Ano5
Introduction Gnathodiaphyseal dysplasia (GDD; OMIM: 166260 ) is a rare skeletal syndrome characterized by sclerosis of limb bones and cemento-osseous lesions in mandibles that occurs in an autosomal dominant pattern ( ). Anoctamin 5 ( ANO5 ; TMEM16E ), encoding a 913 amino-acid protein belonging to the TMEM16 family, was mapped in chromosome 11p14.3–15.1 by linkage analysis and identified to cause GDD ( ; ). Recessive mutations in the ANO5 gene cause two types of muscular dystrophies, proximal limb-girdle muscular dystrophy type 2 (LGMD2L) and Miyoshi muscular dystrophy 3 (MMD3) ( ). Although ANO5 is involved in various disorders, little is known about the molecular function of the ANO5 protein. Considering that ANO5 is located in the endoplasmic reticulum or cell membrane ( ; ), several lines of evidence have manifested that ANO5 functions as a calcium-activated chloride channels (CaCCs) or phospholipid scrambling protein. Heretofore, few preliminary studies on the role of ANO5 in bone remodeling have been performed. Research has shown that ANO5 does not affect osteoblast differentiation and mineralization ( ); on the other hand, XJ Qin demonstrated that Ano5 -deficient mice exhibited suppressed osteogenesis due to abnormal calcium oscillation, which is divergent from the GDD phenotype ( ). In fact, most GDD patients are clinically characterized by elevated serum alkaline phosphatase (ALP) levels, a classical measurement of systematic osteogenic activity, and increased bone mineral density (BMD), as determined by radiographic examinations ( ; ; ). However, a consensus is emerging about the association between ANO5 and osteoclastogenesis. Ano5 knockdown leads to decreased osteoclast differentiation, and its overexpression consistently promotes osteoclast maturation through Akt/nuclear factor of activated T cells 1 (Nfatc1) signaling pathway ( ; ). The serine/threonine kinase AKT is located downstream of PI3K and undergoes dual activation in the structural domain, which is phosphorylated by phosphoinositide-dependent kinase-1 (PDK1), and the regulatory domain is phosphorylated by mammalian target of rapamycin complex 2 (mTORC2) ( ; ). Subsequently, AKT detaches from the cell membrane to regulate downstream signaling molecules, including GSK3β, Akt1/forkhead box class O (FoxO), and mTORC1, which are generally involved in various biological processes, including cell proliferation, survival, protein synthesis, and metabolism ( ). Notably, the modulatory role of Akt signaling in bone homeostasis has drawn substantial concern. Activation of Akt signaling plays a crucial role in enhancing preosteoblast proliferation, initiating osteogenic differentiation, and promoting the maturation of committed osteoblasts, which is essential for bone morphogenetic protein 2 regulatory function for bone remodeling. ( ; ). As a consequence, Akt1 deficiency in mice leads to osteopenia due to the dysregulation of both osteoblasts and osteoclasts ( ). Furthermore, Akt signaling exerts positive effects on osteoclast differentiation and maturation mediated by increased phosphorylation of GSK3β and nuclear localization of NFATc1 ( ). In our previous study, Ano5 Cys360Tyr knockin ( Ano5 KI/KI ) mice equivalent to the p.Cys360Tyr mutation in a Han GDD family that was firstly reported by our group was successfully generated and partially replicated GDD phenotypes characterized by enhanced osteoblast differentiation and bone formation. Additionally, mice displayed inhibited osteoclastogenesis, which was closely related with early signaling pathways regulated by RANKL-RANK-TNF receptor associated factor 6 (TRAF6) cascades, including Akt, NF-κB, p38, and Erk signaling ( ). It is worth noting that the phosphorylation of Akt, a classical molecule associated with bone metabolism, was extremely reduced. In the present study, we investigated the effects of the specific Akt activator SC79 on osteoblastogenesis and osteoclastogenesis of mouse calvarial osteoblasts (mCOBs) and bone marrow-derived macrophages (BMMs) from Ano5 KI/KI mice and surmised that Akt signaling pathway plays a crucial role in skeletal dysfunction of GDD. 

