F).

[0039] NFATc1 (nuclear factor of activated T cells 1) is a major regulator of osteoclast differentiation, regulating multiple osteoclast-specific genes, including TRAP, cathepsin K, calcitonin receptor, and osteoclast-associated receptor (OSCAR). BMMs were isolated from mice and treated with RANKL for 1 hour. The expression of NFATc1 was detected by immunofluorescence. The results showed that the deletion of Gαi1 / 3 inhibited the expression of NFATc1 in BMMs ( Figure 3 G, H).

[0040] To further investigate the effect of Gαi1 / 3 on osteoclast formation and function, we knocked down Gαi1 and Gαi3 in BMMs. Lentiviral particles of Gαi1 shRNA and Gαi3 shRNA were transfected into BMMs. After puromycin selection, stable BMMs, designated "shGαi1 / 3" BMMs, were established. In shC BMMs, RANKL treatment promoted osteoclast formation. After silencing of Gαi1 and Gαi3, RANKL-induced osteoclast development was almost completely blocked. In addition, silencing of Gαi1 / 3 inhibited osteoclast resorption of bone tissue and the expression of NFATc1 in BMMs. It also blocked the expression of Ctsk, Mmp9, and Dc-stamp, which are closely related to osteoclast formation. RANKL (50 ng / mL, 30 minutes) treatment did not alter the expression of Gαi subunits in BMMs ( Figure 5 A). Collectively, these results indicate that Gαi1 / 3 plays a key role in RANKL-induced osteoclast formation and function. Gαi1 / 3 may be a key protein for the treatment and prevention of osteoporosis-related diseases.

[0041] Example 4 Overexpression of Gαi1 and Gαi3 Enhances RANKL-Induced Osteoclast Development and Bone Resorption Function

[0042] Since knockdown or knockout of Gαi1 / 3 significantly inhibited osteoclast development, function, and RANKL-induced signaling, we hypothesized that overexpression of Gαi1 and Gαi3 would have the opposite effect, enhancing osteoclastogenesis and bone resorption function. To test this, lentiviral particles carrying expression vectors of Gαi1 and Gαi3 were co-transfected into BMMs, and then a stable oeGαi1 / 3 cell line was established by puromycin selection. RANKL (50 ng / mL) induced osteoclast differentiation, and osteoclast formation was evaluated by TRAP staining and F-actin staining after 6 days. Overexpression of Gαi1 and Gαi3 promoted the formation of multinucleated osteoclasts. The number of TRAP-positive cells and the area of osteoclasts were both increased ( Figure 4 A-D).

[0043] In addition, overexpression of Gαi1 / 3 enhanced the bone resorption capacity of osteoclasts, resulting in an increase in the area of resorption pits on the bone plate. In addition, after RANKL stimulation, increased expression of NFATc1 was detected in oeGαi1 / 3 BMMs ( Figure 4 E-H).

[0044] Overexpression of Gαi1 / 3 also significantly upregulated the expression of key osteoclast-related genes in BMMs, including Ctsk, Mmp9, and Dc-stamp ( Figure 4 I-K). Given that overexpression of Gαi1 / 3 enhanced RANKL-induced osteoclast formation and function, we further investigated its effect on the molecular signaling pathway in BMMs. Consistent with expectations, the expression of Gαi1 and Gαi3 was significantly increased in oeGαi1 / 3 BMMs, while the expression of Gαi2 remained unchanged ( Figure 4 L, O). In addition, the phosphorylation of p65, Akt-S473, JNK, Erk1 / 2, and p38 induced by RANKL was significantly enhanced in oeGαi1 / 3 BMMs ( Figure 4 O-Q). These findings are consistent with the results in the Gαi1 / 3 knockdown or knockout models, jointly strengthening the key role of Gαi1 / 3 in RANKL-induced osteoclastogenesis and signal transduction.

[0045] Example 5 Gαi1 / 3 is a key protein regulator of RANKL signal transduction

[0046] RANKL binds to RANK on bone marrow monocytes and triggers osteoclastogenesis and promotes bone resorption by activating downstream pathways such as NF-κB and MAPK. To explore the role of Gαi1 / 3 in RANKL-induced signal transduction, BMMs from Gαi1 / 3 DKO mice were used. Western blot analysis confirmed that neither Gαi1 nor Gαi3 was detected in these cells ( Figure 5 A).

