Moesin activation controls bone resorption and tunneling nanotube-dependent osteoclast fusion.

Osteoclasts are multinucleated cells unique in their ability to resorb bone. Osteoclastogenesis involves several steps of actin-driven rearrangements that participate not only in the cell-cell fusion process, but also in the formation of the sealing zone, the adhesive structure determining the resorption area. Despite the importance of these actin cytoskeleton-based processes, their precise mechanisms of regulation are still poorly characterized. Here, we found that moesin, a member of the Ezrin/Radixin/Moesin (ERM) protein family, is activated during osteoclast maturation and plays an instrumental role for both osteoclast fusion and function. In mouse and human osteoclast precursors, moesin is negatively regulated to potentiate their ability to fuse and degrade bone. Accordingly, we demonstrated that moesin depletion decreases membrane-to-cortex attachment and enhances formation of tunneling nanotubes (TNTs), F-actin-containing intercellular bridges that we revealed to trigger osteoclast fusion. In addition, via a ß3-integrin/RhoA/SLK pathway and independently of its role in fusion, moesin regulates the number and organization of sealing zones in mature osteoclast, and thus participates in the control of bone resorption. Supporting these findings, we found that moesin-deficient mice are osteopenic with a reduced density of trabecular bones and increased osteoclast abundance and activity. These findings provide a better understanding of the regulation of osteoclast biology, and open new opportunities to specifically target osteoclast activity in bone disease therapy.

Trauma patient heterotopic ossification diagnosis is associated with increased hospital length of stay.

BACKGROUND: Traumatic heterotopic ossification (tHO) refers to the development of extra-skeletal bone in muscle and soft tissues following tissue insult secondary to surgery or trauma. This presents a persistent clinical concern associated with significant patient morbidity and expense to diagnose and treat. Traumatic HO is a substantial barrier to rehabilitation for trauma-injured patients. As such, the development of tHO after burn and other trauma is hypothesised to prolong inpatient length of stay (LOS) and thus increase health care costs. OBJECTIVE: To investigate the association between an inpatient tHO diagnosis and hospital LOS in trauma patients. METHODS: A retrospective audit of trauma patients over a 14-year period was completed using data from four WA hospitals. Burn and neurological trauma patients diagnosed with tHO as an inpatient (tHO+) and control subjects (tHO-), matched (1:3) by age, gender, and injury severity factors, were identified using medical diagnostic codes. Data relating to patient and injury-related determinants of LOS from tHO+ and tHO- subjects were analysed to model the association of tHO on total hospital length of stay. RESULTS: 188 identified patients were hospitalised due to traumatic injury; 47 patients with tHO following burn injury (n = 17), spinal cord injury (n = 13) and traumatic brain injury (n = 17), and 141 control patients. Those who developed tHO during hospitalisation had a significantly higher median LOS than matched trauma patients who did not develop tHO (142 days vs. 61 days). Multivariate regression analyses identified the following independent predictive factors of a prolonged hospital LOS: tHO diagnosis, mechanical ventilation hours, injury to the hip region and thigh area, other ossification disorder, pressure injury, admission to intensive care unit and deep vein thrombosis. Trauma patients diagnosed with tHO during their hospital admission stayed 1.6 times longer than trauma patients matched for injury severity without a tHO diagnosis (IRR 1.56, 95% CI 1.35-1.79, p<0.001). CONCLUSION: Traumatic heterotopic ossification is an independent explanatory factor for increased hospital LOS in patients following burns, spinal cord, and traumatic brain injury. Early diagnosis may assist in reducing the impact of tHO on acute hospital stay after trauma.

Evaluation of the accuracy of diagnostic coding and clinical documentation for traumatic heterotopic ossification diagnoses in Western Australian hospitals.

