Search

Context X-linked hypophosphatemia (XLH) is a rare genetic disease caused by a primary excess of fibroblast growth factor 23 (FGF23). FGF23 has been associated with inflammation and impaired osteoclastogenesis, but these pathways have not been investigated in XLH. Objective This work aimed to evaluate whether XLH patients display peculiar inflammatory profile and increased osteoclastic activity. Methods We performed a prospective, multicenter, cross-sectional study analyzing transcript expression of 8 inflammatory markers (Il6, Il8, Il1β, CXCL1, CCL2, CXCR3, Il1R, Il6R) by real-time quantitative polymerase chain reaction on peripheral blood mononuclear cells (PBMCs) purified from total blood samples extracted from patients and healthy control individuals. The effect of native/active vitamin D on osteoclast formation was also assessed in vitro from XLH patients’ PBMCs. Results In total, 28 XLH patients (17 children, among them 6 undergoing standard of care [SOC] and 11 burosumab therapy) and 19 controls were enrolled. Expression of most inflammatory markers was significantly increased in PBMCs from XLH patients compared to controls. No differences were observed between the burosumab and SOC subgroups. Osteoclast formation was significantly impaired in XLH patients. XLH mature osteoclasts displayed higher levels of inflammatory markers, being however lower in cells derived from the burosumab subgroup (as opposed to SOC). Conclusion We describe for the first time a peculiar inflammatory profile in XLH. Since XLH patients have a propensity to develop arterial hypertension, obesity, and enthesopathies, and because inflammation can worsen these clinical outcomes, we hypothesize that inflammation may play a critical role in these extraskeletal complications of XLH.

International audience; Purpose of review: To describe the methods that can be used to obtain functional and mature osteoclasts from peripheral blood mononuclear cells (PBMCs) and report the data obtained with this model in two peculiar diseases, namely pediatric chronic kidney disease-associated mineral and bone disorders (CKD-MBD) and nephropathic cystinosis. To discuss future research possibilities in the field.Recent findings: Bone tissue undergoes continuous remodeling throughout life to maintain bone architecture; it involves two processes: bone formation and bone resorption with the coordinated activity of osteoblasts, osteoclasts, and osteocytes. Animal models fail to fully explain human bone pathophysiology during chronic kidney disease, mainly due to interspecies differences. The development of in vitro models has permitted to mimic human bone-related diseases as an alternative to in vivo models. Since 1997, osteoclasts have been generated in cell cultures, notably when culturing PBMCs with specific growth factors and cytokines (i.e., M-CSF and RANK-L), without the need for osteoblasts or stromal cells. These models may improve the global understanding of bone pathophysiology. They can be been used not only to evaluate the direct effects of cytokines, hormones, cells, or drugs on bone remodeling during CKD-MBD, but also in peculiar genetic renal diseases inducing specific bone impairment.

International audience; Active vitamin D analogs and calcimimetics are the main therapies used for treating secondary hyperparathyroidism (SHPT) in patients with chronic kidney disease (CKD). Peripheral blood mononuclear cells of 19 pediatric patients with CKD1-5D and 6 healthy donors (HD) were differentiated into mature osteoclasts with receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The effects of single or combined treatment with active vitamin D (1.25-D) and/or calcimimetic KP2326 were evaluated on osteoclastic differentiation and osteoclastic-mediated bone resorption. Although 1.25-D inhibited osteoclastic differentiation, a significant resistance to 1.25-D was observed when glomerular filtration rate decreased. A significant albeit less important inhibitory effect of KP2326 on osteoclastic differentiation was also found both in cells derived from HD and CKD patients, through a putative activation of the Erk pathway. This inhibitory effect was not modified by CKD stage. Combinatorial treatment with 1.25-D and KP2326 did not result in synergistic effects. Last, KP2326 significantly inhibited osteoclast-mediated bone resorption. Both 1.25-D and KP2326 inhibit osteoclastic differentiation, however, to a different extent. There is a progressive resistance to 1.25-D in advanced CKD that is not found with KP2326. KP2326 also inhibits bone resorption. Given that 1.25-D has no effect on osteoclastic resorption activity and that calcimimetics also have direct anabolic effects on osteoblasts, there is an experimental rationale that could favor the use of decreased doses of 1.25-D with low doses of calcimimetics in SHPT in dialysis to improve the underlying osteodystrophy. However, this last point deserves confirmatory clinical studies. © 2020 American Society for Bone and Mineral Research.

