Calcium Channel TRPV6 is a Novel Regulator in RANKL-induced Osteoclastic Differentiation and Activity through the IGF–PI3K–AKT Pathway

Background: Calcium ion signals are important for osteoclast differentiation. Transient receptor potential vanilloid 6 (TRPV6) is a novel regulator of bone homeostasis. However, it is unclear whether TRPV6 was involved in the process of osteoclast formation. Results: In the present study, we found that knockout of the TRPV6 gene induced osteoporosis in mice, and depletion of TRPV6 did not affect bone formation but signicantly enhanced bone resorption. Further studies showed that TRPV6, distributed on the cell membrane of osteoclasts, was a negative regulator of osteoclast differentiation and function. Mechanistically, TRPV6 suppressed osteoclastogenesis by decreasing the production the IGF1R and AKT ligands to inhibit the IGF–PI3K–AKT signaling pathway. Blocking the IGF–PI3K–AKT pathway signicantly reduced the effect of TRPV6 on osteoclasts. Conclusion: Our study conrmed the important role of TPRV6 in bone metabolism and claried its regulatory role on osteoclasts at the cellular level, which may provide a new strategy for the treatment of osteoporosis. IGF


Background
Osteoporosis is a very common global metabolic bone disease characterized by decreased bone mineral density and bone mass [1,2] . The decreasing of bone mass is related to the abnormal differentiation and proliferation of osteoclasts and the function of bone resorption, so osteoclasts are considered to be a target cell for treating osteoporosis [3][4][5] . Despite the regulation of osteoclast formation and function by numerous cytokines and hormones, the macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) are the most critical molecules for osteoclastogenesis [6][7][8][9] . Calcium ion (Ca 2+ ) channels are also essential for osteoclast differentiation [10] . Extensive studies have indicated that RANKL induced oscillatory changes in intracellular Ca 2+ concentrations and activated nuclear factor of activated T cells c1 (NFATc1), resulting in osteoclast-speci c gene transcription to induce osteoclast differentiation [11,12] . However, the exact calcium signals involved in RANKL-induced osteoclast differentiation need further clari cation.
Transient receptor potential vanilloid (TRPV) is a family of calcium transport proteins. Of which only TRPV5/6 are highly selective calcium channel proteins [13][14][15] . Recent studies have found that TRPV6 is an important calcium channel involved in the regulation of bone metabolism [16][17][18][19] . However, the speci c molecular events remain elusive and need to be further explored.
The IGF signaling pathway widely exists in bone tissue and is involved in the regulation of bone metabolism [20,21] . Many studies have indicated that IGF1R and IGF1 proteins are expressed in osteoclasts, and IGF1R is involved in osteoclast differentiation [22][23][24] . However, whether the IGF signaling pathway is involved in the regulation of osteoclast formation and bone resorption function by TRPV6 remains unclear.
In this study, we observed the potential effect and mechanism of TRPV6 in bone metabolism, osteoclast formation and bone resorption activity. In in vivo studies, we found that the depletion of TRPV6 results in severe osteoporosis in TRPV6 gene knockout mice. Inactivation of TRPV6 did not affect bone formation but signi cantly enhanced bone resorption. The in vitro studies revealed that TRPV6 was an important negative regulator for osteoclast differentiation and bone resorption by inhibiting the IGFIR-PI3K-AKT pathway.

Materials And Methods
In vivo treatment Eight-week-old TRPV6 gene knockout mice and wild-type mice (from the Laboratory Animal Center of the Second Military Medical University) were used in this study. All of the mice were housed in rearing cages ( ve mice per cage) and kept under standard laboratory conditions (12-hour light-dark cycle; 25°C). Experiments were approved by Medical Ethics Committee of the Second Military Medical University.

Skeletal phenotyping
To assess bone density and trabecular micro architecture. The distal femurs were taken for scanning using micro-computed tomographic imaging (SkyScan, Aartselaar, Belgium). The scanning analysis area was the trabecular portion of the proximal femoral growth plate from 2 mm downward. Microarchitectural parameters include bone mineral density (BMD), trabecular number (Tb.N) and Bone Volume/Total Volume (BV/TV).