BACKGROUND: Traumatic heterotopic ossification (tHO) refers to the pathological formation of ectopic bone in soft tissues that can occur following burn, neurological ororthopaedic trauma. As completeness and accuracy of medical diagnostic coding can vary based on coding practices and depend on the institutional culture of clinical documentation, it is important to assess diagnostic coding in that local context. To the authors' knowledge, there is no prior study evaluating the accuracy of medical diagnostic coding or specificity of clinical documentation for tHO diagnoses across Western Australia (WA) trauma centres or across the full range of inciting injury and surgical events. OBJECTIVE: To evaluate and compare the clinical documentation and the diagnostic accuracy of ICD-10-AM coding for tHO in trauma populations across 4 WA hospitals. METHODS: A retrospective data search of the WA trauma database was conducted to identify patients with tHO admitted to WA hospitals following burn, neurological or orthopaedic trauma. Patient demographic and tHO diagnostic characteristics were assessed for all inpatient and outpatient tHO diagnoses. The frequency and distribution of M61 (HO-specific) and broader, musculoskeletal (non-specific) ICD-10-AM codes were evaluated for tHO cases in each trauma population. RESULTS: HO-specific M61 ICD-10-AM codes failed to identify more than a third of true tHO cases, with a high prevalence of non-specific HO codes (19.4 %) and cases identified via manual chart review (25.4 %). The sensitivity of M61 codes for correctly diagnosing tHO after burn injury was 50 %. ROC analysis showed that M61 ICD-10-AM codes as a predictor of a true positive tHO diagnosis were a less than favourable method (AUC=0.731, 95 % CI=0.561-0.902, p = 0.012). Marked variability in clinical documentation for tHO was identified across the hospital network. CONCLUSION: Coding inaccuracies may, in part, be influenced by insufficiencies in clinical documentation for tHO diagnoses, which may have implications for future research and patient care. Clinicians should consistently employ standardised clinical terminology from the point of care to increase the likelihood of accurate medical diagnostic coding for tHO diagnoses.

Age-dependent genetic regulation of osteoarthritis: independent effects of immune system genes.

OBJECTIVES: Osteoarthritis (OA) is a joint disease with a heritable component. Genetic loci identified via genome-wide association studies (GWAS) account for an estimated 26.3% of the disease trait variance in humans. Currently, there is no method for predicting the onset or progression of OA. We describe the first use of the Collaborative Cross (CC), a powerful genetic resource, to investigate knee OA in mice, with follow-up targeted multi-omics analysis of homologous regions of the human genome. METHODS: We histologically screened 275 mice for knee OA and conducted quantitative trait locus (QTL) mapping in the complete cohort (> 8 months) and the younger onset sub-cohort (8-12 months). Multi-omic analysis of human genetic datasets was conducted to investigate significant loci. RESULTS: We observed a range of OA phenotypes. QTL mapping identified a genome-wide significant locus on mouse chromosome 19 containing Glis3, the human equivalent of which has been identified as associated with OA in recent GWAS. Mapping the younger onset sub-cohort identified a genome-wide significant locus on chromosome 17. Multi-omic analysis of the homologous region of the human genome (6p21.32) indicated the presence of pleiotropic effects on the expression of the HLA - DPB2 gene and knee OA development risk, potentially mediated through the effects on DNA methylation. CONCLUSIONS: The significant associations at the 6p21.32 locus in human datasets highlight the value of the CC model of spontaneous OA that we have developed and lend support for an immune role in the disease. Our results in mice also add to the accumulating evidence of a role for Glis3 in OA.

Leveraging osteoclast genetic regulatory data to identify genes with a role in osteoarthritis.

There has been a growing interest in the role of the subchondral bone and its resident osteoclasts in the progression of osteoarthritis (OA). A recent genome-wide association study (GWAS) identified 100 independent association signals for OA traits. Most of these signals are led by noncoding variants, suggesting that genetic regulatory effects may drive many of the associations. We have generated a unique human osteoclast-like cell-specific expression quantitative trait locus (eQTL) resource for studying the genetics of bone disease. Considering the potential role of osteoclasts in the pathogenesis of OA, we performed an integrative analysis of this dataset with the recently published OA GWAS results. Summary data-based Mendelian randomization (SMR) and colocalization analyses identified 38 genes with a potential role in OA, including some that have been implicated in Mendelian diseases with joint/skeletal abnormalities, such as BICRA, EIF6, CHST3, and FBN2. Several OA GWAS signals demonstrated colocalization with more than one eQTL peak, including at 19q13.32 (hip OA with BCAM, PRKD2, and BICRA eQTL). We also identified a number of eQTL signals colocalizing with more than one OA trait, including FAM53A, GCAT, HMGN1, MGAT4A, RRP7BP, and TRIOBP. An SMR analysis identified 3 loci with evidence of pleiotropic effects on OA-risk and gene expression: LINC01481, CPNE1, and EIF6. Both CPNE1 and EIF6 are located at 20q11.22, a locus harboring 2 other strong OA candidate genes, GDF5 and UQCC1, suggesting the presence of an OA-risk gene cluster. In summary, we have used our osteoclast-specific eQTL dataset to identify genes potentially involved with the pathogenesis of OA.