Methods 2.1 Generation of Ano5 KI/KI mice The Ano5 knock-in mouse model (c.1080G > A) was generated by Biocytogen Pharmaceuticals (Beijing) Co., Ltd. A transformation from cysteine into tyrosine at codon 360 of Ano5 was introduced through genomic editing of Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. Adequate measures were taken to minimize pain and discomfort. All protocols were approved by the Institutional Animal Care and Use Committee of the Beijing Stomatological Hospital (approval number: KQYY-202012-005). Ano5 KI/KI mice carrying the p.Cys360Tyr mutation were established through CRISPR Cas9 genomic editing technology. Specifics about the generation and genotype identification of the Ano5 knock-in mouse model were previously described ( ). DNA was extracted from mouse tails according to the instructions of a universal genomic DNA kit (CW2298, CWbio, Jiangsu, China), and then genotyping was performed by polymerase chain reaction (PCR). Mice were housed in a pathogen-free environment under a 12-hour light-dark cycle and fed standard food ad libitum . 2.2 Isolation and culture of mouse osteoclasts Wild type and Ano5 KI/KI male mice of 6–8 weeks old were sacrificed by cervical dislocation after being deep anesthesia with carbon dioxide narcosis. and their tibias and femurs were collected in PBS. Bone marrow was flushed with alpha-modified minimum essential medium (α-MEM) (12,561,056, Gibco-Invitrogen, New York, USA) containing 10 % fetal bovine serum (10,091,148, FBS; New York, Gibco, USA) and incubated at 37 °C with 5 % CO 2 for 1 day. Nonadherent cells were collected and resuspended in α-MEM supplemented with 30 ng/ml macrophage-colony stimulation factor (31,502, M-CSF; PeproTech, Rocky Hill, USA) to promote BMMs proliferation for 3 days. Subsequently, BMMs differentiated and matured under the stimulation of osteoclast differentiation medium consisting of α-MEM supplemented with 30 ng/ml M-CSF and 100 ng/ml receptor activator of nuclear factor-κβ (RANK) ligand (31511C, RANKL; PeproTech, Rocky Hill, USA). The medium was replaced every other day until the formation of mature multinucleated osteoclasts at day 7. 5 μg/ml AKT activator SC79 (S7863, Selleck, Texas, USA) was treated during the entire differentiation time. 2.3 In vitro osteoclastogenesis assay To identify mature osteoclasts, BMMs were planked in 96-well plates at a density of 5000 cells/well and cultured with osteoclast differentiation medium for 7 days. Next, the cells were washed with phosphate-buffered saline (PBS) (61,231,114, Gibco, New York, USA) and fixed in 4 % paraformaldehyde (DF0135, Leagene, Beijing, China) for 20 min. TRAP staining was performed according to the manufacturer's instructions (387 A, Sigma Aldrich, New York, USA). Methyl green staining solution is used to stain the nucleus (G1652, Solarbio, Beijing, China). TRAP-positive multinucleated osteoclasts were observed by microcopy (Olympus BX51, Tokyo, Japan). The area featured by multinucleated osteoclasts (≥ 3 nucleus) and TRAP-positive was analyzed by using Image-Pro plus software 6.0. BMMs were also seeded in 24-well plates at a density of 5 × 10 4 cells/well for phalloidin staining as previously reported. After culture period, the cells were incubated with phalloidin-iFluor 488 (ab176753, Abcam, Cambridge, UK) for 40 min and then with DAPI solution (C0065, Solarbio, Beijing, China) for 5 min to perform nuclear staining. F-actin rings were captured under a fluorescence microscope at Ex/Em = 493/517 nm. 2.4 Preparation of mouse calvarial osteoblasts (mCOBs) culture mCOBs were isolated from postnatal 1- to 3-day-old female and male littermates after decollation as previously described ( ). The calvaria were digested in an enzyme mixture containing 2 mg/ml type I collagenase and 0.25 % trypsin for four cycles of 15 min/cycle. Then, the cells were harvested and cultured in α-MEM with 10 % FBS. For osteogenic induction, mCOBs were then cultured in α-MEM supplemented with 10 % FBS, 1 % streptomycin/penicillin (15,140,122, Gibco, New York, USA), 4 μg/ml dexamethasone, 50 mg/ml ascorbic acid and 4 mM β-glycerophosphate (D4902, A5960, and G9422, Sigma-Aldrich, St. Louis, USA). The cultures were changed every two days. 5 μg/ml SC79 was treated during the entire differentiation time. mCOBs were cultured in 6-well plates at a density of 2 × 10 5 cells/well and used for RNA and protein extraction. 2.5 In vitro osteogenic assay and calcium nodule staining Osteoblasts were seeded in 6-well plates at a density of 2 × 10 5 cells per well. After osteogenic induction for 7 days, cells were stained with the ALP staining kit (A59022, Beyotime, Shanghai, China) after fixation with 4 % paraformaldehyde for 30 min, and ALP activity was determined with the OD values at 405 nm on a multifunction microplate scanner. Alizarin red staining was performed for visualization of calcium deposits as previously reported. Briefly, mature osteoblasts with 21-day osteogenic induction were fixed using 75 % ethanol, and then alizarin red staining solution (1 mg/ml, pH 5.5) (A5533, Sigma-Aldrich, St. Louis, USA) was added. After washing with distilled water, stained cells were photographed. Subsequently, mineralized nodules were incubated with 10 % cetylpyridinium chloride, and absorbance at 562 nm was recorded to examine calcium content. 2.6 Assessment of free calcium in BMMs cytoplasm For the detection of free calcium in the cytoplasm, BMMs were cultured in 96-well plates at a density of 5 × 10 3 cells per well. Live cells at different stages of osteoclast differentiation were incubated with CellProbe Fluo-4 AM (S1060, Beyotime, Shanghai, China) for 30 min, and the fluorescence intensity was detected by a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 516 nm to determine the level of free calcium. 2.7 Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA was isolated using TRIzol reagent (15,596,026, Thermo Fisher Scientific, MA, USA) following the manufacturer's instructions. cDNA synthesis was performed with a SuperRT cDNA Synthesis Kit (CW0471, CWbio, Beijing, China) using one microgram of purified RNA. Synthesized cDNA (1 μl) was used for qRT-PCR analysis with Ultra SYBR Mixture with low ROX (CW3008, CWbio, Beijing, China) on a Bio-Rad thermocycler (Bio-Rad, Hercules, USA) PCR system. The relative expression levels of target genes were calculated using the 2 −ΔΔCt method and normalized to the internal control Actb. The PCR primer sequences are listed in Supplemental Table S1, Additional File 1. 2.8 Western blot analysis Cells were harvested and lysed on ice in RIPA Lysis Buffer (Sigma, USA). Coomassie brilliant blue R-250 (Bio-Rad, California, USA) staining solution was employed to measure the protein concentration. Equal amounts (30 μg) of protein from each sample were separated on a 10 % sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes using a Bio-Rad Trans-Blot SD. Membranes were blocked with 5 % nonfat milk and incubated overnight with primary antibodies at 4 °C. Then, horseradish peroxidase (HRP)-conjugated secondary antibody at a dilution of 1:5000 was used for 1 h. The blots were developed with an enhanced chemiluminescent reagent (P10060, NCM Biotech, Suzhou, China) and quantified with a ChemiDoc Touch Imaging System (Bio-Rad, California, USA). Gapdh or Actb was detected as the housekeeping protein. All primary antibodies used are listed in Supplemental Table S2, Additional File 1. 2.9 Statistical analysis All experiment were performed in triplicate. Statistical analyses were performed using unpaired, two-tailed Student's t- test for comparisons between two groups and one-way ANOVA for comparisons of more than two groups using GraphPad Prism 8.0.2 software (GraphPad Software, La Jolla, CA, USA). The results are presented as the mean ± standard deviation (SD). For all experiments, a P value < 0.05 was considered to indicate a statistically significant difference. 

