Regulation Of Osteoclast Function Research Articles

Tm4sf19 deficiency inhibits osteoclast multinucleation and prevents bone loss

BackgroundMultinucleation is a hallmark of osteoclast formation and has a unique ability to resorb bone matrix. During osteoclast differentiation, the cytoskeleton reorganization results in the generation of actin belts and eventual bone resorption. Tetraspanins are involved in adhesion, migration and fusion in various cells. However, its function in osteoclast is still unclear. In this study, we identified Tm4sf19, a member of the tetraspanin family, as a regulator of osteoclast function. Materials and methodsWe investigate the effect of Tm4sf19 deficiency on osteoclast differentiation using bone marrow-derived macrophages obtained from wild type (WT), Tm4sf19 knockout (KO) and Tm4sf19 LELΔ mice lacking the large extracellular loop (LEL). We analyzed bone mass of young and aged WT, KO and LELΔ mice by μCT analysis. The effects of Tm4sf19 LEL-Fc fusion protein were accessed in osteoclast differentiation and osteoporosis animal model. ResultsWe found that deficiency of Tm4sf19 inhibited osteoclast function and LEL of Tm4sf19 was responsible for its function in osteoclasts in vitro. KO and LELΔ mice exhibited higher trabecular bone mass compared to WT mice. We found that Tm4sf19 interacts with integrin αvβ3 through LEL, and that this binding is important for cytoskeletal rearrangements in osteoclast by regulating signaling downstream of integrin αvβ3. Treatment with LEL-Fc fusion protein inhibited osteoclast function in vitro and administration of LEL-Fc prevented bone loss in an osteoporosis mouse model in vivo. ConclusionWe suggest that Tm4sf19 regulates osteoclast function and that LEL-Fc may be a promising drug to target bone destructive diseases caused by osteoclast hyper-differentiation.

MiR-92a-1-5p enriched prostate cancer extracellular vesicles regulate osteoclast function via MAPK1 and FoxO1

BackgroundWe have previously reported that extracellular vesicles (EVs) derived from osteoblastic, osteoclastic and mixed prostate cancer cells promote osteoclast differentiation and inhibit osteoblast differentiation via transferring miR-92a-1-5p. In the present study, we focused on engineering miR-92a-1-5p into EVs and determining any therapeutic roles and mechanisms of the engineered EVs.MethodsA stable prostate cancer cell line (MDA PCa 2b) overexpressing miR-92a-1-5p was constructed by lentivirus, and EVs were isolated by ultracentrifugation. The overexpression of miR-92a-1-5p in both cells and EVs was tested using qPCR. Osteoclast function was evaluated by Trap staining, mRNA expression of osteoclastic markers ctsk and trap, immunolabeling of CTSK and TRAP and microCT using either in vitro and in vivo assays. Target gene of miR-92a-1-5p was proved by a dual-luciferase reporter assay system. siRNAs were designed and used for transient expression in order to determine the role of downstream genes on osteoclast differentiation.ResultsStable overexpression cells of miRNA-92a-5p was associated with EVs upregulating this microRNA, as confirmed by qPCR. Further, miR-92a-1-5p enriched EVs promote osteoclast differentiation in vitro by reducing MAPK1 and FoxO1 expression, associated with increased osteoclast function as shown by TRAP staining and mRNA expression of osteoclast functional genes. siRNA targeting MAPK1 or FoxO1 resulted in similar increase in osteoclast function. In vivo, the miR-92a-1-5p enriched EVs given via i.v. injection promote osteolysis, which was associated with reduction of MAPK1 and FoxO1 expression in bone marrow.ConclusionThese experiments suggest that miR-92a-1-5p enriched EVs regulate osteoclast function via reduction of MAPK1 and FoxO1.Graphical

Magnesium alloys for orthopedic applications:A review on the mechanisms driving bone healing

Magnesium (Mg) alloys have attracted a wealth of attention in orthopedic fields for their superior mechanical properties, degradability, and excellent biocompatibility. Consistently, to resolve the issues on rapid degradation, more studies are dedicated to the researches on the composition design, preparation and processing, surface modification, the degradation modes of Mg alloys. Nevertheless, the mechanisms by which Mg alloys promote bone healing remain elusive. This review gives an account of specific mechanisms on Mg alloys promoting bone healing from four aspects, immunomodulatory, angiogenesis, osteogenesis and regulation of osteoclast function. We highlight the regulation of Mg alloys on the functional status and interactions of numerous cells that are involved in bone healing, including immune cells, osteogenic-related cells, osteoclasts, endothelial cells (ECs), nerve cells, etc., and summarize the signaling pathways involved, with the aim to provide the basis and support on future investigation on mechanisms on Mg alloys driving bone regeneration. More importantly, it provides a rationale and a general new basis for the application of Mg alloys in orthopedic fields.