Bone Trans-omics: Integrating Omics to Unveil Mechanistic Molecular Networks Regulating Bone Biology and Disease.

PURPOSE OF REVIEW: Recent advancements in "omics" technologies and bioinformatics have afforded researchers new tools to study bone biology in an unbiased and holistic way. The purpose of this review is to highlight recent studies integrating multi-omics data gathered from multiple molecular layers (i.e.; trans-omics) to reveal new molecular mechanisms that regulate bone biology and underpin skeletal diseases. RECENT FINDINGS: Bone biologists have traditionally relied on single-omics technologies (genomics, transcriptomics, proteomics, and metabolomics) to profile measureable differences (both qualitative and quantitative) of individual molecular layers for biological discovery and to investigate mechanisms of disease. Recently, literature has grown on the implementation of integrative multi-omics to study bone biology, which combines computational and informatics support to connect multiple layers of data derived from individual "omic" platforms. This emerging discipline termed "trans-omics" has enabled bone biologists to identify and construct detailed molecular networks, unveiling new pathways and unexpected interactions that have advanced our mechanistic understanding of bone biology and disease. While the era of trans-omics is poised to revolutionize our capacity to answer more complex and diverse questions pertinent to bone pathobiology, it also brings new challenges that are inherent when trying to connect "Big Data" sets. A concerted effort between bone biologists and interdisciplinary scientists will undoubtedly be needed to extract physiologically and clinically meaningful data from bone trans-omics in order to advance its implementation in the field.

Circulating lipocalin-2 and features of metabolic syndrome in community-dwelling older women: A cross-sectional study.

Lipocalin-2 (LCN2) is released by several cell types including osteoblasts and adipocytes and has been suggested as a marker of renal dysfunction, metabolic syndrome (MetS) and type 2 diabetes (T2D). Whether LCN2 is linked to these diseases in older women remains unknown. This study investigated whether LCN2 is related to features of MetS and T2D in older women. This cross-sectional study included 705 non-diabetic women (mean age 75.1 ± 2.6 years) for MetS analysis and 76 women (mean age 75.4 ± 2.8 years) with T2D. Total circulating LCN2 levels were analysed using a two-step chemiluminescent microparticle monoclonal immunoassay. MetS was determined by a modified National Cholesterol Education Program Adult Treatment Panel III classification. Multivariable-adjusted logistic regression analysis was used to assess odds ratios between LCN2 quartiles and MetS. Women in the highest LCN2 quartile had approximately 3 times greater risk for MetS compared to women in the lowest quartile (OR 3.05; 95%CI 1.86-5.02). Women with T2D or MetS scores of ≥ 3 had higher LCN2 levels compared to women with a MetS score of 0 (p < 0.05). Higher LCN2 correlated with higher body mass index, fat mass, triglycerides and glycated haemoglobin and lower high-density lipoprotein cholesterol and estimated glomerular filtration rate (p < 0.05). Higher circulating levels of LCN2 are associated with worsened cardio-metabolic risk factors and increased odds of MetS and T2D in older women. Whether it can be used as a biomarker for identifying those at risk for MetS and T2D should be explored further.

Sugar transporter Slc37a2 regulates bone metabolism in mice via a tubular lysosomal network in osteoclasts.