Histological analysis
The harvested femurs were xed in 4% paraformaldehyde solution, and decalci ed in EDTA buffer saline solution. Sagittal tissue sections (10 μm thick) were prepared for immunohistochemical. HE staining, Masson trichromic staining and TRAP staining were performed afterward to assess the histological changes.

Dynamic bone formation
The femurs from mice were dehydrated with different concentrations of alcohol (70% to 100%), and embedded in methyl-methacrylate (MMA; Sigma-Aldrich, St. Louis, MO, USA). The bone tissue was then cut and polished to a thickness of about 10 mm. The sections were imaged using a uorescent microscope (Carl Zeiss, Oberkochen, Germany). The mineral apposition rate (MAR) was calculated using histomorphometric data.

Measurements of plasma ALP and CTX-1
Plasma ALP and CTX-1 concentrations were determined using ELISA kits (mlbio, Shanghai, China). Samples were incubated on microporous plates for 2 hours. Next, the same volume of primary antibody against ALP or CTX-1 was added to each sample and incubated for another hour. This was followed by a half-hour incubation with secondary antibodies. Finally, a uorescence microplate reader was used detect OD values (450 nm).

Cell culture
Bone marrow cells were isolated from the femur and tibiae of C57BL/6 or TRPV6 gene knockout C57BL/6 mice. Cells were cultured in a-MEM medium supplemented with M-CSF for 1 day. The unadherent cells were harvested and cultured in a medium containing M-CSF (50 ng/mL) for 3 days. Then the adherent cells, which were preosteoclasts, were collected. Next, a-MEM medium containing M-CSF (30 ng/mL) and RANKL (50 ng/mL) was added and allowed to incubate for 7 days to generate mature osteoclasts.
Tartrate-resistant acid phosphatase staining TRAP staining is the most common staining method to mark mature osteoclasts. A leukocyte acid phosphatase kit was used to x and stain the cells according to the manufacturer's instructions. Mature osteoclasts (with more than three TRAP-positive nuclei) appeared dark red and were counted by an optical microscope.

Pit formation assay
Pit formation assay was used to assess bone resorption activity. Bovine cortical bone was polished into thin slices (200 µm thick). Cell slices were then cultured on 24-well plates and induced by M-CSF and RANKL. Seven days later, the sections were placed in sterile water and washed using ultrasound for 10 min to remove the cells. This was followed by staining with toluidine blue for 5 min. The percentage of pit areas was quanti ed by the ImageJ software.

Cell immuno uorescence
BMMs were induced by M-CSF and RANKL for 7 days. Then The culture medium was washed with PBS and the cells were xed with 4% paraformaldehyde for 20 min. Afterwards, 0.2% triton-x100 was applied for 5 min to induce cell permeability. After washing the permeable solution with PBS, the permeable solution was blocked with 5% BSA for half an hour. Additionally, the primary antibodies (TRPV6, IGFIR or p-AKT) were added to incubate the cells overnight at 4°C. Then, the PBS washed off the primary and upper corresponding secondary antibodies for 30 min at 37°C. After the second antibody was washed, the cells were nally xed with an anti-fading xing medium. Fluorescence was observed by confocal laser scanning microscope (Leica, Germany).

Lentiviral transduction and oligonucleotide transfection
Osteoclast precursors were seeded into the 6-well plates with a density of 2×10 5 and transfection began when the cell fusion rate reached approximately 60%. Cells were infected with TRPV6 shRNA lentiviral particles or TRPV6 lentiviral activation particles (Santa Cruz, CA, USA) separately for 24 hours. The culture medium was then replaced with a normal osteoclast induction medium.