Generation of Ano5 KI/KI mice The Ano5 knock-in mouse model (c.1080G > A) was generated by Biocytogen Pharmaceuticals (Beijing) Co., Ltd. A transformation from cysteine into tyrosine at codon 360 of Ano5 was introduced through genomic editing of Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. Adequate measures were taken to minimize pain and discomfort. All protocols were approved by the Institutional Animal Care and Use Committee of the Beijing Stomatological Hospital (approval number: KQYY-202012-005). Ano5 KI/KI mice carrying the p.Cys360Tyr mutation were established through CRISPR Cas9 genomic editing technology. Specifics about the generation and genotype identification of the Ano5 knock-in mouse model were previously described ( ). DNA was extracted from mouse tails according to the instructions of a universal genomic DNA kit (CW2298, CWbio, Jiangsu, China), and then genotyping was performed by polymerase chain reaction (PCR). Mice were housed in a pathogen-free environment under a 12-hour light-dark cycle and fed standard food ad libitum . 

Isolation and culture of mouse osteoclasts Wild type and Ano5 KI/KI male mice of 6–8 weeks old were sacrificed by cervical dislocation after being deep anesthesia with carbon dioxide narcosis. and their tibias and femurs were collected in PBS. Bone marrow was flushed with alpha-modified minimum essential medium (α-MEM) (12,561,056, Gibco-Invitrogen, New York, USA) containing 10 % fetal bovine serum (10,091,148, FBS; New York, Gibco, USA) and incubated at 37 °C with 5 % CO 2 for 1 day. Nonadherent cells were collected and resuspended in α-MEM supplemented with 30 ng/ml macrophage-colony stimulation factor (31,502, M-CSF; PeproTech, Rocky Hill, USA) to promote BMMs proliferation for 3 days. Subsequently, BMMs differentiated and matured under the stimulation of osteoclast differentiation medium consisting of α-MEM supplemented with 30 ng/ml M-CSF and 100 ng/ml receptor activator of nuclear factor-κβ (RANK) ligand (31511C, RANKL; PeproTech, Rocky Hill, USA). The medium was replaced every other day until the formation of mature multinucleated osteoclasts at day 7. 5 μg/ml AKT activator SC79 (S7863, Selleck, Texas, USA) was treated during the entire differentiation time. 

In vitro osteoclastogenesis assay To identify mature osteoclasts, BMMs were planked in 96-well plates at a density of 5000 cells/well and cultured with osteoclast differentiation medium for 7 days. Next, the cells were washed with phosphate-buffered saline (PBS) (61,231,114, Gibco, New York, USA) and fixed in 4 % paraformaldehyde (DF0135, Leagene, Beijing, China) for 20 min. TRAP staining was performed according to the manufacturer's instructions (387 A, Sigma Aldrich, New York, USA). Methyl green staining solution is used to stain the nucleus (G1652, Solarbio, Beijing, China). TRAP-positive multinucleated osteoclasts were observed by microcopy (Olympus BX51, Tokyo, Japan). The area featured by multinucleated osteoclasts (≥ 3 nucleus) and TRAP-positive was analyzed by using Image-Pro plus software 6.0. BMMs were also seeded in 24-well plates at a density of 5 × 10 4 cells/well for phalloidin staining as previously reported. After culture period, the cells were incubated with phalloidin-iFluor 488 (ab176753, Abcam, Cambridge, UK) for 40 min and then with DAPI solution (C0065, Solarbio, Beijing, China) for 5 min to perform nuclear staining. F-actin rings were captured under a fluorescence microscope at Ex/Em = 493/517 nm. 