Osteoclasts are giant bone-digesting cells that harbor specialized lysosome-related organelles termed secretory lysosomes (SLs). SLs store cathepsin K and serve as a membrane precursor to the ruffled border, the osteoclast's 'resorptive apparatus'. Yet, the molecular composition and spatiotemporal organization of SLs remains incompletely understood. Here, using organelle-resolution proteomics, we identify member a2 of the solute carrier 37 family (Slc37a2) as a SL sugar transporter. We demonstrate in mice that Slc37a2 localizes to the SL limiting membrane and that these organelles adopt a hitherto unnoticed but dynamic tubular network in living osteoclasts that is required for bone digestion. Accordingly, mice lacking Slc37a2 accrue high bone mass owing to uncoupled bone metabolism and disturbances in SL export of monosaccharide sugars, a prerequisite for SL delivery to the bone-lining osteoclast plasma membrane. Thus, Slc37a2 is a physiological component of the osteoclast's unique secretory organelle and a potential therapeutic target for metabolic bone diseases.

Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia.

Context: In the clinic it is important to differentiate primary hyperparathyroidism (PHPT) from the more benign, inherited disorder, familial hypocalciuric hypercalcemia (FHH). Since the conditions may sometimes overlap biochemically, identification of calcium-sensing receptor (CASR) gene variants causative of FHH (but not PHPT) is the most decisive diagnostic aid. When novel variants are identified, bioinformatics and functional assessment are required to establish pathogenicity. Objective: We identified 3 novel CASR transmembrane domain missense variants, Thr699Asn, Arg701Gly, and Thr808Pro, in 3 probands provisionally diagnosed with FHH and examined the variants using bioinformatics and functional analysis. Methods: Bioinformatics assessment utilized wANNOVAR software. For functional characterization, each variant was cloned into a mammalian expression vector; wild-type and variant receptors were transfected into HEK293 cells, and their expression and cellular localization were assessed by Western blotting and confocal immunofluorescence, respectively. Receptor activation in HEK293 cells was determined using an IP-One ELISA assay following stimulation with Ca++ ions. Results: Bioinformatics analysis of the variants was unable to definitively assign pathogenicity. Compared with wild-type receptor, all variants demonstrated impaired expression of mature receptor reaching the cell surface and diminished activation at physiologically relevant Ca++ concentrations. Conclusion: Three CASR missense variants identified in probands provisionally diagnosed with FHH result in receptor inactivation and are therefore likely causative of FHH. Inactivation may be due to inadequate processing/trafficking of mature receptor and/or conformational changes induced by the variants affecting receptor signaling. This study demonstrates the value of functional studies in assessing genetic variants identified in hypercalcemic patients.

Long-term exposure of mice to 890 ppm atmospheric CO2 alters growth trajectories and elicits hyperactive behaviours in young adulthood.

Atmospheric carbon dioxide (CO2 ) levels are currently at 418 parts per million (ppm), and by 2100 may exceed 900 ppm. The biological effects of lifetime exposure to CO2 at these levels is unknown. Previously we have shown that mouse lung function is altered by long-term exposure to 890 ppm CO2 . Here, we assess the broader systemic physiological responses to this exposure. Mice were exposed to either 460 or 890 ppm from preconception to 3 months of age, and assessed for effects on developmental, renal and osteological parameters. Locomotor, memory, learning and anxiety-like behaviours of the mice were also assessed. Exposure to 890 ppm CO2 increased birthweight, decreased female body weight after weaning, and, as young adults, resulted in reduced engagement in memory/learning tasks, and hyperactivity in both sexes in comparison to controls. There were no clear anxiety, learning or memory changes. Renal and osteological parameters were minimally affected. Overall, this study shows that exposure of mice to 890 ppm CO2 from preconception to young adulthood alters growth and some behaviours, with limited evidence of compensatory changes in acid-base balance. These findings highlight the potential for a direct effect of increased atmospheric CO2 on mammalian health outcomes. KEY POINTS: Long-term exposure to elevated levels of atmospheric CO2 is an uncontrolled experiment already underway. This is the first known study to assess non-respiratory physiological impacts of long-term (conception to young adulthood) exposure of mice to CO2 at levels that may arise in the atmosphere due to global emissions. Exposure to elevated CO2 , in comparison to control mice, altered growth patterns in early life and resulted in hyperactive behaviours in young adulthood. Renal and bone parameters, which are important to balance acid-base levels to compensate for increased CO2 exposure, remained relatively unaffected. This work adds to the body of evidence regarding the effects of carbon emissions on mammalian health and highlights a potential future burden of disease.