Quanti cation of mRNA and qPCR
Total RNA was isolated from cells using TRIzol reagent (Takara Biotechnology, Japan), and cDNA was synthesized from RNA with the miScript Reverse Transcription Kit (Takara, Tokyo, Japan) according the manufacturer's protocols. qRT-PCR was carried out with the SYBR Green PCR kit (Takara Biotechnology, Japan) and sequence detection was performed using an ABI 7500 Sequencing Detection System (Applied Biosystems, Foster City, CA). β-actin was used as the housekeeping gene. The primers used are described in Table 1.

Western blot analysis
Proteins the from osteoclast lysates were introduced to 10% SDS-PAGE, and were subsequently electroblotted onto a polyvinylidene uoride membrane. The membrane was blocked with 5% fat-free milk and incubated with appropriate antibodies. Chemiluminescence reagents (Thermo Scienti c, Rockford, IL) were used to detected antigen-antibody complexes. ImageJ software was used to quantify the immunoblots.

Statistical analysis
Data are shown as mean ± SD. SPSS24.0 statistical software was used for statistical analysis. The two tailed non-paired Student's t test and one-way ANOVA were used to compare differences. Statistical signi cance was set at P<0.05 and very signi cance was P<0.01.

Results
Knockout of the TRPV6 gene induces osteoporosis in mice TRPV6 gene knockout mice were used in this study to investigate the biological role of TRPV6 in bone metabolism. To con rm whether the TRPV6 depletion mouse model was successfully constructed, we compared the transcription levels of mRNA TRPV6 in bone marrow monocytes from wild-type mice and TRPV6 −/− mice. The result showed that the expression of TRPV6 was signi cantly depleted in TRPV6 −/− mice. Furthermore, there were no signi cant differences in the expression of mRNA TRPV2, mRNA TRPV4 and mRNA TRPV5 in bone marrow monocytes from TRPV6 −/− mice and WT mice (Fig. 1A). At 12 weeks old, mice were euthanized and femur cancellous bone was analyzed by μCT. The results revealed that TRPV6 −/− mice had remarkably decreased BMD (Fig. 1B). Representative μCT images of trabecular bone are presented in Fig. 1C. Analysis of the trabecular structure revealed that the Tb.N and BV/TV were signi cantly lower in TRPV6 −/− mice ( Fig. 1D-E), which is consistent with the results of HE staining (Fig.  1F).
In TRPV6 knockout mice, bone absorption was enhanced and bone formation remained unchanged Abnormal bone formation or resorption leads osteoporosis. At the beginning of this study of the effect of TRPV6 deletion on osteogenic function, the number of osteoblasts (N.Ob/BS) in TRPV6 −/− mice was similar to that in WT mice, which was calculated using Masson trichromic staining ( Fig. 2A, B). Similar results were attained for the tetracycline double-standard staining, and the mineral apposition rate (MAR) was comparable between the two groups (Fig. 2C, D). In addition, there was no signi cant difference in serum ALP concentration between TRPV6 −/− mice and WT mice (Fig. 2E). TRAP staining in the bone sections further revealed the effect of TRPV6 depletion on osteoclastogenesis (Fig. 2F). Quanti cation showed that the number and surface area of osteoclasts in TRPV6 −/− mice increased signi cantly (Fig.   2G). Compared with WT mice, TRPV6 −/− mice had higher levels of CTX-1. To summarize, these results suggest that bone absorption was enhanced and bone formation remained unchanged in TRPV6 −/− mice.

TRPV6 negatively regulates osteoclast differentiation and fusion, inhibiting osteoclast formation
The results of western blotting suggest that TRPV6 is expressed in osteoclasts. The expression of TRPV6 in osteoclasts was reduced during cell differentiation (Fig. 3A). In addition, immunocytochemical uorescence indicated that TRPV6 mainly distributes on the membrane of mature osteoclast (Fig. 3B). For deeper insight into the role of TRPV6 on osteoclast function, TRAP staining was rst applied, in cultures derived from TRPV6 −/− mice, and the number of TRAP + multinucleated osteoclasts were signi cantly higher (Fig. 3C-D). A bone resorption lacuna experiment result showed that resorption pits were signi cantly enhanced in TRPV6 −/− osteoclasts ( Fig. 3E-F). In addition, PCR results revealed that the mRNA levels of marker genes for osteoclastogenesis (cathepsin k, DC-STAMP, Atp6v0d2 and TRAP) were obviously upregulated in TRPV6-depleted osteoclasts ( Fig. 3G-J).