PGE2 activates EP4 in subchondral bone osteoclasts to regulate osteoarthritis

Prostaglandin E2 (PGE2), a major cyclooxygenase-2 (COX-2) product, is highly secreted by the osteoblast lineage in the subchondral bone tissue of osteoarthritis (OA) patients. However, NSAIDs, including COX-2 inhibitors, have severe side effects during OA treatment. Therefore, the identification of novel drug targets of PGE2 signaling in OA progression is urgently needed. Osteoclasts play a critical role in subchondral bone homeostasis and OA-related pain. However, the mechanisms by which PGE2 regulates osteoclast function and subsequently subchondral bone homeostasis are largely unknown. Here, we show that PGE2 acts via EP4 receptors on osteoclasts during the progression of OA and OA-related pain. Our data show that while PGE2 mediates migration and osteoclastogenesis via its EP2 and EP4 receptors, tissue-specific knockout of only the EP4 receptor in osteoclasts (EP4LysM) reduced disease progression and osteophyte formation in a murine model of OA. Furthermore, OA-related pain was alleviated in the EP4LysM mice, with reduced Netrin-1 secretion and CGRP-positive sensory innervation of the subchondral bone. The expression of platelet-derived growth factor-BB (PDGF-BB) was also lower in the EP4LysM mice, which resulted in reduced type H blood vessel formation in subchondral bone. Importantly, we identified a novel potent EP4 antagonist, HL-43, which showed in vitro and in vivo effects consistent with those observed in the EP4LysM mice. Finally, we showed that the Gαs/PI3K/AKT/MAPK signaling pathway is downstream of EP4 activation via PGE2 in osteoclasts. Together, our data demonstrate that PGE2/EP4 signaling in osteoclasts mediates angiogenesis and sensory neuron innervation in subchondral bone, promoting OA progression and pain, and that inhibition of EP4 with HL-43 has therapeutic potential in OA.

Tetraspanin 7 regulates osteoclast function through association with the RANK/αvβ3 integrin complex.

Actin rings are unique structures that facilitate the attachment of osteoclasts to the bone matrix during bone resorption. Previous studies have shown that tetraspanin7 (TSPAN7) plays an important role in the reorganization of the cytoskeleton necessary for the bone-resorbing activity of osteoclasts. However, questions remain as to the mechanisms by which TSPAN7 regulates this cytoskeletal rearrangement. In this study, we investigated the roles of TSPAN7 in osteoclasts by deleting the Tm4sf2 gene in mice, which encodes TSPAN7. The Tm4sf2 global knockout model showed protective effects on pathological bone loss, but no discernible changes in bone phenotypes under physiological conditions. In vitro study revealed that ablation of Tm4sf2 caused significant defects in integrin-mediated actin ring formation, thereby leading to significantly decreased bone resorption. Additionally, we demonstrated an association between TSPAN7 and the receptor activator of nuclear factor-кB/αvβ3 integrin. Overall, our findings suggest that TSPAN7 acts as a novel modulator regulating the bone-resorbing function of osteoclasts.

ELMO1 signaling is a promoter of osteoclast function and bone loss

Osteoporosis affects millions worldwide and is often caused by osteoclast induced bone loss. Here, we identify the cytoplasmic protein ELMO1 as an important ‘signaling node’ in osteoclasts. We note that ELMO1 SNPs associate with bone abnormalities in humans, and that ELMO1 deletion in mice reduces bone loss in four in vivo models: osteoprotegerin deficiency, ovariectomy, and two types of inflammatory arthritis. Our transcriptomic analyses coupled with CRISPR/Cas9 genetic deletion identify Elmo1 associated regulators of osteoclast function, including cathepsin G and myeloperoxidase. Further, we define the ‘ELMO1 interactome’ in osteoclasts via proteomics and reveal proteins required for bone degradation. ELMO1 also contributes to osteoclast sealing zone on bone-like surfaces and distribution of osteoclast-specific proteases. Finally, a 3D structure-based ELMO1 inhibitory peptide reduces bone resorption in wild type osteoclasts. Collectively, we identify ELMO1 as a signaling hub that regulates osteoclast function and bone loss, with relevance to osteoporosis and arthritis.