Perspective of the GEMSTONE Consortium on Current and Future Approaches to Functional Validation for Skeletal Genetic Disease Using Cellular, Molecular and Animal-Modeling Techniques.

The availability of large human datasets for genome-wide association studies (GWAS) and the advancement of sequencing technologies have boosted the identification of genetic variants in complex and rare diseases in the skeletal field. Yet, interpreting results from human association studies remains a challenge. To bridge the gap between genetic association and causality, a systematic functional investigation is necessary. Multiple unknowns exist for putative causal genes, including cellular localization of the molecular function. Intermediate traits ("endophenotypes"), e.g. molecular quantitative trait loci (molQTLs), are needed to identify mechanisms of underlying associations. Furthermore, index variants often reside in non-coding regions of the genome, therefore challenging for interpretation. Knowledge of non-coding variance (e.g. ncRNAs), repetitive sequences, and regulatory interactions between enhancers and their target genes is central for understanding causal genes in skeletal conditions. Animal models with deep skeletal phenotyping and cell culture models have already facilitated fine mapping of some association signals, elucidated gene mechanisms, and revealed disease-relevant biology. However, to accelerate research towards bridging the current gap between association and causality in skeletal diseases, alternative in vivo platforms need to be used and developed in parallel with the current -omics and traditional in vivo resources. Therefore, we argue that as a field we need to establish resource-sharing standards to collectively address complex research questions. These standards will promote data integration from various -omics technologies and functional dissection of human complex traits. In this mission statement, we review the current available resources and as a group propose a consensus to facilitate resource sharing using existing and future resources. Such coordination efforts will maximize the acquisition of knowledge from different approaches and thus reduce redundancy and duplication of resources. These measures will help to understand the pathogenesis of osteoporosis and other skeletal diseases towards defining new and more efficient therapeutic targets.

Osteoblast-derived EGFL6 couples angiogenesis to osteogenesis during bone repair.

Rationale: Angiogenesis and osteogenesis are highly coupled processes which are indispensable to bone repair. However, the underlying mechanism(s) remain elusive. To bridge the gap in understanding the coupling process is crucial to develop corresponding solutions to abnormal bone healing. Epidermal growth factor-like protein 6 (EGFL6) is an angiogenic factor specifically and distinctively up-regulated during osteoblast differentiation. In contrast with most currently known osteoblast-derived coupling factors, EGFL6 is highlighted with little or low expression in other cells and tissues. Methods: In this study, primary bone marrow mesenchymal stem cells (MSCs) and osteoblastic cell line (MC3T3-E1) were transduced with lentiviral silencing or overexpression constructs targeting EGFL6. Cells were induced by osteogenic medium, followed by the evaluation of mineralization as well as related gene and protein expression. Global and conditional knockout mice were established to examine the bone phenotype under physiological condition. Furthermore, bone defect models were created to investigate the outcome of bone repair in mice lacking EGFL6 expression. Results: We show that overexpression of EGFL6 markedly enhances osteogenic capacity in vitro by augmenting bone morphogenic protein (BMP)-Smad and MAPK signaling, whereas downregulation of EGFL6 diminishes osteoblastic mineralization. Interestingly, osteoblast differentiation was not affected by the exogenous addition of EGFL6 protein, thereby indicating that EGFL6 may regulate osteoblastic function in an intracrine manner. Mice with osteoblast-specific and global knockout of EGFL6 surprisingly exhibit a normal bone phenotype under physiological conditions. However, EGFL6-deficiency leads to compromised bone repair in a bone defect model which is characterized by decreased formation of type H vessels as well as osteoblast lineage cells. Conclusions: Together, these data demonstrate that EGFL6 serves as an essential regulator to couple osteogenesis to angiogenesis during bone repair.

The SQSTM1/p62 UBA domain regulates Ajuba localisation, degradation and NF-κB signalling function.