Preparation of mouse calvarial osteoblasts (mCOBs) culture mCOBs were isolated from postnatal 1- to 3-day-old female and male littermates after decollation as previously described ( ). The calvaria were digested in an enzyme mixture containing 2 mg/ml type I collagenase and 0.25 % trypsin for four cycles of 15 min/cycle. Then, the cells were harvested and cultured in α-MEM with 10 % FBS. For osteogenic induction, mCOBs were then cultured in α-MEM supplemented with 10 % FBS, 1 % streptomycin/penicillin (15,140,122, Gibco, New York, USA), 4 μg/ml dexamethasone, 50 mg/ml ascorbic acid and 4 mM β-glycerophosphate (D4902, A5960, and G9422, Sigma-Aldrich, St. Louis, USA). The cultures were changed every two days. 5 μg/ml SC79 was treated during the entire differentiation time. mCOBs were cultured in 6-well plates at a density of 2 × 10 5 cells/well and used for RNA and protein extraction. 

In vitro osteogenic assay and calcium nodule staining Osteoblasts were seeded in 6-well plates at a density of 2 × 10 5 cells per well. After osteogenic induction for 7 days, cells were stained with the ALP staining kit (A59022, Beyotime, Shanghai, China) after fixation with 4 % paraformaldehyde for 30 min, and ALP activity was determined with the OD values at 405 nm on a multifunction microplate scanner. Alizarin red staining was performed for visualization of calcium deposits as previously reported. Briefly, mature osteoblasts with 21-day osteogenic induction were fixed using 75 % ethanol, and then alizarin red staining solution (1 mg/ml, pH 5.5) (A5533, Sigma-Aldrich, St. Louis, USA) was added. After washing with distilled water, stained cells were photographed. Subsequently, mineralized nodules were incubated with 10 % cetylpyridinium chloride, and absorbance at 562 nm was recorded to examine calcium content. 

Assessment of free calcium in BMMs cytoplasm For the detection of free calcium in the cytoplasm, BMMs were cultured in 96-well plates at a density of 5 × 10 3 cells per well. Live cells at different stages of osteoclast differentiation were incubated with CellProbe Fluo-4 AM (S1060, Beyotime, Shanghai, China) for 30 min, and the fluorescence intensity was detected by a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 516 nm to determine the level of free calcium. 

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA was isolated using TRIzol reagent (15,596,026, Thermo Fisher Scientific, MA, USA) following the manufacturer's instructions. cDNA synthesis was performed with a SuperRT cDNA Synthesis Kit (CW0471, CWbio, Beijing, China) using one microgram of purified RNA. Synthesized cDNA (1 μl) was used for qRT-PCR analysis with Ultra SYBR Mixture with low ROX (CW3008, CWbio, Beijing, China) on a Bio-Rad thermocycler (Bio-Rad, Hercules, USA) PCR system. The relative expression levels of target genes were calculated using the 2 −ΔΔCt method and normalized to the internal control Actb. The PCR primer sequences are listed in Supplemental Table S1, Additional File 1. 

Western blot analysis Cells were harvested and lysed on ice in RIPA Lysis Buffer (Sigma, USA). Coomassie brilliant blue R-250 (Bio-Rad, California, USA) staining solution was employed to measure the protein concentration. Equal amounts (30 μg) of protein from each sample were separated on a 10 % sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes using a Bio-Rad Trans-Blot SD. Membranes were blocked with 5 % nonfat milk and incubated overnight with primary antibodies at 4 °C. Then, horseradish peroxidase (HRP)-conjugated secondary antibody at a dilution of 1:5000 was used for 1 h. The blots were developed with an enhanced chemiluminescent reagent (P10060, NCM Biotech, Suzhou, China) and quantified with a ChemiDoc Touch Imaging System (Bio-Rad, California, USA). Gapdh or Actb was detected as the housekeeping protein. All primary antibodies used are listed in Supplemental Table S2, Additional File 1. 

Statistical analysis All experiment were performed in triplicate. Statistical analyses were performed using unpaired, two-tailed Student's t- test for comparisons between two groups and one-way ANOVA for comparisons of more than two groups using GraphPad Prism 8.0.2 software (GraphPad Software, La Jolla, CA, USA). The results are presented as the mean ± standard deviation (SD). For all experiments, a P value < 0.05 was considered to indicate a statistically significant difference. 