Tumor- and osteoclast-derived NRP2 in prostate cancer bone metastases

Understanding the role of neuropilin 2 (NRP2) in prostate cancer cells as well as in the bone microenvironment is pivotal in the development of an effective targeted therapy for the treatment of prostate cancer bone metastasis. We observed a significant upregulation of NRP2 in prostate cancer cells metastasized to bone. Here, we report that targeting NRP2 in cancer cells can enhance taxane-based chemotherapy with a better therapeutic outcome in bone metastasis, implicating NRP2 as a promising therapeutic target. Since, osteoclasts present in the tumor microenvironment express NRP2, we have investigated the potential effect of targeting NRP2 in osteoclasts. Our results revealed NRP2 negatively regulates osteoclast differentiation and function in the presence of prostate cancer cells that promotes mixed bone lesions. Our study further delineated the molecular mechanisms by which NRP2 regulates osteoclast function. Interestingly, depletion of NRP2 in osteoclasts in vivo showed a decrease in the overall prostate tumor burden in the bone. These results therefore indicate that targeting NRP2 in prostate cancer cells as well as in the osteoclastic compartment can be beneficial in the treatment of prostate cancer bone metastasis.

Osteoclast-derived small extracellular vesicles induce osteogenic differentiation via inhibiting ARHGAP1

Activated osteoclasts release large amounts of small extracellular vesicles (sEVs) during bone remodeling. However, little is known about whether osteoclast-derived sEVs affect surrounding cells. In this study, osteoclasts were generated by stimulating bone marrow macrophages (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear actor κB ligand (RANKL). We performed microarray analysis of sEV-microRNAs (miRNAs)s secreted from osteoclast at different stages and identified four miRNAs that were highly expressed in mature osteoclast-derived sEVs. One of these miRNAs, miR-324, significantly induced osteogenic differentiation and mineralization of primary mesenchymal stem cells (MSCs) in vitro by targeting ARHGAP1, a negative regulator of osteogenic differentiation. We next fabricated an sEV-modified scaffold by coating decalcified bone matrix (DBM) with osteoclast-derived sEVs, and the pro-osteogenic regeneration activities of the sEV-modified scaffold were validated in a mouse calvarial defect model. Notably, miR-324-enriched sEV-modified scaffold showed the highest capacity on bone regeneration, whereas inhibition of miR-324 in sEVs abrogated these effects. Taken together, our findings suggest that miR-324-contained sEVs released from mature osteoclast play an essential role in the regulation of osteogenic differentiation and potentially bridge the coupling between osteoclasts and MSCs.

Kynurenine Promotes RANKL-Induced Osteoclastogenesis In Vitro by Activating the Aryl Hydrocarbon Receptor Pathway.

There is increasing evidence of the involvement of the tryptophan metabolite kynurenine (KYN) in disrupting osteogenesis and contributing to aging-related bone loss. Here, we show that KYN has an effect on bone resorption by increasing osteoclastogenesis. We have previously reported that in vivo treatment with KYN significantly increased osteoclast number lining bone surfaces. Here, we report the direct effect of KYN on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in Raw 264.7 macrophage cells, and we propose a potential mechanism for these KYN-mediated effects. We show that KYN/RANKL treatment results in enhancement of RANKL-induced osteoclast differentiation. KYN drives upregulation and activation of the key osteoclast transcription factors, c-fos and NFATc1 resulting in an increase in the number of multinucleated TRAP+ osteoclasts, and in hydroxyapatite bone resorptive activity. Mechanistically, the KYN receptor, aryl hydrocarbon receptor (AhR), plays an important role in the induction of osteoclastogenesis. We show that blocking AhR signaling using an AhR antagonist, or AhR siRNA, downregulates the KYN/RANKL-mediated increase in c-fos and NFATc1 and inhibits the formation of multinucleated TRAP + osteoclasts. Altogether, this work highlights that the novelty of the KYN and AhR pathways might have a potential role in helping to regulate osteoclast function with age and supports pursuing additional research to determine if they are potential therapeutic targets for the prevention or treatment of osteoporosis.

Central Regulatory Role of Cytokines in Periodontitis and Targeting Options.