The LIM-domain containing protein Ajuba and the scaffold protein SQSTM1/p62 regulate signalling of NF-κB, a transcription factor involved in osteoclast differentiation and survival. The ubiquitin-associated domain of SQSTM1/p62 is frequently mutated in patients with Paget's disease of bone. Here, we report that Ajuba activates NF-κB activity in HEK293 cells, and that co-expression with SQSTM1/p62 inhibits this activation in an UBA domain-dependent manner. SQSTM1/p62 regulates proteins by targeting them to the ubiquitin-proteasome system or the autophagy-lysosome pathway. We show that Ajuba is degraded by autophagy, however co-expression with SQSTM1/p62 (wild type or UBA-deficient) protects Ajuba levels both in cells undergoing autophagy and those exposed to proteasomal stress. Additionally, in unstressed cells co-expression of SQSTM1/p62 reduces the amount of Ajuba present in the nucleus. SQSTM1/p62 with an intact ubiquitin-associated domain forms holding complexes with Ajuba that are not destined for degradation yet inhibit signalling. Thus, in situations with altered levels and localization of SQSTM1/p62 expression, such as osteoclasts in Paget's disease of bone and various cancers, SQSTM1/p62 may compartmentalize Ajuba and thereby impact its cellular functions and disease pathogenesis. In Paget's, ubiquitin-associated domain mutations may lead to increased or prolonged Ajuba-induced NF-κB signalling leading to increased osteoclastogenesis. In cancer, Ajuba expression promotes cell survival. The increased levels of SQSTM1/p62 observed in cancer may enhance Ajuba-mediated cancer cell survival.

Osteal macrophages support osteoclast-mediated resorption and contribute to bone pathology in a postmenopausal osteoporosis mouse model.

Osteal macrophages (osteomacs) support osteoblast function and promote bone anabolism, but their contribution to osteoporosis has not been explored. Although mouse ovariectomy (OVX) models have been repeatedly used, variation in strain, experimental design and assessment modalities have contributed to no single model being confirmed as comprehensively replicating the full gamut of osteoporosis pathological manifestations. We validated an OVX model in adult C3H/HeJ mice and demonstrated that it presents with human postmenopausal osteoporosis features with reduced bone volume in axial and appendicular bone and bone loss in both trabecular and cortical bone including increased cortical porosity. Bone loss was associated with increased osteoclasts on trabecular and endocortical bone and decreased osteoblasts on trabecular bone. Importantly, this OVX model was characterized by delayed fracture healing. Using this validated model, we demonstrated that osteomacs are increased post-OVX on both trabecular and endocortical bone. Dual F4/80 (pan-macrophage marker) and tartrate-resistant acid phosphatase (TRAP) staining revealed osteomacs frequently located near TRAP+ osteoclasts and contained TRAP+ intracellular vesicles. Using an in vivo inducible macrophage depletion model that does not simultaneously deplete osteoclasts, we observed that osteomac loss was associated with elevated extracellular TRAP in bone marrow interstitium and increased serum TRAP. Using in vitro high-resolution confocal imaging of mixed osteoclast-macrophage cultures on bone substrate, we observed macrophages juxtaposed to osteoclast basolateral functional secretory domains scavenging degraded bone byproducts. These data demonstrate a role for osteomacs in supporting osteoclastic bone resorption through phagocytosis and sequestration of resorption byproducts. Overall, our data expose a novel role for osteomacs in supporting osteoclast function and provide the first evidence of their involvement in osteoporosis pathogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).

The oncogene AAMDC links PI3K-AKT-mTOR signaling with metabolic reprograming in estrogen receptor-positive breast cancer.

Adipogenesis associated Mth938 domain containing (AAMDC) represents an uncharacterized oncogene amplified in aggressive estrogen receptor-positive breast cancers. We uncover that AAMDC regulates the expression of several metabolic enzymes involved in the one-carbon folate and methionine cycles, and lipid metabolism. We show that AAMDC controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC and modulating the transcriptional activity of AAMDC-dependent promoters. High AAMDC expression is associated with sensitization to dactolisib and everolimus, and these PI3K-mTOR inhibitors exhibit synergistic interactions with anti-estrogens in IntClust2 models. Ectopic AAMDC expression is sufficient to activate AKT signaling, resulting in estrogen-independent tumor growth. Thus, AAMDC-overexpressing tumors may be sensitive to PI3K-mTORC1 blockers in combination with anti-estrogens. Lastly, we provide evidence that AAMDC can interact with the RabGTPase-activating protein RabGAP1L, and that AAMDC, RabGAP1L, and Rab7a colocalize in endolysosomes. The discovery of the RabGAP1L-AAMDC assembly platform provides insights for the design of selective blockers to target malignancies having the AAMDC amplification.

