Osteoclastogenesis inhibition by mutated IGSF23 results in human osteopetrosis

Abstract Objectives Osteopetrosis is a rare inherited skeletal disease characterized by increased bone mineral density due to the loss of osteoclast function or differentiation potential. Materials and Methods The study involved a Chinese patient with osteopetrosis (the proband) and her immediate family members and 180 controls without osteopetrosis. Bone density of the femoral neck, lumbar spine and total body was measured using dual‐energy x‐ray absorptiometry. Osteoclast differentiation by the participants’ peripheral blood mononuclear cells (PBMCs) was investigated using tartrate‐resistant acid phosphatase (TRAP) staining. Osteoblast differentiation was examined with Alizarin Red S staining. Reverse transcription‐quantitative PCR was used to amplify immunoglobulin superfamily member 23 (IGSF23), c‐FOS and nuclear factor of activated T cells 1 (NFATC1). Results We found a homozygous mutation (c.295C>T) in the IGSF23 gene in two osteopetrosis samples. The mutation led to the formation of a stop codon, causing loss of the immunoglobulin‐like domain and the whole transmembrane domain. PBMCs from the proband (IGSF23 −/−) exhibited poor ability for differentiating into mature osteoclasts in vitro. Overexpression of IGSF23 rescued the ability of IGSF23 −/− PBMCs to differentiate into osteoclasts. Moreover, knockdown of IGSF23 reversed the bone loss in OVX mice by injecting AAV‐shIGSF23 into mice femoral bone marrow cavity. Furthermore, we also found that the IGSF23 mutation led to decreased c‐Fos and NFATC1 expression levels by inhibiting the mitogen‐activated protein kinase signalling pathways. Conclusions IGSF23‐mediated osteoclast differentiation of PBMCs may serve as a potential target in osteoporosis therapy.

IKBKG and ITGB3 have been reported to associated with human osteopetrosis. [10][11][12][13] Most of these mutations cause osteoclast-rich osteosclerosis characterized by increasing the amount of osteoclasts but the activity of bone resorption is weakened or lost. 11 However, the mutation of RANKL (TNFSF11) or its receptor RANK (TNFRSF11A) cause a kind of more rarely osteopetrosis characterized by lacking of mature osteoclasts. 14,15 In this study, we found a novel pathogenic gene mutation (IGSF23) from an autosomal recessive osteopetrosis pedigree, the mutation of IGSF23 lead to osteopetrosis as a result of preventing osteoclastogenesis of PBMCs. 16

| Patients
The family members ( Figure 1) and 180 normal controls involved in this study were recruited by Second Xiangya Hospital. All subjects were Han ethnicity and were recruited from a local population of Hunan Province located in central south China.

| Cells culture and vectors
Peripheral blood mononuclear cells were isolated and purified from blood samples as previous described. 17 Briefly, the 25 mL diluted blood samples was added with 12 mL Ficoll ® PM 400 Histopaque ® -1077 (Sigma), and then centrifugated at 1000 g for F I G U R E 1 The characteristic of the Chinese autosomal recessive osteopetrosis family. A, Affected family members are indicated by black symbols. B, X-rays showed the characteristic of tibia bone and spine from proband (II:1) and unaffected family members. C, Osteoclasts stained by TRAP staining analysis from the proband and unaffected family members, arrows indicate osteoclasts (TRAP-positive). D, Quantitative analysis of TRAP staining analysis. **P < .01 10 minutes. After centrifugation, PBMCs was adsorbed with a transpirator and seeded on 10-cm tissue culture dish with 15 mL α-MEM (Gibco) containing 10% foetal bovine serum (FBS) and 1% streptomycin and penicillin. Primary osteoclasts were isolated from human vertebral cancellous bone as previous described. 18 Osteoblasts (passage 3) were regarded as a gift from Department of Endocrinology Research Center of Xiangya Hospital. Osteoclasts and osteoblasts were cultured in α-MEM complete medium at 37°C and 5% CO 2 incubator (Thermo Fisher Scientific, Inc).

| Bone density measurements
The femoral neck, lumbar spine and total body bone density were measured by dual-energy x-ray absorptiometry. The results are expressed as Z-scores. 19

| Mutational analysis
Peripheral blood was collected from all family members and normal controls, and the blood genomic DNA was extracted with FlexiGene @ DNA kit (Qiagen). 20 The exons of candidate genes for osteopetrosis were amplified and sequenced by DNA sequencing instrument (llumine 2500). Specific primers for amplification of IGSF23 exons are provided in Table S1.

| Protein fractionation
The PBMCs were collected after treated with M-CSF and RANKL for 3 days. Cells were fractionated into cytosol, membrane and nuclei using Qproteome cell compartment kit (Qiagen) according to the manufacturer's instructions. And then, the cytosol, membrane and nuclei fraction were subjected to Western blotting analysis.