[0047] To evaluate RANKL-mediated signal transduction, BMMs were treated with RANKL (50 ng / mL) for 1, 5, 10, 20, or 60 minutes, and the activation of intracellular signaling proteins was analyzed. In WT BMMs, RANKL significantly increased the phosphorylation of p65 (Ser-2448), Akt-S473, JNK, Erk1 / 2, and p38, indicating that RANKL activated intracellular NF-κB, Akt, and MAPK signal transduction. However, in Gαi1 / 3-DKO BMMs, RANKL-activated NF-κB, Akt, and MAPK signal transduction was blocked at all tested time points ( Figure 5B-E). Among the tested time points, 30-minute RANKL treatment induced the strongest activation of these pathways in WT BMMs ( Figure 5 B-E).

[0048] To further investigate the effect of RANKL concentration, BMMs were treated with increasing concentrations of RANKL for 30 minutes. In WT BMMs, phosphorylation of p65 (Ser-2448), Akt-S473, JNK, Erk1 / 2, and p38 increased in a dose-dependent manner, indicating activation of the NF-κB, Akt, and MAPK cascades ( Figure 5 F-H). Notably, RANKL-induced activation of the NF-κB, Akt, and MAPK cascades was completely abolished in Gαi1 / 3-DKO BMMs ( Figure 5 F-J). The total levels of p65 (Ser-2448), Akt-S473, JNK, Erk1 / 2, and p38 were comparable in these BMMs ( Figure 5 I-J).

[0049] Evaluation of the individual role of Gαi1 or Gαi3 in RANKL-induced signaling showed that single knockout (SKO) of Gαi1 or Gαi3 in BMMs only partially reduced the phosphorylation of p65, Akt-S473, JNK, and Erk1 / 2 induced by RANKL (50 ng / mL) ( Figure 5 K, L). Only in Gαi1 / 3-DKO BMMs was RANKL-activated signaling completely blocked ( Figure 5 K,L). Gαi2 SKO had no significant effect on RANKL-induced signaling (data not shown). To further verify these findings, we examined whether RANKL-activated signaling was inhibited. After knocking down Gαi1 / 3 (shGαi1 / 3) in BMMs, RANKL-induced downstream signaling was significantly inhibited. Phosphorylation of p65, Akt-S473, JNK, Erk1 / 2, and p38 induced by RANKL was almost completely blocked in shGαi1 / 3 BMMs. Collectively, these results indicate that Gαi1 / 3 is a key regulator of RANKL-mediated signal transduction and plays an important role in the activation of the NF-κB, Akt, and MAPK pathways during osteoclastogenesis.

[0050] Example 6 Gαi1 / 3 Mediates RANK-TRAF6 Binding via the 173 Asp Residue, Regulating RANKL Signaling and Osteoclastogenesis

[0051] RANK-TRAF6 binding is crucial for the activation of RANKL signaling and subsequent osteoclastogenesis. To determine the role of Gαi1 / 3 in this process, we performed co-immunoprecipitation (Co-IP) experiments and extracted proteins after RANKL treatment (5 minutes) of BMMs. The results showed that RANKL stimulation promoted the formation of the RANK-Gαi1 / 3-TRAF6 complex, indicating the role of Gαi1 / 3 in promoting the RANK-TRAF6 interaction ( Figure 6 A).

[0052] To further verify this observation, confocal microscopy of BMMs showed that membrane-bound RANK bound to TRAF6 upon RANKL stimulation. However, the deletion of Gαi1 / 3 disrupted this interaction and prevented RANK-TRAF6 binding ( Figure 6 B). Consistently, in situ proximity ligation assay (PLA) confirmed that the direct interaction between endogenous RANK and TRAF6 was abolished in Gαi1 / 3-deficient BMMs ( Figure 6 C). These findings indicate that Gαi1 / 3 plays a key role in mediating RANK-TRAF6 binding, thereby affecting RANKL signaling and osteoclast function.

[0053] To characterize the molecular basis of the Gαi1 / 3-RANK interaction, we constructed truncated mutant plasmids using Gαi3 as a model ( Figure 6 D). Co-IP experiments confirmed the strong interaction between Gαi3 and RANK, further revealing that the G2 domain of Gαi3 is crucial for this interaction ( Figure 6 E). To identify the specific amino acids involved, mutation analysis of the 173-175 region showed that mutations in this sequence disrupted the Gαi3-RANK interaction, indicating that this region is crucial for binding ( Figure 6 F).