Results 3.1 Activation of Akt reversed Ano5 KI/KI osteoclast differentiation To further confirm the association of Akt signaling pathway with inhibition of osteoclast differentiation caused by the Ano5 Cys360Tyr mutation, the specific Akt activator SC79 was used to treat BMMs from Ano5 KI/KI mice. Taken into account the evidence that clinical penetrance between female and male patients with GDD did not show different, wild type and Ano5 KI/KI BMMs were cultured from male mice to avoid disturbance of hormone fluctuation. The suppression of Akt phosphorylation was obviously reversed in response to SC79, especially in 15 and 30 min after RANKL induction ( a). Importantly, compared with Ano5 KI/KI group, the number of TRAP-positive multinucleated osteoclasts with SC79 treatment became more and even up to be comparable with wild-type group, along with sizable enlargement of the spread area in Ano5 KI/KI BMMs cultures ( b-c). These results indicated that Akt activation could facilitate Ano5 Cys360Tyr osteoclast differentiation and maturation. 3.2 Akt activator promoted actin ring formation in Ano5 KI/KI osteoclasts An intact filamentous actin (F-actin) ring structure is a vital process by which differentiated osteoclasts activate bone resorption by attaching to the bone surface and forming a sealing zone ( ). F-actin is composed of a double-helical polymer of monomeric actin (G-actin) in a globular configuration through burning up energy, and phalloidin can specifically bind to F-actin ( ). Phalloidin staining was performed to explore the reverse effect of the Akt activator on osteoclast differentiation and bone resorption. SC79 supplementation into mutant BMMs cultures remarkably rescued actin ring formation and osteoclasts maturation ( a-c). Consistently, the Akt activator induced the expression of dendritic cell-specific transmembrane protein ( Dc-stamp ) in Ano5 KI/KI BMMs, which is essential for cell-cell fusion and actin ring formation in osteoclasts ( d) ( ). There is no significant difference at day 3, SC79 stimulation also enhanced the transcription level of the vacuolar (H + ) ATPase (v-ATPase) V0 domain ( Atp6v0d2 ) at other stages of osteoclast differentiation ( c). Interestingly, it has been reported that Atp6v0d2-deficient mice exhibited representative disruption of osteoclasts fusion similar to Dc-stamp knockout mice. In addition, Atp6v0d2 triggers proton transport to the resorptive lacunae and thus preserves the acidic environment needed for bone degradation ( ; ). 3.3 SC79 positively regulated the expression of osteoclastogenesis-related factors in Ano5 KI/KI BMMs cultures Following the formation of an actin ring-rich sealing zone and the presence of a ruffled membrane, bone matrix degradation is induced by osteoclast-specific genes ( ). As shown in a, the decreased expression of Trap , cathepsin K ( Ctsk ), and matrix metalloproteinase-9 ( Mmp9 ) in Ano5 KI/KI BMMs cultures was switched back by SC79 at various differential stages. Furthermore, these factors have multiple sites recognized by Nfatc1, which is known as a significant regulator of osteoclastogenesis, and the reduction in Nfatc1 was also reversed by SC79 stimulation ( b). Notably, the level of cytosolic calcium taking part in robust Nfatc1 activation was obviously decreased, which was also rescued due to SC79 intervention ( c). In brief, these results suggested the negative effects of the p.Cys360Tyr mutation on osteoclast differentiation and function could be rectified by Akt activation. 3.4 SC79 inhibited osteogenic differentiation and mineralization mediated by the p.Cys360Tyr mutation in Ano5 Bone metabolism relies on the delicate balance between bone resorption mediated by osteoclasts and bone formation mediated by osteoblasts. In light of decreased Akt phosphorylation in Ano5 KI/KI osteoclasts, we further examined the effects of Akt signaling pathway on the alterations in Ano5 KI/KI osteoblast differentiation and mineralization caused by the p.Cys360Tyr mutation. Western blot analysis revealed that the level of Akt phosphorylation was comparable between wild-type and Ano5 KI/KI osteoblasts at day 0 ( a). However, Akt phosphorylation was significantly suppressed in Ano5 KI/KI mature osteoblasts after 14 days of osteogenic induction, and this effect was rescued by 5 μg/ml SC79 ( b-c). To investigate the intrinsic role of Akt activation on GDD-related enhanced osteogenesis, ALP and alizarin red staining assays were carried out. SC79 stimulation in Ano5 KI/KI osteoblasts diminished excessive ALP activity, which is an early marker of osteogenic differentiation ( d). Furthermore, extracellular matrix mineralization, the most vital phenomenon in the late stage of bone formation, was detected by alizarin red staining accompanied by the measurement of calcium content, which showed that many fewer mineralized nodules and calcium deposition were formed after Ano5 KI/KI cells were cultured with the Akt activator SC79 ( e). 3.5 Akt activation negatively regulated the expression of osteogenic-related factors in Ano5 KI/KI mCOBs Subsequently, the influences of the Akt activator on transcription levels of bone-forming related factors in different stages of Ano5 KI/KI mCOB cultures were explored. We found that up-regulation of Runx2 , a key regulator of osteoblast differentiation, was reversed almost to the level in Ano5 +/+ group by SC79 in both undifferentiated and mature Ano5 KI/KI mCOB ( ). Osteopontin (OPN), an important noncollagen protein in bone tissue, is synthesized by osteogenic progenitor cells and bone cells ( ), and the upregulated OPN expression was also inhibited by the Akt activator ( ). Additionally, osteocalcin ( Ocn ) and collagen type I alpha 1 ( Col1a1 ), which are classical markers of late osteogenic factors and matrix maturation, showed similar trends ( ). Overall, SC79 partially attenuated the enhanced osteogenesis caused by the GDD-related Ano5 mutation. Osteoblast could regulate osteoclastogenesis through regulating the expression of RANKL and OPG that is coordinated to regulate bone resorption and density positively and negatively by controlling the activation state of RANK on osteoclasts. Our previous study have found reduced osteoclastogenesis in Ano5 KI/KI BMMs partly attributed to elevated Opg/Rankl ratio in osteoblast ( ). Rankl transcription level with SC79 treatment was equivalent to Ano5 KI/KI group ( ). However, SC79 significantly attenuated Opg expression, which not only promotes bone formation but completes with RANKL to bind with RANK to inhibit osteoclast differentiation and maturation. Consistently, the ratio of Opg to Rankl was obviously declined, implying Akt activation could further rescue osteoclastogenesis in GDD via the effect of osteoblast-mediated osteoclastogenesis ( ). 