Periodontitis is an immune-inflammatory disease that leads to the progressive destruction of bone and connective tissue in the periodontal area. The cytokine network plays a primary role in tissue homeostasis, the recruitment of immune cells to control the pathogenic impact and the regulation of osteoclastic function, thus modulating the intensity and duration of the immune response. This review provides an update on the main cytokines implicated in the pathogenesis and progression of periodontitis and their targeting potential in order to enrich current treatment options. A structured search of bibliographic databases (PubMed, MEDLINE, Scopus) was performed for peer-reviewed cytokine studies focused on periodontitis the last ten years. A qualitative content analysis was performed in screened papers and a critical discussion of main findings is provided. An altered cytokine profile has been detected in periodontitis patients and the interplay of pro-inflammatory and/or anti-inflammatory cytokines has been associated with disease pathogenesis. Among the most prominent pro-inflammatory cytokines, TNF-α, IL-1β, IL-17, IL-6 and the chemokines CXCL-6, CXCL-8 are overexpressed in periodontitis patients and correlate with disease progression. On the other hand, the anti-inflammatory IL-4 and IL- 11 levels are reduced while IL-12 and IFN-γ expression play a dual role in periodontal disease. Current periodontitis treatment strategies include selective antibiotics, antimicrobial photodynamic therapy and probiotics, which can modulate the cytokine network and when applied in combination with specific anti-cytokine agents can exert additional beneficial effects. It is evident that cytokines play a central regulatory role in the inflammatory process and immune cell response that underlies bone destruction in periodontitis. Specific cytokine targeting should be considered as a complementary therapeutic scheme to current periodontal management.

Optineurin regulates osteoblastogenesis through STAT1

A sophisticated and delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts regulates bone metabolism. Optineurin (OPTN) is a gene involved in primary open-angle glaucoma and amyotrophic lateral sclerosis. Although its function has been widely studied in ophthalmology and neurology, recent reports have shown its possible involvement in bone metabolism through negative regulation of osteoclast differentiation. However, little is known about the role of OPTN in osteoblast function. Here, we demonstrated that OPTN controls not only osteoclast but also osteoblast differentiation. Different parameters involved in osteoblastogenesis and osteoclastogenesis were assessed in Optn−/- mice. The results showed that osteoblasts from Optn−/- mice had impaired alkaline phosphatase activity, defective mineralized nodules, and inability to support osteoclast differentiation. Moreover, OPTN could bind to signal transducer and activator of transcription 1 (STAT1) and regulate runt-related transcription factor 2 (RUNX2) nuclear localization by modulating STAT1 levels in osteoblasts. These data suggest that OPTN is involved in bone metabolism not only by regulating osteoclast function but also by regulating osteoblast function by mediating RUNX2 nuclear translocation via STAT1.

Modulation of Transient Receptor Potential Channels 3 and 6 Regulates Osteoclast Function with Impact on Trabecular Bone Loss

Enhanced osteoclast formation and function is a fundamental cause of alterations to bone structure and plays an important role in several diseases impairing bone quality. Recent work revealed that TRP calcium channels 3 and 6 might play a special role in this context. By analyzing the bone phenotype of TRPC6-deficient mice we detected a regulatory effect of TRPC3 on osteoclast function. These mice exhibit a significant decrease in bone volume per tissue volume, trabecular thickness and -number together with an increased number of osteoclasts found on the surface of trabecular bone. Primary bone marrow mononuclear cells from TRPC6-deficient mice showed enhanced osteoclastic differentiation and resorptive activity. This was confirmed in vitro by using TRPC6-deficient RAW 264.7 cells. TRPC6 deficiency led to an increase of TRPC3 in osteoclasts, suggesting that TRPC3 overcompensates for the loss of TRPC6. Raised intracellular calcium levels led to enhanced NFAT-luciferase reporter gene activity in the absence of TRPC6. In line with these findings inhibition of TRPC3 using the specific inhibitor Pyr3 significantly reduced intracellular calcium concentrations and normalized osteoclastic differentiation and resorptive activity of TRPC6-deficient cells. Interestingly, an up-regulation of TRPC3 could be detected in a cohort of patients with low bone mineral density by comparing micro array data sets of circulating human osteoclast precursor cells to those from patients with high bone mineral density, suggesting a noticeable contribution of TRP calcium channels on bone quality. These observations demonstrate a novel regulatory function of TRPC channels in the process of osteoclastic differentiation and bone loss.