International audience; Lysophosphatidic acid (LPA) is a natural bioactive phospholipid with pleiotropic activities affecting multiple tissues, including bone. LPA exerts its biological functions by binding to G-protein coupled LPA receptors (LPA1-6) to stimulate cell migration, proliferation, and survival. It is largely produced by autotaxin (ATX), a secreted enzyme with lysophospholipase D activity that converts lysophosphatidylcholine (LPC) into active LPA. Beyond its enzymatic activity, ATX serves as a docking molecule facilitating the efficient delivery of LPA to its specific cell surface receptors. Thus, LPA effects are the result of local production by ATX in a given tissue or cell type. As a consequence, the ATX/LPA axis should be considered as an entity to better understand their roles in physiology and pathophysiology and to propose novel therapeutic strategies. Herein, we provide not only an extensive overview of the relevance of the ATX/LPA axis in bone cell commitment and differentiation, skeletal development, and bone disorders, but also discuss new working hypotheses emerging from the interplay of ATX/LPA with well-established signaling pathways regulating bone mass.

International audience; Bone complications of cystinosis have been recently described. The main objectives of this paper were to determine in vitro the impact of CTNS mutations and cysteamine therapy on human osteoclasts and to carry out a genotype-phenotype analysis related to osteoclastic differentiation. Human osteoclasts were differentiated from peripheral blood mononuclear cells (PBMCs) and were treated with increasing doses of cysteamine (0, 50, 200 µM) and then assessed for osteoclastic differentiation. Results are presented as median (min-max). A total of 17 patients (mainly pediatric) were included, at a median age of 14 (2–61) years, and a eGFR of 64 (23–149) mL/min/1.73 m2. Most patients (71%) were under conservative kidney management (CKM). The others were kidney transplant recipients. Three functional groups were distinguished for CTNS mutations: cystinosin variant with residual cystin efflux activity (RA, residual activity), inactive cystinosin variant (IP, inactive protein), and absent protein (AP). PBMCs from patients with residual cystinosin activity generate significantly less osteoclasts than those obtained from patients of the other groups. In all groups, cysteamine exerts an inhibitory effect on osteoclastic differentiation at high doses. This study highlights a link between genotype and osteoclastic differentiation, as well as a significant impact of cysteamine therapy on this process in humans.

International audience; Rheumatoid arthritis is characterized by synovial inflammation and irreversible bone erosions, both highlighting the immense reciprocal relationship between the immune and bone systems, designed osteoimmunology two decades ago. Osteoclast-mediated resorption at the interface between synovium and bone is responsible for the articular bone erosions. The main triggers of this local bone resorption are autoantibodies directed against citrullinated proteins, as well as pro-inflammatory cytokines and the receptor activator of nuclear factor-κB ligand, that regulate both the formation and activity of the osteoclast, as well as immune cell functions. In addition, local bone loss is due to the suppression of osteoblast-mediated bone formation and repair by inflammatory cytokines. Similarly, inflammation affects systemic bone remodeling in rheumatoid arthritis with the net increase in bone resorption, leading to systemic osteoporosis. This review summarizes the substantial progress that has been made in understanding the pathophysiology of systemic and local bone loss in rheumatoid arthritis.

International audience; Lysphosphatidic acid (LPA) is a major natural bioactive lipid mediator whose biological functions affect multiple organs. These include bone as demonstrated by global Lpar1-knockout mice (Lpar1-/-) which present a bone growth defect. LPA acts on all bone cells including osteoblasts, that are responsible for bone formation, and osteoclasts, which are specialized cells that resorb bone. LPA appears as a potential new coupling molecule during bone remodeling. LPA1 is the most ubiquitous LPA receptor among the six LPA receptor family members (LPA1-6). To better understand the specific role of LPA via its receptor LPA1 in osteoblastic cell lineage we generated osteoblast-specific Lpar1 knockout mice (Lpar1-∆Ob) by crossing Lpar1flox/flox and Osx:Cre+ mouse lines. Lpar1-∆Ob mice do not recapitulate the bone defects of Lpar1-/- mice but revealed reduced bone mineralization and decreased cortical thickness, as well as increased bone porosity associated with an augmentation in the lacunae areas of osteocyte and their apoptotic yield. In vitro, primary Lpar1-∆Ob and immortalized cl1-Ob-Lpar1-/- osteoblasts revealed a remarkable premature expression of alkaline phosphatase, reduced cell proliferation associated with decreased YAP-P nuclear accumulation, and reduced mineralization activity. Osteocyte specification is markedly impaired as demonstrated by reduced expression of early (E11) and late (DMP1, DKK1, SOST) osteocyte markers ex vivo in enriched osteocytic fractions of Lpar1-∆Ob mouse bone explants. In addition, E11 expression and dendrite formation induced by FGF2 are markedly impaired in both primary Lpar1-∆Ob and immortalized cl1-Ob-Lpar1-/- osteoblasts. Taken together these results suggest a new role for LPA in bone mass control via bone mineralization and osteocyte function.