Silencing of TRPV6 enhanced osteoclast formation
To further verify the role of TRPV6 on osteoclast formation and bone resorption, we silenced and overexpressed TRPV6 gene in osteoclasts by lentivirus transfection. Almost all cells expressed GFP, indicating that the lentivirus transfection rate was more than 95% (Fig. 4A). Western blotting con rmed that TRPV6 was effectively silenced and overexpressed (Fig. 4B). The mRNA levels of cathepsin k, DC-STAMP, Atp6v0d2 and TRAP were all increased in osteoclasts infected with lenti-shRNA-TRPV6, whereas were all decreased in the group of osteoclast infected with lenti-pMX-TRPV6 (Fig. 4C-F). Next, we assessed the differentiation of osteoclasts with silenced or overexpressed TRPV6 by TRAP staining assay. The number of stained multinuclear TRAP + osteoclasts was obviously increased in osteoclasts with silenced TRPV6 in a time-dependent manner, whereas osteoclasts showed decreases in the TRPV6overexpressed group (Fig. 4G-H). The above results strongly suggest that TRPV6 was a negative regulator of osteoclast differentiation and function.
The IGF pathway is involved in the negative regulation of osteoclast formation and resorption by TRPV6 Our previous study revealed that the RANKL-induced [Ca 2+ ]i oscillation response was not signi cantly affected by inhibition of TRPV6 [16] . As a non-ca 2+ oscillating signaling pathway, the IGF pathway plays an important role in osteoclast formation [25][26][27] . Therefore, we speculated that the IGF signaling pathway was potentially involved in the regulation of osteoclast differentiation and bone absorption by TRPV6. The results showed that the levels of IGF1R and IGFBP1 mRNA and protein in osteoclasts were signi cantly increased after TRPV6 gene silencing (Fig. 5A-D), which con rmed our speculation. Next, we used an IGF1R antagonist NVP-AEW541 to block the IGF signaling pathway, TRAP staining demonstrated that the inhibitory effect of TRPV6 on osteoclast differentiation was weakened in osteoclasts with blockers ( Fig. 5E-F). In accordance with the results for TRAP staining, the IGF1R blocker signi cantly inhibited the induction of the bone resorption of osteoclasts by silencing of TRPV6, as evidenced by the pit formation assay (Fig. 5G-H). These results strongly suggest that TRPV6 negatively regulated osteoclast formation and bone resorption by inhibiting the IGF pathway.
TRPV6 negatively regulates osteoclast formation and resorption by inhibiting the IGF1R-PI3K-AKT pathway As assessed by immuno uorescence, we found that osteoclasts highly expressed TRPV6 and IGF1R in the membrane and cytoplasm [ Fig. 6A]. To further explore the downstream signaling of the IGF pathway, BMMs isolated from TRPV6 -/and WT mice were induced by RANKL and M-CSF for 7 days to form mature osteoclasts. The levels of phosphorylated markers P85/p-P85, PDK1/p-PDK1 and AKT/p-AKT in the PI3K-AKT pathway were detected by western blotting. The results showed that the ratios of P85/p-P85, PDK1/p-PDK1 and AKT/p-AKT were increased in osteoclasts isolated from TRPV6 -/mice compared with WT mice [ Fig. 6B-E]. Consistent with the western blotting results, immuno uorescence showed that the ratio of PDK1/p-PDK1 of osteoclasts isolated from TRPV6 -/mice was higher than that of osteoclasts isolated from WT mice [ Fig. 6F-G]. Next, we used NVP-AEW541 to block the IGF signaling pathway and western blotting revealed that there was no signi cant difference in the level of AKT/p-AKT between the osteoclasts derived from TRPV6 -/mice and WT mice [ Fig. 6H-I].