MicroRNA-25-3p regulates osteoclasts through nuclear factor I X

Osteoporosis is a bone metabolic disease, characterized by loss of bone density leading to fractures. Its incidence increases with age and affects patient quality of life. Although osteoclasts play a significant role in osteoporosis, their underlying regulatory mechanisms remain unclear. In this study, we found that microRNA (miR)-25-3p negatively regulates osteoclast function through nuclear factor I X (NFIX). Overexpression of NFIX promoted osteoclast proliferation and increased the expression of the osteoclast differentiation and activity markers tartrate-resistant acid phosphatase and cathepsin K. MiR-25-3p transfection inhibited NFIX expression, which in turn inhibited osteoclast proliferation. Collectively, our results suggest that miR-25-3p promotes osteoclast activity by regulating the expression of NFIX. Therefore, targeting miR-25-3p in osteoclasts could be a promising strategy for treating skeletal disorders involving reduced bone formation.

RACK1 interaction with c-Src is essential for osteoclast function

The scaffolding protein receptor for activated C-kinase 1 (RACK1) mediates receptor activator of nuclear factor κΒ ligand (RANKL)-dependent activation of p38 MAPK in osteoclast precursors; however, the role of RACK1 in mature osteoclasts is unclear. The aim of our study was to identify the interaction between RACK1 and c-Src that is critical for osteoclast function. A RACK1 mutant protein (mutations of tyrosine 228 and 246 residues to phenylalanine; RACK1 Y228F/Y246F) did not interact with c-Src. The mutant retained its ability to differentiate into osteoclasts; however, the integrity of the RANKL-mediated cytoskeleton, bone resorption activity, and phosphorylation of c-Src was significantly decreased. Importantly, lysine 152 (K152) within the Src homology 2 (SH2) domain of c-Src is involved in RACK1 binding. The c-Src K152R mutant (mutation of lysine 152 into arginine) impaired the resorption of bone by osteoclasts. These findings not only clarify the role of the RACK1-c-Src axis as a key regulator of osteoclast function but will also help to develop new antiresorption therapies to prevent bone loss-related diseases.

Highly Dispersed Fullerenols Hamper Osteoclast Ruffled Border Formation by Perturbing Ca2+ Bundles.

Osteoporosis, a common and serious bone disorder affecting aged people and postmenopausal women, is characterized by osteoclast overactivity. One therapeutic strategy is suppressing the bone resorption function of hyperactive osteoclasts, but there is no effective drug in clinical practice so far. Herein, it is demonstrated that fullerenols suppress the bone resorption of osteoclasts by inhibiting ruffled borders (RBs) formation. The RBs formation, which is supported by well-aligned actin bundles (B-actins), is a critical event for osteoclast bone resorption. To facilitate this function, osteoclast RBs dynamics is regulated by variable microenvironments to bundle F-actins, protrude cell membrane, and so on. B-actin perturbation by fullerenols is determined here, offering an opportunity to regulate osteoclast function by destroying RBs. In vivo, the therapeutic effect of fullerenols on overactive osteoclasts is confirmed in a mouse model of lipopolysaccharide-induced bone erosion. Collectively, the findings suggest that fullerenols adhere to F-actin surfaces and inhibit RBs formation in osteoclasts, mainly through hampering Ca2+ from bundling F-actins, and this is likely due to the stereo-hindrance effect caused by adherent fullerenols.

LRRK1 regulation of actin assembly in osteoclasts involves serine 5 phosphorylation of L-plastin.

Mice with disruption of Lrrk1 and patients with nonfunctional mutant Lrrk1 exhibit severe osteopetrosis phenotypes because of osteoclast cytoskeletal dysfunction. To understand how Lrrk1 regulates osteoclast function by modulating cytoskeleton rearrangement, we examined the proteins that are differentially phosphorylated in wild‐type mice and Lrrk1‐deficient osteoclasts by metal affinity purification coupled liquid chromatography/mass spectrometry (LC/MS) analyses. One of the candidates that we identified by LC/MS is L‐plastin, an actin bundling protein. We found that phosphorylation of L‐plastin at serine (Ser) residues 5 was present in wild‐type osteoclasts but not in Lrrk1‐deficient cells. Western blot analyses with antibodies specific for Ser5 phosphorylated L‐plastin confirmed the reduced L‐plastin Ser5 phosphorylation in Lrrk1 knockout (KO) osteoclasts. micro computed tomography (Micro‐CT) analyses revealed that the trabecular bone volume of the distal femur was increased by 27% in the 16 to 21‐week‐old L‐plastin KO females as compared with the wild‐type control mice. The ratio of bone volume to tissue volume and connectivity density were increased by 44% and 47% (both P < 0.05), respectively, in L‐plastin KO mice. Our data suggest that targeted disruption of L‐plastin increases trabecular bone volume, and phosphorylation of Ser5 in L‐plastin in the Lrrk1 signaling pathway may in part contribute to actin assembly in mature osteoclasts.