| Osteoclast differentiation and TRAP staining
Peripheral blood mononuclear cells were cultured and seeded into 48-well plates with glass coverslips containing 10% FBS, 50 ng/mL M-CSF (R&D) and 100 ng/mL RANKL (R&D). Cells were treated for 7 days, osteoclasts containing more than three nuclei were generated and further identified by tartrate-resistant acid phosphatase (TRAP) staining. 21 TRAP staining was performed as previous. 22 Images were captured with a Diaphot Inverted Microscope and Camera System (Leica). TRAP-positive multinuclear cells were observed and counted in 5 random regions per sample. 23

| RT-qPCR analysis
Total RNA of tissues or cells was extracted using Trizol reagent (Invitrogen), and transcribed into cDNA by using SuperScript II Kit

| Western blot analysis
Tissues and cells were collected and lysed with 400 μL RIPA buffer containing 1× protease inhibitors (Roche) on ice for 30 minutes.
After 30 minutes, the samples were centrifugated, denaturated and separated by 12% SDS gel electrophoresis as previous. 28 The rabbit F I G U R E 3 Expression pattern of IGSF23. A, Various adult human tissues were homogenated and isolated the total RNA for qPCR analysis. B, Various adult human tissues were homogenated and lysated for Western blot analysis incubated with IGSF23 antibody. C, Total RNA was isolated from osteoblasts, PBMCs and osteoclasts and was subjected to qPCR analysis. D, Osteoblasts, PBMCs and osteoclasts were collected and lysated for Western blot analysis incubated with IGSF23 antibody. E, PBMCs were collected for qPCR analysis after induced by M-CSF/RANKL. F, PBMCs were collected for Western blot analysis incubated with IGSF23 antibody. G, PBMCs fractionation were separated and subjected to Western blots analysis incubated with IGSF23 antibody. Three replicates were performed for RT-qPCR and Western blotting analysis. *P < .05, **P < .01 anti-IGSF23 polyclonal antibody was raised against the linear peptide sequence MQPTEAEPMEPDPTLS of the predicted human IGSF23 protein (NP_001192209), β-actin, RANK, c-Fos, NFATc1, p-ERK, ERK, p38, p-p38 antibodies were purchased from Cell Signaling Technology; mSin3A (Abcam), 5'NT, D4-GDI and TRAP antibodies were purchased from Abcam.

| Toluidine blue staining
Peripheral blood mononuclear cells were cultured in 24-well plates with bovine bone slices containing 10% FBS, 50 ng/mL M-CSF and 100 ng/mL RANKL. After treating for 21 days, the slices were cleaned of cells and stained with 0.1% toluidine blue, and observed by conventional light microscopy. Bone resorption activity was evaluated by measurement of the area of bone resorption pits using image analysis software (Image J).

| Statistical analysis
All data were analysed using SPSS 22.0 software (SPSS Inc). The comparison for multiple groups was performed by one-way ANOVA analysis, and differences were considered significant at P < .05. 29-33

| Clinical examination
The proband (II:1) was a 41-year-old woman admitted to hospital with sacroiliac joint pain. Osteopetrosis was diagnosed by X-ray analysis. The study of pedigree showed that osteopetrosis in two siblings, but another four family members did not exhibit any symptoms of the disease (Figure 1 A). Skeletal radiographic examinations showed that the proband bone density was significantly increasing in the tibia bone and spine compared to her sister (II:2, an age-matched healthy individual). Besides, her spine appeared with a "sandwich" appearance in the vertebrae ( Figure 1B).
Moreover, the BMD in the proband femoral neck, lumbar spine and total body was dramatically increased when compared to unaffected family members (Table 1) by dual-energy x-ray absorptiometry analysis.
But, both of them the growth parameters (height and weight), blood glucose, liver and kidney function were normal and none of them had any history of fractures. And the levels of calcium, phosphate, PTH and 25-Hydroxyvitamin D were also normal in serum (Table 2). However, the serum bone resorption biomarker TRAP-5b and bone formation biomarkers BAP were lower in affected samples compared to unaffected family members (Table 2). Furthermore, the amount of osteoclasts was fewer in the proband compared to her sister (II:2) by TRAP staining (Figure 1C,D).