Discussion
In the present study, we demonstrated that TRPV6 was a critical negative regulator in RANKL-induced osteoclast differentiation and activity. Our studies showed that TRPV6 decreased osteoclast formation and bone resorption by inhibiting the IGF-PI3K-AKT signaling pathway (Fig. 7).
Bone metabolism maintains bone mass through complex regulation of the balance between bone resorption and formation [28][29][30][31] . As a highly selective calcium channel in the TRPV subfamily, TRPV6 is an important protein in osteoclasts and participates in the regulation of osteoclast differentiation and bone resorption [16] . Bianco et al [32] found that TRPV6 -/mice had obvious bone metabolism disorders, manifested as intestinal calcium absorption dysfunction and decreased bone density. Lieben et al reported that the bone mass of TRPV6 -/mice fed a low calcium diet decreased signi cantly and bone resorption and bone formation were enhanced simultaneously, with bone resorption being more active than bone formation [33] . In this study, we found that BMD, Tb.N and BV/TV were obviously decreased in TRPV6-depleted mice, suggesting that TRPV6 is involved in the regulation of bone metabolism. Furthermore, Masson trichromic staining showed that N.Ob/BS was not decreased in TRPV6 -/-. Similar results were revealed by tetracycline double-standard staining, MAR was comparable between the two groups; however, TRPV6 -/mice had signi cantly more TRAP positive cells in the metaphyseal region of femoral bone sections than WT mice. Therefore, based on the above ndings, we believed that TRPV6 contributes to bone homeostasis through the regulation of osteoclast differentiation and activity.
Previous studies have con rmed that in the presence of M-CSF and RANKL su cient to maintain cell survival and induce complete osteoclastic differentiation [34,35] . Several reports showed that TRPV6 regulates a variety of cellular functions, including differentiation, proliferation and apoptosis, by affecting intracellular Ca 2+ concentration [36] . The increased concentration of Ca 2+ in osteoclasts causes the cell pseudopodia to retract, restrict its movement, destroy the absorption skeleton structure, and thus inhibit bone absorption [37] . In our study, we found that TRPV6 expression was decreased in a time-dependent manner during the process of osteoclast differentiation, and the distribution of TRPV6 in osteoclasts was identi ed as being mainly distributed on the cell membrane. In addition, TRAP staining and a bone absorption pit experiment showed that the differentiation and fusion of osteoclasts isolated from TRPV6 -/mice was signi cantly quicker than that from WT mice. We also found that silencing TRPV6 in osteoclasts signi cantly increased osteoclastogenesis. Thus, it is likely that the inhibitory role of TRPV6 in osteoclastic resorption is caused by decreasing osteoclastogenesis.
Both independent signaling pathways and calcineurin-dependent pathways contribute to NFATc1 activation, leading to e cient osteoclastogenesis [38,39] . Our previous research found that non-Ca 2+ oscillation signaling pathways contribute to TRPV6 de ciency-induced osteoclastogenesis [16] . As an important non-Ca 2+ oscillating pathway, the IGF signaling pathway is widely expressed in bone tissue and participates in the regulation of bone metabolism [40,41] . Dai et al . [42] found that TRPV5 negatively regulated the proliferation of NaR cells by inhibiting the IGF signaling pathway in culture medium with a normal calcium concentration. Our results showed that silencing of TRPV6 signi cantly increased the expression of two key proteins of the IGF signaling pathway, IGF1R and IGFBP1, in osteoclasts. Blocking IGF1R suppresses the increase of osteoclastic differentiation induced by inhibition of TRPV6. Taken together, we presume that TRPV6 is responsible for osteoclast formation and resorption through inhibition of the IGF signaling pathway.
As a downstream signaling pathway of the cascade reaction of the IGF signaling pathway, PI3K-AKT plays an important role in the activation of osteoclasts. AKT promotes osteoclast survival by regulating cell cytoskeletal replacement and movement, and knockdown AKT reduces the expression of osteocalcin [43] . Lee et al. [44] revealed that PI3K, p38 and extracellular signal-regulated kinase pathways were involved in osteoclast differentiation. Xing et al. reported that the PI3K-AKT signaling pathway was involved in RANKL-independent osteoclastogenesis [45] . In the present study, TRPV6 inhibited the phosphorylation level of PI3K-AKT in osteoclasts and reduced the expression of P85/p-P85, PDK1/p-PDK1 and AKT/p-AKT. The effect of TRPV6 was eliminated after pretreatment with an IGF1R inhibitor (NVP-AEW541).
In conclusion, our study con rmed the important role of TPRV6 in bone metabolism, clari ed its regulatory role on osteoclasts at the cellular level, and revealed the molecular mechanism of TRPV6's negative regulation of osteoclast formation and bone absorption. These results may provide a new strategy for the treatment of osteoporosis.