Membrane Transport Proteins in Osteoclasts: The Ins and Outs.

During bone resorption, the osteoclast must sustain an extraordinarily low pH environment, withstand immense ionic pressures, and coordinate nutrient and waste exchange across its membrane to sustain its unique structural and functional polarity. To achieve this, osteoclasts are equipped with an elaborate set of membrane transport proteins (pumps, transporters and channels) that serve as molecular 'gatekeepers' to regulate the bilateral exchange of ions, amino acids, metabolites and macromolecules across the ruffled border and basolateral domains. Whereas the importance of the vacuolar-ATPase proton pump and chloride voltage-gated channel 7 in osteoclasts has long been established, comparatively little is known about the contributions of other membrane transport proteins, including those categorized as secondary active transporters. In this Special Issue review, we provide a contemporary update on the 'ins and outs' of membrane transport proteins implicated in osteoclast differentiation, function and bone homeostasis and discuss their therapeutic potential for the treatment of metabolic bone diseases.

Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption.

Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.

A missense mutation sheds light on a novel structure-function relationship of RANKL.

The tumor necrosis factor (TNF)-like core domain of receptor activator of nuclear factor-κB ligand (RANKL) is a functional domain critical for osteoclast differentiation. One of the missense mutations identified in patients with osteoclast-poor autosomal recessive osteopetrosis (ARO) is located in residue methionine 199 that is replaced with lysine (M199K) amid the TNF-like core domain. However, the structure-function relationship of this mutation is not clear. Sequence-based alignment revealed that the fragment containing human M199 is highly conserved and equivalent to M200 in rat. Using site-directed mutagenesis, we generated three recombinant RANKL mutants M200K/A/E (M200s) by replacing the methionine 200 with lysine (M200K), alanine (M200A), and glutamic acid (M200E), representative of distinct physical properties. TRAcP staining and bone pit assay showed that M200s failed to support osteoclast formation and bone resorption, accompanied by impaired osteoclast-related signal transduction. However, no antagonistic effect was found in M200s against wild-type rat RANKL. Analysis of the crystal structure of RANKL predicted that this methionine residue is located within the hydrophobic core of the protein, thus, likely to be crucial for protein folding and stability. Consistently, differential scanning fluorimetry analysis suggested that M200s were less stable. Western blot analysis analyses further revealed impaired RANKL trimerization by M200s. Furthermore, receptor-ligand binding assay displayed interrupted interaction of M200s to its intrinsic receptors. Collectively, our studies revealed the molecular basis of human M199-induced ARO and elucidated the indispensable role of rodent residue M200 (equivalent to human M199) for the RANKL function.

The molecular structure and function of sorting nexin 10 in skeletal disorders, cancers, and other pathological conditions.

SNX10 is a member of the phox homology domain-containing family of phosphoinositide-binding proteins. Intracellularly, SNX10 localizes to endosomes where it mediates intracellular trafficking, endosome organization, and protein localization to the centrosome and cilium. It is highly expressed in bone and the gut where it participates in bone mineral and calcium homeostasis through the regulation of osteoclastic bone resorption and gastric acid secretion, respectively. Not surprisingly, patients harboring mutations in SNX10 mutation manifest a phenotype of autosomal recessive osteopetrosis or malignant infantile osteopetrosis, which is clinically characterized by dense bones with increased cortical bone into the medullary space with bone marrow occlusion or depletion, bone marrow failure, and anemia. Accordingly, SNX10 mutant osteoclasts exhibit impaired bone resorptive capacity. Beyond the skeleton, there is emerging evidence implicating SNX10 in cancer development, metabolic disorders, inflammation, and chaperone-mediated autophagy. Understanding the structural basis through which SNX10 exerts its diverse biological functions in both cell and tissue-specific manners may therefore inform new therapeutic opportunities toward the treatment and management of SNX10-related diseases.