| Genetic linkage analysis and IGSF23 mutation detection
Previous studies demonstrated that 11 genes mutation, including TCIRG1, CLCN7, LRP5, IGSF23, OSTM1, CAII, PLEKHM1, TNFSF11, TNFRSF11A, CTSK, IKBKG and ITGB3 are reported to associate with osteopetrosis. So, we plan to find the genes mutation in the pathogenic family. However, the genes mutation of TCIRG1, CLCN7, LRP5, OSTM1, CAII, PLEKHM1, TNFSF11, TNFRSF11A, CTSK, IKBKG and ITGB3 are not found. Next, the coding region was screened by the high-throughput sequencing approach. We identified a nonsense mutation (c.295C>T) in exon 2 of IGSF23 ( Figure 2A). The mutation of IGSF23 led to the arginine residue (CGA) at codon 99 of IGSF23 protein is substituted by a stop codon TGA (p.R99X; Figure A,B), which predicted to generate a mutated IGSF23 protein with only 98 amino acid residues. Interesting, the arginine residue at the position 99 of IGSF23 protein is highly conserved in mouse, rat, pan troglodytes and canis familiaris ( Figure 2C). Lastly, we found that the p.R99X mutation showed a perfect co-segregation associated with the osteopetrosis phenotype by sequencing of IGSF23 from other five samples. That is, all affected individuals were homozygous mutation, their parents were heterozygous mutation, and two other sisters were either heterozygous mutation (II:3) or normal (II:2). Interestingly, the type of mutation was absent in 180 unrelated normal samples.

| IGSF23 expression pattern and subcellular localization
To confirm IGSF23 gene expression pattern and its subcellular localization, total RNA and protein were isolated from multiple adult human tissues (Clontech Laboratories) are performed. The result of qRT-PCR and Western blot analysis showed that IGSF23 was especially expressed in bone and small intestine, but was not found in other tissues including brain, liver, heart, kidney, lung, prostate, skeletal muscle, fat, spleen, testis and thyroid gland ( Figure A,B). To further understand the expression pattern of IGSF23, the total RNA and protein were isolated from primary human osteoblasts, PBMCs (pre-osteoclasts) and osteoclasts, qRT-PCR and Western blot analysis showed that IGSF23 was especially high expressed in osteoclasts, but was low expressed in osteoblasts and PBMCs ( Figure 3C,D). Interestingly, the mRNA levels and protein expression of IGSF23 in PBMCs were These data demonstrated that IGSF23 expressed in membrane and cytosol, IGSF23 may be involved in the process of osteoclastogenesis.

| IGSF23 regulates M-CSF/RANKL-induced osteoclastogenesis of PBMCs
To investigate whether IGSF23 involved in the process of osteoclas-

| Silencing of IGSF23 reversed the bone loss in OVX mice
Osteoporosis is caused by bone resorption that exceeds bone formation. [34][35][36] Considering that the mutation of IGSF23 inhibited the osteoclast differentiation, which indicates that inhibition of IGSF23 may be regarded as a putative strategy for treating osteoporosis. To investigate the therapeutic effects of silencing IGSF23 in osteoclast differentiation, adeno-associated virus (AAV)-shIGSF23 was injected into the femoral bone marrow cavity of OVX mice twice per month for 3 months (n = 6).
The result showed that intra-bone marrow injection of AAV-shIGSF23 significantly decreased the mRNA levels of IGSF23 by RT-qPCR analysis ( Figure 6B). Moreover, silencing of IGSF23 increased the bone density, trabecular bone volume per tissue volume, trabecular thickness and showed lower trabecular separation compared with vehicle-treated mice ( Figure 6A,C-F). Furthermore, the number of osteoclasts on the bone surface was much lesser in mice treated with AAV-shIGSF23 relative to controls ( Figure 6G). These results revealed that silencing of IGSF23 inhibited the formation of osteoclast and reversed the bone loss in OVX mice.

| IGSF23 promotes osteoclasts differentiation of PBMCs via MAPK signalling pathways
Previous studies demonstrated that RANK signalling pathways are crucial for RANKL-stimulated osteoclast differentiation. 11 We next have been shown to be associated with osteopetrosis or osteopetrosis-like phenotypes. 10 However, none of these mutations was detected in this study, which indicates a novel gene mutation may emerge. In our study, we found a new mutation of IGSF23, which The mechanism of IGSF23 involvement in osteoclast differentiation was then investigated. Osteoclast differentiation is depended on the activation of RANK which binds to the RANKL. 40 And then, RANK-RANKL stimulates some transcription factors expression for osteoclast differentiation by activating MAPK or NF-κB pathway. [40][41][42] Among them, c-Fos and NFATc1 are regarded as critical transcription factors for osteoclastogenesis. 43,44 Previous studies showed that c-Fos and NFATc1 knockout mice develop severe osteopetrotic phenotype with complete deficiency of osteoclasts. 43 Our results in this study demonstrate that PBMCs from the proband (IGSF23 mutation) lost the osteoclastogenic potential, because the mutation of IGSF23 lead to decrease the phosphorylation of ERK1/2 and p38 and then inhibit the expression of c-Fos and NFATc1.
In conclusion, this study identified a novel gene mutation of IGSF23, which caused a rare form of autosomal recessive osteopetrosis. IGSF23 is selectively expressed in osteoclasts and promotes M-CSF and RANKL-stimulated osteoclast differentiation via activating MAPK signalling pathways. Therefore, IGSF23 may be regarded as a new target for osteoporosis.

ACK N OWLED G EM ENTS
We thank the family for their cooperation.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.