[0054] In addition, three-point mutant plasmids targeting the 173-175 sequence were generated, and Co-IP experiments showed that Asp173 of Gαi3 was crucial for RANK binding ( Figure 6 G). To functionally validate these findings, we employed a dominant-negative (DN) strategy, in which the DN-Gαi1 / 3 construct replaced the conserved 173-Asp residue with Thr. Co-transfection of the DN-Gαi1 and DN-Gαi3 constructs into WT BMMs showed a significant reduction in RANK-TRAF6 binding in DN-Gαi1 / 3 BMMs ( Figure 6 H).

[0055] Importantly, RANKL-induced osteoclastogenesis and bone resorption were abolished in DN-Gαi1 / 3 BMMs, indicating the crucial role of Gαi1 / 3 in osteoclast differentiation and function. In addition, DN-Gαi1 / 3 significantly reduced the expression of key osteoclast-related genes, including Ctsk, Mmp9, and Dc-stamp, which are essential for osteoclast differentiation and resorptive activity. Furthermore, RANKL-induced activation of the NF-κB, Akt, and MAPK cascades was significantly inhibited in DN-Gαi1 / 3 BMMs, further confirming the crucial role of Gαi1 / 3 in RANKL-mediated signaling.

[0056] In summary, our results indicate that the 173 Asp residue of Gαi1 / 3 is a key site mediating RANK-TRAF6 binding. Mutation of this residue significantly disrupted RANKL-induced signaling, osteoclast differentiation, and bone resorption, highlighting Gαi1 / 3 as a key regulator of osteoclastogenesis.

[0057] Example 7 Inhibition of Gαi1 / 3 exhibits anti-osteoporotic effects comparable to those of denosumab analogs

[0058] Denosumab is a clinically approved monoclonal antibody that effectively treats osteoporosis by binding to RANKL, thereby preventing its interaction with RANK on osteoclast precursor cells and inhibiting osteoclastogenesis and bone resorption. However, as a humanized antibody, denosumab may trigger immune responses in mice after long-term use. To address this limitation, we employed a denosumab analog (IK22 / 5, 8 mg / kg, anti-mouse RANKL antibody) to evaluate its anti-osteoporotic effects as a denosumab substitute.

[0059] Osteoporosis was induced in Gαi1 / 3oc+ and Gαi1 / 3oc- mice and then treated with the denosumab analog. After 6 weeks, micro-CT was used to analyze the bone mass at the femoral metaphysis. 3D reconstruction images showed that the denosumab analog delayed bone loss in Gαi1 / 3oc+ mice, and the process of bone loss was also slowed after Gαi1 / 3 knockdown. Notably, the denosumab analog did not exhibit additional anti-osteoporotic effects in Gαi1 / 3oc- mice ( Figure 7 A).

[0060] In addition, compared with the control group of Gαi1 / 3oc- OVX mice, the bone morphometric parameters, including BMD, BV / TV, Tb.N, Tb.Th, and Tb.Sp, did not change significantly in the denosumab analog group ( Figure 7B - G). To further evaluate osteoclast activity, histological analysis of the femur was performed. In the control group of Gαi1 / 3oc - OVX mice, the number of Trap+ osteoclasts on the bone tissue surface was reduced, and the denosumab analogue did not further reduce the osteoclasts on the bone surface ( Figure 7 H - I). These findings indicate that inhibiting Gαi1 / 3 has an anti - osteoporosis effect comparable to that of the denosumab analogue.

[0061] Discussion

[0062] Our study highlights the crucial role of Gαi1 and Gαi3, key members of the Gαi protein family, in the pathogenesis of osteoporosis. Increasing evidence suggests that Gαi proteins are involved in regulating immune cell function and inflammatory responses. In addition, dysregulation within the Gαi subfamily may affect bone metabolism. However, the exact role of Gαi proteins in osteoporosis remains unclear. In this study, we demonstrated that Gαi proteins play a crucial role in osteoclast differentiation and bone resorption, and investigated the molecular mechanisms by which Gαi mediates the occurrence and development of osteoporosis. In addition, we compared the effects of modulating Gαi protein activity with existing therapeutic drugs (such as denosumab) in the management of osteoporosis. Importantly, in vivo inhibition of Gαi function significantly delayed the onset of osteoporosis, producing an effect comparable to denosumab treatment.