Background and Aims Active vitamin D analogs and calcimimetics are cornerstones for managing secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD). Their direct effects on bone cells remain to be determined. Method Peripheral blood mononuclear cells (PBMCs) of 19 pediatric CKD patients and 6 healthy donors (HD) were differentiated into osteoclasts in presence of M-CSF and RANKL. Effect of combined or single treatment with active vitamin D (1.25-D) and/or the calcimimetic KP2326 were evaluated onto osteoclast differentiation and osteoclast mediated bone resorption. Results 1.25-D inhibited osteoclastic differentiation, a significant resistance to 1.25-D was observed when CKD worsens. A significant albeit less important inhibitory effect of KP2326 on osteoclastic differentiation was also found both in cells derived from HD and CKD patients, through an activation of the Erk pathway. This inhibitory effect was not modified by CKD stage. Combinatorial treatment with 1.25-D and KP2326 did not result in synergistic effects. Last, KP2326 significantly inhibited human osteoclast-mediated bone resorption. Conclusion Both 1.25-D and KP2326 inhibit osteoclastic differentiation, however to a different extent. Whilst 1.25-D has no significant effect on bone resorption, KP2326 inhibits bone resorption. Recent data showed that calcimimetics also have a direct anabolic effect on bone, through the stimulation of osteoblastic differentiation and mineralization in human mesenchymal stem cells in vitro. All these results provide a strong rationale for a global positive effect of calcimimetics on bone remodeling. Calcimimetics also significantly decrease FGF23 levels. In the setting of global systematic deleterious effects of high FGF23 levels in CKD, and keeping in mind that active vitamin D analogs stimulate FGF 23 production, all these data could favor the use of decreased doses of 1.25-D with low-doses of calcimimetics in SHPT in dialysis, the combination of these two therapies already being proposed in the 2017 K-DIGO guidelines.

International audience; Patients with chronic kidney disease (CKD) display significant mineral and bone disorders (CKD-MBD) that induce significant cardiovascular, growth and bone comorbidities. Nephropathic cystinosis is an inherited metabolic disorder caused by the lysosomal accumulation of cystine due to mutations in the CTNS gene encoding cystinosin, and leads to end-stage renal disease within the second decade. The cornerstone of management relies on cysteamine therapy to decrease lysosomal cystine accumulation in target organs. However, despite cysteamine therapy, patients display severe bone symptoms, and the concept of "cystinosis metabolic bone disease" is currently emerging. Even though its exact pathophysiology remains unclear, at least five distinct but complementary entities can explain bone impairment in addition to CKD-MBD: long-term consequences of renal Fanconi syndrome, malnutrition and copper deficiency, hormonal disturbances, myopathy, and intrinsic/iatrogenic bone defects. Direct effects of both CTNS mutation and cysteamine on osteoblasts and osteoclasts are described. Thus, the main objective of this manuscript is not only to provide a clinical update on bone disease in cystinosis, but also to summarize the current experimental evidence demonstrating a functional impairment of bone cells in this disease and to discuss new working hypotheses that deserve future research in the field.