Activation of Akt reversed Ano5 KI/KI osteoclast differentiation To further confirm the association of Akt signaling pathway with inhibition of osteoclast differentiation caused by the Ano5 Cys360Tyr mutation, the specific Akt activator SC79 was used to treat BMMs from Ano5 KI/KI mice. Taken into account the evidence that clinical penetrance between female and male patients with GDD did not show different, wild type and Ano5 KI/KI BMMs were cultured from male mice to avoid disturbance of hormone fluctuation. The suppression of Akt phosphorylation was obviously reversed in response to SC79, especially in 15 and 30 min after RANKL induction ( a). Importantly, compared with Ano5 KI/KI group, the number of TRAP-positive multinucleated osteoclasts with SC79 treatment became more and even up to be comparable with wild-type group, along with sizable enlargement of the spread area in Ano5 KI/KI BMMs cultures ( b-c). These results indicated that Akt activation could facilitate Ano5 Cys360Tyr osteoclast differentiation and maturation. 

Akt activator promoted actin ring formation in Ano5 KI/KI osteoclasts An intact filamentous actin (F-actin) ring structure is a vital process by which differentiated osteoclasts activate bone resorption by attaching to the bone surface and forming a sealing zone ( ). F-actin is composed of a double-helical polymer of monomeric actin (G-actin) in a globular configuration through burning up energy, and phalloidin can specifically bind to F-actin ( ). Phalloidin staining was performed to explore the reverse effect of the Akt activator on osteoclast differentiation and bone resorption. SC79 supplementation into mutant BMMs cultures remarkably rescued actin ring formation and osteoclasts maturation ( a-c). Consistently, the Akt activator induced the expression of dendritic cell-specific transmembrane protein ( Dc-stamp ) in Ano5 KI/KI BMMs, which is essential for cell-cell fusion and actin ring formation in osteoclasts ( d) ( ). There is no significant difference at day 3, SC79 stimulation also enhanced the transcription level of the vacuolar (H + ) ATPase (v-ATPase) V0 domain ( Atp6v0d2 ) at other stages of osteoclast differentiation ( c). Interestingly, it has been reported that Atp6v0d2-deficient mice exhibited representative disruption of osteoclasts fusion similar to Dc-stamp knockout mice. In addition, Atp6v0d2 triggers proton transport to the resorptive lacunae and thus preserves the acidic environment needed for bone degradation ( ; ). 

SC79 positively regulated the expression of osteoclastogenesis-related factors in Ano5 KI/KI BMMs cultures Following the formation of an actin ring-rich sealing zone and the presence of a ruffled membrane, bone matrix degradation is induced by osteoclast-specific genes ( ). As shown in a, the decreased expression of Trap , cathepsin K ( Ctsk ), and matrix metalloproteinase-9 ( Mmp9 ) in Ano5 KI/KI BMMs cultures was switched back by SC79 at various differential stages. Furthermore, these factors have multiple sites recognized by Nfatc1, which is known as a significant regulator of osteoclastogenesis, and the reduction in Nfatc1 was also reversed by SC79 stimulation ( b). Notably, the level of cytosolic calcium taking part in robust Nfatc1 activation was obviously decreased, which was also rescued due to SC79 intervention ( c). In brief, these results suggested the negative effects of the p.Cys360Tyr mutation on osteoclast differentiation and function could be rectified by Akt activation. 