[0063] Gαi proteins were initially classified based on their ability to inhibit adenylate cyclase. Epinephrine, acetylcholine, dopamine, and serotonin utilize Gαi proteins to trigger physiological responses. After binding to GPCRs, Gαi proteins are activated and subsequently release Gβγ subunits, which directly couple to downstream signaling pathways. Previous studies have shown that G proteins are key immunomodulatory factors that regulate the migration, activation, survival, proliferation, differentiation, and cytokine secretion of immune cells, including macrophages. In our study, we observed an increase in Gαi3 expression during the differentiation of mononuclear macrophages into osteoclasts, and a significant increase in Gαi1 / 3 expression in the bone marrow of osteoporosis patients. Consistently, in the OVX mouse model, compared with the control group, the formation of osteoclasts and bone mass loss were significantly reduced in Gαi1 / 3 - DKO and Gαi1 / 3oc - mice, thus confirming that Gαi proteins are key regulators of osteoclast function and bone metabolism. Although single - cell sequencing did not show a significant change in Gαi1 expression, differences were detected in the bulk analysis of osteoporosis patients, which may be due to single - cell resolution and computational limitations. Gαi1 and Gαi3 have 85 - 95% amino acid sequence identity and exhibit considerable structural similarity. Both proteins play a crucial role in the development of osteoporosis, and it remains challenging to determine the major contributor. This issue warrants further investigation.

[0064] RANKL is a key molecule that links the skeletal and immune systems. RANKL stimulates the activation of downstream signaling pathways of RANK, including NF-κB and MAPK. This triggers the differentiation of macrophages into osteoclasts. Subsequently, multinucleated osteoclasts are formed by the fusion of pre-osteoclasts, preparing for bone resorption. This study reveals a new mechanism by which Gαi1 / 3 plays a role in RANKL-mediated signal transduction, which is the basis of osteoclast formation. We demonstrate that Gαi1 / 3 is required for RANKL-induced activation of NF-κB and MAPK. In BMDMs, human macrophages, and MEFs, Gαi1 / 3 knockout (KO), shRNA, CRISPR-induced Gαi1 / 3 gene editing, or dominant-negative Gαi1 / 3 mutations strongly inhibited RANKL-induced activation of NF-κB and MAPK. Conversely, overexpression of Gαi1 / 3 enhanced RANKL-induced activation of NF-κB and MAPK. Functional studies showed that Gαi1 / 3-deficient BMDMs were resistant to RANKL-induced osteoclast formation and bone resorption, while Gαi1 / 3-overexpressing BMDMs showed enhanced osteoclast fusion and bone resorption upon RANKL stimulation. Our results support that Gαi1 / 3 is essential for RANKL-RANK signaling, osteoclastogenesis, and bone resorption. RANKL is a key central regulator of the differentiation of monocytes / macrophages into osteoclasts. M-CSF is a key cytokine that induces the expression of RANK on the cell membranes of monocytes / macrophages and osteoclast precursors. Osteoclast precursors expressing RANK bind to RANKL, thereby inducing osteoclast differentiation.

[0065] Although Gαi1 / 3 is crucial for osteoclast differentiation and RANKL signaling, its mechanism of action remains unclear. Previous studies have shown that Gαi1 / 3 interacts with the receptors CD146 and TrkB to activate downstream signaling. Our study confirmed that RANKL promotes the binding of Gαi1 / 3 to RANK-TRAF6, and the deletion of Gαi1 / 3 prevents the formation of this complex, hindering downstream signaling. Gαi1 / 3 plays a key role in the binding of RANK-TRAF6, and inhibitory Gα subunits have 85-95% sequence identity and overlapping expression patterns. We performed a mutational analysis of Gαi3 and found that Asp173 is crucial for mediating the interaction with RANK. In addition, a dominant-negative mutation of Asp173 (DN-Gαi1 / 3) inhibited the binding of RANK-TRAF6, thereby suppressing RANKL-induced signaling and osteoclast differentiation. This enhanced our understanding of the role of Gαi1 / 3 in osteoporosis and paved the way for the development of anti-osteoporosis therapies. Although the in vivo significance of Asp173 in this context has not been verified, future studies should address this limitation and explore the potential of small molecules targeting Gαi1 / 3 in the treatment of osteoporosis.

[0066] Denosumab, a humanized monoclonal antibody against RANKL, is a commonly used anti-osteoporosis therapeutic agent. Our results showed that denosumab analogs had a stronger anti-osteoporosis effect in mice with normal osteoclast-specific Gαi1 / 3 expression (Gαi1 / 3oc+). In contrast, osteoporosis in Gαi1 / 3oc- mice was inhibited, but no additional anti-osteoporosis effect of denosumab analogs was observed. These findings suggest that targeted inhibition of Gαi1 / 3 may be as effective as denosumab in the treatment of osteoporosis. Although denosumab is effective, it rapidly activates osteoclasts after discontinuation, accelerating bone loss and increasing the risk of fractures. Elderly, dialysis-dependent female patients are prone to hypocalcemia after denosumab treatment. These limitations restrict the widespread use of denosumab. Targeting Gαi1 / 3 may lead to more effective anti-osteoporosis treatment with fewer side effects. The combination of Gαi1 / 3 inhibitors and denosumab may enhance the anti-osteoporosis effect with fewer side effects. Studies on the effects of denosumab usually require huRANKL transgenic mice. In the absence of such models, we used a mouse-specific RANKL antibody as a substitute for denosumab, which may not fully reproduce the clinical anti-osteoporosis effect of denosumab.