WITHDRAWN: Catalytically inactive phosphatase MTMR12 is a novel regulator of osteoclast function through F-actin ring formation

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P2X7 receptor regulates osteoclast function and bone loss in a mouse model of osteoporosis

Post-menopausal osteoporosis is a condition that affects millions worldwide and places a huge socio-economic burden on society. Previous research has shown an association of loss of function SNPs in the gene for the purinergic receptor P2X7R with low bone mineral density, increased rates of bone loss and vertebral fractures in post-menopausal women. In this study we use a mouse model of oestrogen deficiency-induced bone loss and the BALB/cJ P2X7R−/− to show that absence of the P2X7R resulted in increased bone loss. Osteoclast precursors were isolated from both BALB/cJ P2X7R−/− and BALB/cJ P2X7R+/+ mice and then cultured in vitro to form mature resorbing osteoclasts. The BALB/cJ P2X7R−/− derived precursors generated slightly more osteoclasts but with a significant reduction in the amount of resorption per osteoclast. Furthermore, when using modified culture conditions osteoclast activity was additionally increased in the absence of the P2X7R suggest that P2X7R may regulate the lifespan and activity of osteoclasts. Finally using mechanical loading as an anabolic stimulus for bone formation, we demonstrated that the increased oestrogen-deficient bone loss could be rescued, even in the absence of P2X7R. This study paves the way for clinical intervention for women with post-menopausal osteoporosis and P2XR7 loss of function polymorphisms.

Histochemical evidence of IGF2 mRNA-binding protein 2-mediated regulation of osteoclast function and adhesive ability.

Insulin-like growth factor 2 (IGF2) messenger RNA-binding proteins (IMPs) are a family of oncofetal RNA-binding proteins that play important roles in cell migration, renewal, and metabolism. IMP2 gene expression may be important in determining IGF2 levels and might, thereby, be central to bone metabolism. In our present study, IMP2-deficient mice exhibited more immature bone structures, characterized by abundant residual cartilage cores; growth plates containing more rich cartilage matrix, which was arranged irregularly; and a significantly thicker hypertrophic chondrocyte layer in the femoral metaphysis, compared with wild-type mice. These abnormalities were associated with profound effects on the size and morphology of osteoclasts. Specifically, the osteoclasts exhibited various polymorphisms, failed to form resorption lacunae, and were detached from the bone surface. Consistent with these findings, IMP2 deficiency reduced the expression of two important proteases (cathepsin K and matrix metallopeptidase 9) as well as that of C-SRC, a critical regulator of ruffled border formation in osteoclasts, indicating impaired osteoclastic activity. IMP2-deficient mice also displayed inhibited osteoclast adhesion owing to defects in the CD44-osteopontin signaling pathway. In summary, we used IMP2-deficient mice as a model to determine whether IMP2 plays a role during bone metabolism. Our results indicate that IMP2 deficiency delayed bone remodeling by significantly inhibiting the activity of osteoclasts and impairing their adhesion.

Analysis of two transcript isoforms of vacuolar ATPase subunit H in mouse and zebrafish

ATP6V1H encodes the subunit H of vacuolar ATPase (V-ATPase) and has been recently proved to regulate osteoclast function. The alternative splicing of ATP6V1H gene results in two isoforms, and it is not clear whether and how the two isoforms function differently. In this report, we used bioinformatics methods to compare the differences of two isoforms in different species. The distributions and amounts of two isoforms were analyzed in eleven kinds of mouse tissues and mouse osteoclasts using RT-PCR, Q-PCR, western blot and immunohistochemical staining methods, respectively. In order to observe the in vivo biological differences of two isoforms during development, the zebrafish mRNA of two wild type atp6v1h transcripts as well as their mutant forms were also injected into zebrafish embryos, respectively. Bioinformatic analysis revealed that two isoforms were quite different in many ways, especially in protein size, internal space, phosphorylation state and H-bond binding. The amounts of two transcripts and the ratio of long and short transcript varied a lot from tissue to tissue or cell to cell, and osteoclasts were the cells only expressing long isoform among the tissues or cells we detected. The in vivo selective expression of two subunit H splice variants showed their different effects on the craniofacial development of zebrafish. The short isoform reduced the size of zebrafish head and did not play a complete function compared with the long isoform. We propose that long isoform of subunit H is necessary for the normal craniofacial bone development and the lack of short transcript might be necessary for the normal osteoclastic function.