Declarations
Ethics approval and consent to participate: Experiments were approved by Medical Ethics Committee of the Second Military Medical University.
Consent for publication: Not available.
Availability of data and materials: All data generated or analyzed during this study are included in this published article (and its additional les).
Competing interests: The authors declare that they have no competing interests.
Funding: This study was sponsored by the National Natural Science Foundation of China (No. 81672202 and No.81874003), the "Outstanding Young Scholar Program" and the "Doctoral innovation fund" supported by Second Military Medical University, and the "Research Clinician Program" supported by Shanghai Changzheng Hospital.
Authors' contributions: JM conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; LZ, ZBZ and TFX: collection and/ or assembly of data, data analysis; LY : data analysis ; XY, AMC: conception and design, data analysis and interpretation, nancial support, manuscript writing; TWY: conception and design, data analysis and interpretation, nancial support, manuscript writing, nal approval of manuscript. All authors read and approved the nal manuscript. Furthermore, there were no signi cant differences in the expression of mRNA TRPV2, mRNA TRPV4 and mRNA TRPV5 in bone marrow monocytes from TRPV6−/− mice and WT mice (Fig. 1A). At 12 weeks old, mice were euthanized and femur cancellous bone was analyzed by μCT. The results revealed that Abnormal bone formation or resorption leads osteoporosis. At the beginning of this study of the effect of TRPV6 deletion on osteogenic function, the number of osteoblasts (N.Ob/BS) in TRPV6−/− mice was similar to that in WT mice, which was calculated using Masson trichromic staining ( Fig. 2A, B). Similar results were attained for the tetracycline double-standard staining, and the mineral apposition rate (MAR) was comparable between the two groups (Fig. 2C, D). In addition, there was no signi cant difference in serum ALP concentration between TRPV6−/− mice and WT mice (Fig. 2E). TRAP staining in the bone sections further revealed the effect of TRPV6 depletion on osteoclastogenesis (Fig. 2F). Quanti cation showed that the number and surface area of osteoclasts in TRPV6−/− mice increased signi cantly (Fig.  2G). Compared with WT mice, TRPV6−/− mice had higher levels of CTX-1. To summarize, these results suggest that bone absorption was enhanced and bone formation remained unchanged in TRPV6−/− mice.

Figure 3
The results of western blotting suggest that TRPV6 is expressed in osteoclasts. The expression of TRPV6 in osteoclasts was reduced during cell differentiation (Fig. 3A). In addition, immunocytochemical uorescence indicated that TRPV6 mainly distributes on the membrane of mature osteoclast (Fig. 3B).
For deeper insight into the role of TRPV6 on osteoclast function, TRAP staining was rst applied, in cultures derived from TRPV6−/− mice, and the number of TRAP+ multinucleated osteoclasts were signi cantly higher (Fig. 3C-D). A bone resorption lacuna experiment result showed that resorption pits were signi cantly enhanced in TRPV6−/− osteoclasts (Fig. 3E-F). In addition, PCR results revealed that the mRNA levels of marker genes for osteoclastogenesis (cathepsin k, DC-STAMP, Atp6v0d2 and TRAP) were obviously upregulated in TRPV6-depleted osteoclasts (Fig. 3G-J).