Bone metastasis affects >70% of patients with advanced breast cancer. However, the molecular mechanisms underlying this process remain unclear. On the basis of analysis of clinical datasets, and in vitro and in vivo experiments, we report that the ZNF217 oncogene is a crucial mediator and indicator of bone metastasis. Patients with high ZNF217 mRNA expression levels in primary breast tumours had a higher risk of developing bone metastases. MDA-MB-231 breast cancer cells stably transfected with ZNF217 (MDA-MB-231-ZNF217) showed the dysregulated expression of a set of genes with bone-homing and metastasis characteristics, which overlapped with two previously described 'osteolytic bone metastasis' gene signatures, while also highlighting the bone morphogenetic protein (BMP) pathway. The latter was activated in MDA-MB-231-ZNF217 cells, and its silencing by inhibitors (Noggin and LDN-193189) was sufficient to rescue ZNF217-dependent cell migration, invasion or chemotaxis towards the bone environment. Finally, by using non-invasive multimodal in vivo imaging, we found that ZNF217 increases the metastatic growth rate in the bone and accelerates the development of severe osteolytic lesions. Altogether, the findings of this study highlight ZNF217 as an indicator of the emergence of breast cancer bone metastasis; future therapies targeting ZNF217 and/or the BMP signalling pathway may be beneficial by preventing the development of bone metastases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

International audience; OBJECTIVE : The severity of rheumatoid arthritis (RA) correlates directly with bone erosions arising from osteoclast (OC) hyperactivity. Despite the fact that inflammation may be controlled in patients with RA, those in a state of sustained clinical remission or low disease activity may continue to accrue erosions, which supports the need for treatments that would be suitable for long-lasting inhibition of OC activity without altering the physiologic function of OCs in bone remodeling. Autotaxin (ATX) contributes to inflammation, but its role in bone erosion is unknown. METHODS : ATX was targeted by inhibitory treatment with pharmacologic drugs and also by conditional inactivation of the ATX gene Ennp2 in murine OCs (ΔATXC tsk ). Arthritic and erosive diseases were studied in human tumor necrosis factor-transgenic (hTNF+/- ) mice and mice with K/BxN serum transfer-induced arthritis. Systemic bone loss was also analyzed in mice with lipopolysaccharide (LPS)-induced inflammation and estrogen deprivation. Joint inflammation and bone erosion were assessed by histology and micro-computed tomography. The role of ATX in RA was also examined in OC differentiation and activity assays. RESULTS : OCs present at sites of inflammation overexpressed ATX. Pharmacologic inhibition of ATX in hTNF+/- mice, as compared to vehicle-treated controls, significantly mitigated focal bone erosion (36% decrease; P < 0.05) and systemic bone loss (43% decrease; P < 0.05), without affecting synovial inflammation. OC-derived ATX was revealed to be instrumental in OC bone resorptive activity and was up-regulated by the inflammation elicited in the presence of TNF or LPS. Specific loss of ATX in OCs from mice subjected to ovariectomy significantly protected against the systemic bone loss and erosion that had been induced with LPS and K/BxN serum treatments (30% reversal of systemic bone loss [P < 0.01]; 55% reversal of erosion [P < 0.001]), without conferring bone-protective properties. CONCLUSION : Our results identify ATX as a novel OC factor that specifically controls inflammation-induced bone erosions and systemic bone loss. Therefore, ATX inhibition offers a novel therapeutic approach for potentially preventing bone erosion in patients with RA.

The two SIBLING (Small Integrin Binding Ligand N-linked Glycoproteins), bone sialoprotein (BSP) and osteopontin (OPN) are expressed in osteoblasts and osteoclasts. In mature BSP knockout (KO, -/-) mice, both bone formation and resorption as well as mineralization are impaired. OPN-/- mice display impaired resorption, and OPN is described as an inhibitor of mineralization. However, OPN is overexpressed in BSP-/- mice, complicating the understanding of their phenotype. We have generated and characterized mice with a double KO (DKO) of OPN and BSP, to try and unravel their respective contributions. Despite the absence of OPN, DKO bones are still hypomineralized. The SIBLING, matrix extracellular phosphoglycoprotein with ASARM motif (MEPE) is highly overexpressed in both BSP-/- and DKO and may impair mineralization through liberation of its ASARM (Acidic Serine-Aspartate Rich MEPE associated) peptides. DKO mice also display evidence of active formation of trabecular, secondary bone as well as primary bone in the marrow-ablation repair model. A higher number of osteoclasts form in DKO marrow cultures, with higher resorption activity, and DKO long bones display a localized and conspicuous cortical macroporosity. High bone formation and resorption parameters, and high cortical porosity in DKO mice suggest an active bone modeling/remodeling, in the absence of two key regulators of bone cell performance. This first double KO of SIBLING proteins thus results in a singular, non-trivial phenotype leading to reconsider the interpretation of each single KO, concerning in particular matrix mineralization and the regulation of bone cell activity.