SC79 inhibited osteogenic differentiation and mineralization mediated by the p.Cys360Tyr mutation in Ano5 Bone metabolism relies on the delicate balance between bone resorption mediated by osteoclasts and bone formation mediated by osteoblasts. In light of decreased Akt phosphorylation in Ano5 KI/KI osteoclasts, we further examined the effects of Akt signaling pathway on the alterations in Ano5 KI/KI osteoblast differentiation and mineralization caused by the p.Cys360Tyr mutation. Western blot analysis revealed that the level of Akt phosphorylation was comparable between wild-type and Ano5 KI/KI osteoblasts at day 0 ( a). However, Akt phosphorylation was significantly suppressed in Ano5 KI/KI mature osteoblasts after 14 days of osteogenic induction, and this effect was rescued by 5 μg/ml SC79 ( b-c). To investigate the intrinsic role of Akt activation on GDD-related enhanced osteogenesis, ALP and alizarin red staining assays were carried out. SC79 stimulation in Ano5 KI/KI osteoblasts diminished excessive ALP activity, which is an early marker of osteogenic differentiation ( d). Furthermore, extracellular matrix mineralization, the most vital phenomenon in the late stage of bone formation, was detected by alizarin red staining accompanied by the measurement of calcium content, which showed that many fewer mineralized nodules and calcium deposition were formed after Ano5 KI/KI cells were cultured with the Akt activator SC79 ( e). 

Akt activation negatively regulated the expression of osteogenic-related factors in Ano5 KI/KI mCOBs Subsequently, the influences of the Akt activator on transcription levels of bone-forming related factors in different stages of Ano5 KI/KI mCOB cultures were explored. We found that up-regulation of Runx2 , a key regulator of osteoblast differentiation, was reversed almost to the level in Ano5 +/+ group by SC79 in both undifferentiated and mature Ano5 KI/KI mCOB ( ). Osteopontin (OPN), an important noncollagen protein in bone tissue, is synthesized by osteogenic progenitor cells and bone cells ( ), and the upregulated OPN expression was also inhibited by the Akt activator ( ). Additionally, osteocalcin ( Ocn ) and collagen type I alpha 1 ( Col1a1 ), which are classical markers of late osteogenic factors and matrix maturation, showed similar trends ( ). Overall, SC79 partially attenuated the enhanced osteogenesis caused by the GDD-related Ano5 mutation. Osteoblast could regulate osteoclastogenesis through regulating the expression of RANKL and OPG that is coordinated to regulate bone resorption and density positively and negatively by controlling the activation state of RANK on osteoclasts. Our previous study have found reduced osteoclastogenesis in Ano5 KI/KI BMMs partly attributed to elevated Opg/Rankl ratio in osteoblast ( ). Rankl transcription level with SC79 treatment was equivalent to Ano5 KI/KI group ( ). However, SC79 significantly attenuated Opg expression, which not only promotes bone formation but completes with RANKL to bind with RANK to inhibit osteoclast differentiation and maturation. Consistently, the ratio of Opg to Rankl was obviously declined, implying Akt activation could further rescue osteoclastogenesis in GDD via the effect of osteoblast-mediated osteoclastogenesis ( ). 