[0067] This study emphasizes the necessity of further exploring the long-term anti-osteoporotic effects and potential side effects of Gαi1 / 3 inhibition. This study was only conducted in mice, and its results cannot be directly generalized to primates. Clinical translation remains distant. However, the main contribution is the identification of a new target for anti-osteoporotic drug development, which may improve the diagnosis and treatment of osteoporosis.

[0068] Single-cell RNA sequencing and experiments revealed elevated levels of Gαi subunits in osteoclasts and bone marrow in osteoporosis patients. Deletion of Gαi1 / 3 significantly inhibited osteoclast differentiation and bone resorption. We elucidated the mechanism of osteoporosis mediated by Gαi1 / 3 through promoting RANK-TRAF6 binding. Notably, the Asp173 residue in Gαi1 / 3 was identified as crucial for its interaction with RANK. These findings may guide the future development of anti-osteoporotic drugs. Ultimately, Gαi1 / 3 emerges as a promising therapeutic target for clinical application.

[0069] Conclusion

[0070] The research of this invention demonstrates that Gαi1 and Gαi3 are key regulators of osteoclast differentiation and bone resorption in osteoporosis. Using in vitro and in vivo models, it was demonstrated that deletion of Gαi1 / 3 disrupted RANKL-induced NF-κB and MAPK activation by impairing the formation of the RANK-TRAF6 complex, and Asp173 was identified as the key residue mediating this interaction. These findings enhance the understanding of the molecular mechanisms of osteoporosis and highlight the potential of targeting Gαi1 / 3 as an alternative therapeutic strategy to existing treatments such as denosumab. Despite being limited by the model system and sample size, the results of this invention provide valuable insights for future studies to validate these mechanisms in a clinical setting and guide policy decisions for osteoporosis management.

[0071] As can be seen from the above embodiments, this invention provides an application of the Gαi gene as a therapeutic target for osteoporosis. This invention studied the expression of Gαi in osteoporosis patients and osteoclasts / osteoclast progenitor cells and evaluated its effects on osteoclast formation and bone metabolism. By elucidating the specific mechanism of Gαi regulating bone metabolism and related signaling pathways, we confirmed that Gαi is a key mediator of RANKL signal transduction. In addition, we demonstrated that a specific functional domain of Gαi plays a key role in osteoclast differentiation. Notably, we found that the anti-osteoporotic effect of denosumab was impaired in the absence of Gαi, suggesting that Gαi is indispensable for its therapeutic effect. Our research results emphasize Gαi as a potential target for the diagnosis and treatment of osteoporosis, paving the way for the development of novel therapeutic strategies.

[0072] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of the Gαi gene as a therapeutic target for osteoporosis.

2. The application according to claim 1, wherein The types of the Gαi gene are Gαi1 and / or Gαi3.

3. Use of the Gαi protein as a diagnostic marker for osteoporosis.

4. The application according to claim 3, characterized in that, The types of the Gαi protein are Gαi1 and / or Gαi3.

5. Use of the Gαi protein in regulating osteoclast function and bone metabolism.

6. The application according to claim 5, characterized in that, The regulation of osteoclast function is the regulation of osteoclast differentiation, and the regulation of bone metabolism is the regulation of bone resorption; The types of the Gαi protein are Gαi1 protein and / or Gαi3 protein.

7. A method for regulating osteoclast differentiation and bone resorption, characterized in that, By knocking out or inhibiting the expression of the Gαi1 and / or Gαi3 gene, or inhibiting the activity of the Gαi1 and / or Gαi3 protein, the formation of the RANK-TRAF6 complex is blocked, thereby reducing osteoclast differentiation and bone resorption.

8. A Gαi1 / 3 protein inhibitor for preventing or treating osteoporosis, characterized in that, The Gαi1 / 3 protein inhibitor targets the Asp173 residue of the Gαi1 / 3 protein.

9. Use of an inhibitor of the Gαi protein in the preparation of a drug for preventing or treating osteoporosis.

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