Osteoclasts are the main cells responsible for the resorption of mineralized extracellular matrices. They are the major targets for anti-resorptive therapies to manage osteoporosis, a major public health problem. Osteoclasts are giant multinucleated cells that can organize their a unique adhesion structure based on a belt of podosomes, which is the keystone of the bone resorption apparatus. We combined differential transcriptomics and siRNA screening approaches to get a broader view of cytoskeletal regulators that participate in the control of osteoclast cytoskeleton and identify novel regulators of bone resorption by osteoclasts. We identified 20 new candidate regulators of osteoclasts cytoskeleton including Fkbp15, Spire1, Tacc2 and RalA, for which we confirmed they are necessary for proper organization of the podosome belt. We also showed that Anillin, well known for its role during cytokinesis, is essential in osteoclasts for correct podosome patterning and efficient bone resorption. In particular, Anillin controls the levels of the GTPase RhoA, a known regulator of osteoclast cytoskeleton and resorption activity. Finally, we set up and validated an automated imaging strategy based on open-source software for automatic and objective measurement of actin cytoskeleton organization in osteoclasts. We provide these pipelines that are useful to automatically assess the effect of collections of siRNAs or chemical compounds on osteoclast cytoskeleton or differentiation.

Autotaxin (ATX) promotes cancer cell metastasis through the production of lysophosphatidic acid (LPA). ATX binds to αvβ3 integrins controlling metastasis of breast cancer cells. We screened a series of cancer cell lines derived from diverse human and mouse solid tumors for the capacity of binding to ATX and found only a modest correlation with their level of αvβ3 integrin expression. These results strongly suggested the existence of another cell surface ATX-interacting factor. Indeed, ATXα has been shown to bind heparan-sulfate chains because of its unique polybasic insertion sequence, although the biological significance is unknown. We demonstrated here, that among all cell surface heparan-sulfate proteoglycans, syndecan-4 (SDC4) was essential for cancer cell interaction with ATXβ but was restrained by heparan-sulfate chains. In addition, exogenous ATXβ-induced MG63 osteosarcoma cell proliferation required physical interaction of ATXβ with the cell surface via an SDC4-dependent mechanism. In a preclininal mouse model, targeting SDC4 on 4T1 mouse breast cancer cells inhibited early bone metastasis formation. Furthermore, SDC4-prometastatic activity was totally abolished in absence of ATX expression. In conclusion our results determined that ATX and SDC4 are engaged in a reciprocal collaboration for cancer cell metastasis providing the rational for the development of novel anti-metastasis therapies.

Background Bone impairment is a poorly described complication of nephropathic cystinosis (NC). The objectives of this study were to evaluate in vitro effects of cystinosin (CTNS) mutations on bone resorption and of cysteamine treatment on bone cells [namely human osteoclasts (OCs) and murine osteoblasts]. Methods Human OCs were differentiated from peripheral blood mononuclear cells (PBMCs) of patients and healthy donors (HDs). Cells were treated with increasing doses of cysteamine in PBMCs or on mature OCs to evaluate its impact on differentiation and resorption, respectively. Similarly, cysteamine-treated osteoblasts derived from murine mesenchymal stem cells were assessed for differentiation and activity with toxicity and proliferation assays. Results CTNS was expressed in human OCs derived from HDs; its expression was regulated during monocyte colony-stimulating factor- and receptor activator of nuclear factor-κB-dependent osteoclastogenesis and required for efficient bone resorption. Cysteamine had no impact on osteoclastogenesis but inhibited in vitro HD osteoclastic resorption; however, NC OC-mediated bone resorption was impaired only at high doses. Only low concentrations of cysteamine (50 μM) stimulated osteoblastic differentiation and maturation, while this effect was no longer observed at higher concentrations (200 µM). Conclusion CTNS is required for proper osteoclastic activity. In vitro low doses of cysteamine have beneficial antiresorptive effects on healthy human-derived OCs and may partly correct the CTNS-induced osteoclastic dysfunction in patients with NC. Moreover, in vitro low doses of cysteamine also stimulate osteoblastic differentiation and mineralization, with an inhibitory effect at higher doses, likely explaining, at least partly, the bone toxicity observed in patients receiving high doses of cysteamine.