Discussion In the present study, the Akt activator SC79 promoted the maturation of multinucleated osteoclasts and the formation of intact actin rings in Ano5 KI/KI BMMs cultures, suggesting that Akt activation could effectively protect Ano5 KI/KI osteoclast function. Furthermore, we observed that abnormally enhanced capacity of osteoblast differentiation and matrix mineralization in Ano5 KI/KI mCOB cultures were reversed by an Akt activator, which indicated that Akt signaling is closely associated with aberrant bone remodeling in GDD. Autosomal dominant mutations in ANO5 are responsible for GDD, which is mainly characterized by enlargement of the mandible, osteomyelitis of the jaws, and cortical thickening of limb bones. In a previous study, we successfully established an Ano5 Cys360Tyr knockin mouse model expressing the human mutation p.Cys360Tyr in Ano5, which significantly exhibited typical traits of patients with GDD. Furthermore, Ano5 KI/KI mCOB displayed enhanced osteoblastogenesis consistent with our previous study in vitro and in another Ano5 knockout mouse model ( ; ; ). The phenotype of impaired osteoclast differentiation and bone resorption in Ano5 KI/KI mice is highly correlated with the other Ano5 knockout mice reported by the X, Li group. However, it is currently unclear how ANO5 dominant mutations lead to the bone disorder of GDD. Increasing evidence in recent years has indicated that ANO5 is closely associated with osteoclast differentiation and maturation. Consistent with the fact that Ano5 overexpression regulates osteoclast maturation through Akt signaling ( ), our previous research indicated that the p.Cys360Tyr mutation in Ano5 significantly inhibited Akt activation. To further determine the association between Akt signaling and abnormal osteoclastogenesis in GDD, SC79 was utilized and rescued the inhibition of TRAP − positive multinucleated osteoclast formation in Ano5 KI/KI BMMs cultures. The transcription level of Trap was also increased with SC79 stimulation. Importantly, enhanced expression of Nftac1 , a vital transcription factor for osteoclastogenesis, was also observed after Akt activation. The classic NFATc1 activation pathway requires an elevation of the intracellular calcium concentration, which then activates calcium/calmodulin-dependent protein phosphatase calcineurin and in turn leads to robust nuclear translocation of NFATC1 ( ). Furthermore, a recent study highlighted that Ano5 deletion in mice significantly diminished calcium oscillations in osteoclasts, which resulted in reduced RANKL-NFATc1 signaling ( ). Similarly, we found that the free calcium levels in the cytoplasm of Ano5 KI/KI osteoclasts were lower than those of wild-type osteoclasts and were surprisingly upregulated by SC79 stimulation, which may be attributed to reciprocal feedback between Akt activation and calcium signaling. We found Akt activation also could rescue osteoclastogenesis in GDD via declining Opg/Rankl ratio in osteoblast. Our functional evidence further indicated that inhibited osteoclasts differentiation in GDD is closely related with diminished calcium/Nfatc1 cascade pathway that is regulated by inactive Akt signaling. In the process of osteoclastic bone absorption, multinucleated osteoclasts undergo rearrangements of the actin cytoskeleton to constitute intact actin rings to form a sealed compartment between the bone surface and basal membrane ( ). Our in vitro data revealed the inhibitory effects of the p.Cys360Tyr mutation on F-actin formation was rescued by Akt activation as well, which was validated by upregulation of Atp6v0d2 and Dc-stamp . Furthermore, the expression of Mmp9 and Ctsk , responsible for the degradation of bone matrix, was enhanced after SC79 stimulation, which further implied that Akt activation could restore the bone resorption ability of Ano5 KI/KI osteoclasts. These results underscored that inhibition of Akt signaling is a crucial molecule in the abnormal osteoclastogenesis of GDD. Bone homeostasis also relies on osteoblasts mediating bone formation, and Akt signaling plays a critical role in cell viability, inhibiting apoptosis, and cell differentiation ( ). Consistent with the change in osteoclasts, the phosphorylation level of Akt was decreased in mature osteoblasts from Ano5 KI/KI mice. Meanwhile, our results suggested that SC79 inhibited the ALP activity and mineralized nodules formation of Ano5 KI/KI osteoblasts, implying that it is crucial for both the early and late stages of osteogenesis. Furthermore, Akt activation obviously suppressed the expression of Runx2 , a crucial regulator for the proliferation of osteoblast progenitors and their differentiation into osteoblasts and involved in inducing the transcription of Ocn and Col1a1 , which closely take part in the process of bone matrix formation ( ). Canonical AKT signaling is involved in promoting osteoblast proliferation and differentiation through mTOR and Gsk3β/β-catenin cascades ( ). Nevertheless, the levels of Gsk3β and downstream β-catenin were comparable between Ano5 KI/KI and Ano5 +/+ mCOBs in our study (Fig. S1, Additional File 1), which indicates that the decrease in Akt phosphorylation regulated osteogenesis by means other than interference of the Gsk3β/β-catenin pathway. Several studies have yielded similar findings to ours and showed that inhibition of Akt phosphorylation with imatinib stimulates osteogenesis in MSCs, but the reason is not well understood ( ). Surprisingly, Akt/mTOR signaling could inhibit the autophagy process that is required to maintain osteoblast differentiation and bone matrix secretion. Studies have pointed out that intracellular components of crystal-like structures in the cytoplasm are mainly located in autophagosome-like vesicles, and more convincingly, the apatite crystal components in the vesicles have been identified ( ; ). We hypothesized that increased osteogenesis caused by the mutation may be due to the reinforcement of autophagic activity. However, our in vitro data showed that the p.Cys360Tyr mutation in Ano5 had no relevant effect on the protein level of LC3II, one of the widely used markers for autophagy, which was further confirmed by comparable levels of Atg5 , Atg7 , Atg12 , and Beclin-1 (Fig. S2, Additional File 1). The enhanced osteoblastogenesis in Ano5 KI/KI mCOBs is independent of autophagy and Gsk3β signaling, while specific factors mechanically contributing to excessive osteoblast differentiation and bone matrix secretion mediated by reduced Akt phosphorylation in the GDD disorder model need to be further explored. 

Conclusion Our present study reveals that Akt activation modulates abnormal bone remodeling in the GDD model by stimulating osteoclast differentiation and inhibiting osteoblast function. Although the detailed mechanism has not been fully elucidated and further research is needed to verify our findings in vivo , this study not only provides a deeper understanding of the pathogenesis of GDD but also lays a new theoretical foundation for targeted molecular therapy. 

Hongyu Li: Writing – review & editing, Writing – original draft, Funding acquisition, Formal analysis, Data curation, Conceptualization. Shengnan Wang: Data curation. Shuai Zhang: Formal analysis. Rui Dong: Formal analysis. Congcong Miao: Methodology. Zhenchuan Tian: Methodology. Ying Hu: Writing – review & editing, Writing – original draft, Funding acquisition, Conceptualization. 

All methods were carried out in accordance with relevant guidelines and regulations. This study is reported in accordance with the ARRIVE guidelines. Overall animal experimental designs and protocals were approved by Institutional Animal Care and Use Committee of the Beijing Stomatological Hospital (approval number: KQYY-202012-005). 

Not applicable. 

This study was funded by the National Natural Science Foundation of China (Grant #82071103, Grant#81570958) and Young Scientist Program of Beijing Stomatological Hospital, Capital Medical University, YSP202317. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