Figure 4
To further verify the role of TRPV6 on osteoclast formation and bone resorption, we silenced and overexpressed TRPV6 gene in osteoclasts by lentivirus transfection. Almost all cells expressed GFP, indicating that the lentivirus transfection rate was more than 95% (Fig. 4A). Western blotting con rmed that TRPV6 was effectively silenced and overexpressed (Fig. 4B). The mRNA levels of cathepsin k, DC-STAMP, Atp6v0d2 and TRAP were all increased in osteoclasts infected with lenti-shRNA-TRPV6, whereas were all decreased in the group of osteoclast infected with lenti-pMX-TRPV6 (Fig. 4C-F). Next, we assessed the differentiation of osteoclasts with silenced or overexpressed TRPV6 by TRAP staining assay. The number of stained multinuclear TRAP+ osteoclasts was obviously increased in osteoclasts with silenced TRPV6 in a time-dependent manner, whereas osteoclasts showed decreases in the TRPV6overexpressed group (Fig. 4G-H). The above results strongly suggest that TRPV6 was a negative regulator of osteoclast differentiation and function.
Our previous study revealed that the RANKL-induced [Ca2+]i oscillation response was not signi cantly affected by inhibition of TRPV6 [16]. As a non-ca2+ oscillating signaling pathway, the IGF pathway plays an important role in osteoclast formation [25][26][27]. Therefore, we speculated that the IGF signaling pathway was potentially involved in the regulation of osteoclast differentiation and bone absorption by TRPV6. The results showed that the levels of IGF1R and IGFBP1 mRNA and protein in osteoclasts were signi cantly increased after TRPV6 gene silencing (Fig. 5A-D), which con rmed our speculation. Next, we used an IGF1R antagonist NVP-AEW541 to block the IGF signaling pathway, TRAP staining demonstrated that the inhibitory effect of TRPV6 on osteoclast differentiation was weakened in osteoclasts with blockers ( Fig. 5E-F). In accordance with the results for TRAP staining, the IGF1R blocker signi cantly inhibited the induction of the bone resorption of osteoclasts by silencing of TRPV6, as evidenced by the pit formation assay (Fig. 5G-H). These results strongly suggest that TRPV6 negatively regulated osteoclast formation and bone resorption by inhibiting the IGF pathway. As assessed by immuno uorescence, we found that osteoclasts highly expressed TRPV6 and IGF1R in the membrane and cytoplasm [ Fig. 6A]. To further explore the downstream signaling of the IGF pathway, BMMs isolated from TRPV6-/-and WT mice were induced by RANKL and M-CSF for 7 days to form mature osteoclasts. The levels of phosphorylated markers P85/p-P85, PDK1/p-PDK1 and AKT/p-AKT in the PI3K-AKT pathway were detected by western blotting. The results showed that the ratios of P85/p-P85, PDK1/p-PDK1 and AKT/p-AKT were increased in osteoclasts isolated from TRPV6-/-mice compared with WT mice [ Fig. 6B-E]. Consistent with the western blotting results, immuno uorescence showed that the ratio of PDK1/p-PDK1 of osteoclasts isolated from TRPV6-/-mice was higher than that of osteoclasts isolated from WT mice [ Fig. 6F-G]. Next, we used NVP-AEW541 to block the IGF signaling pathway and western blotting revealed that there was no signi cant difference in the level of AKT/p-AKT between the osteoclasts derived from TRPV6-/-mice and WT mice [ Fig. 6H-I].

Figure 7
In the present study, we demonstrated that TRPV6 was a critical negative regulator in RANKL-induced osteoclast differentiation and activity. Our studies showed that TRPV6 decreased osteoclast formation and bone resorption by inhibiting the IGF-PI3K-AKT signaling pathway (Fig. 7).