Introduction Pierson syndrome is caused by a mutation of LAMB2, encoding for laminin β2. Clinical phenotype is variable but usually associates congenital nephrotic syndrome (CNS) and ocular abnormalities. Neuromuscular impairment has also been described. Methods We report on a 15-year old girl, suffering from Pierson Syndrome, who developed severe bone deformations during puberty. This patient initially displayed CNS and microcoria, leading to the clinical diagnosis of Pierson syndrome. Genetic analysis revealed a truncating mutation and a splice site mutation of LAMB2. The patient received a renal transplantation (R-Tx) at the age of 3. After R-Tx, renal evolution was simple, the patient receiving low-dose corticosteroids, tacrolimus and mycophenolate mofetil. At the age of 12, bone deformations progressively appeared. At the time of bone impairment, renal function was subnormal (glomerular filtration rate using iohexol clearance 50 mL/min per 1.73 m2), and parameters of calcium/phosphate metabolism were normal (calcium 2.45 mmol/L, phosphorus 1.30 mmol/L, PTH 81 ng/L, ALP 334 U/L, 25OH-D 73 nmol/L). Radiographs showed major deformations such as scoliosis, genu varum and diffuse epiphyseal abnormalities. A high resolution scanner (HR-pQCT) was performed, demonstrating a bone of “normal low” quantity and quality; major radial and cubital deformations were observed. Stainings of laminin β2 were performed on bone and renal samples from the patient and healthy controls: as expected, laminin β2 was expressed in the control kidney but not in the patient's renal tissue, and a similar pattern was observed in bone. Conclusion This is the first case of skeletal impairment ever described in Pierson syndrome. Integrin α3β1, receptor for laminin β2, are found in podocytes and osteoblasts, and the observation of both the presence of laminin β2 staining in healthy bone and its absence in the patient's bone raises the question of a potential role of laminin β2 in bone physiology.

Vitamin D and FGF23 play a major role in calcium/phosphate balance. Vitamin D may control bone resorption but the potential role of FGF23 has never been evaluated. The objective of this study was therefore to compare the effects of vitamin D and FGF23 on osteoclast differentiation and activity in human monocyte-derived osteoclasts. Human monocytes, purified from blood of healthy donors, were incubated with M-CSF and RANKL to obtain mature multinucleated osteoclasts (MNC). Experiments were carried out to assess the effects of FGF23 as compared to native vitamin D (25-D) and active vitamin D (1,25-D) on osteoclast differentiation and on bone-resorbing osteoclast activity. Additional experiments with the pan fibroblast growth factor receptor inhibitor (FGFR-i) were performed. Phosphorylation Akt and Erk pathways were analyzed by Western blot analyses. Both 1,25-D and FGF23, to a lesser extent, significantly inhibited osteoclastogenesis at early stages; when adding FGFR-i, osteoclast formation was restored. Biochemical experiments showed an activation of the Akt and Erk pathways under FGF23 treatment. In contrast, in terms of activity, 1,25-D had no effect on resorption, whereas FGF23 slightly but significantly increased bone resorption; 25-D had no effects on either differentiation or on activity. These data show that 1,25-D inhibits osteoclastogenesis without regulating osteoclast-mediated bone resorption activity; FGF23 has biphasic effects on osteoclast physiology, inhibiting osteoclast formation while stimulating slightly osteoclast activity. These results may be of importance and taken into account in chronic kidney disease when therapies modulating FGF23 are available.

Two-step transcriptomic profiling of bone-resorbing OCs versus nonresorbing MGCs generated a list of 115 genes potentially involved in bone resorption. Of these, RhoE was investigated. Its role in podosome dynamics is central for OC migration, SZ formation, and, ultimately, bone resorption., The function of osteoclasts (OCs), multinucleated giant cells (MGCs) of the monocytic lineage, is bone resorption. To resorb bone, OCs form podosomes. These are actin-rich adhesive structures that pattern into rings that drive OC migration and into “sealing-zones” (SZs) that confine the resorption lacuna. Although changes in actin dynamics during podosome patterning have been documented, the mechanisms that regulate these changes are largely unknown. From human monocytic precursors, we differentiated MGCs that express OC degradation enzymes but are unable to resorb the mineral matrix. We demonstrated that, despite exhibiting bona fide podosomes, these cells presented dysfunctional SZs. We then performed two-step differential transcriptomic profiling of bone-resorbing OCs versus nonresorbing MGCs to generate a list of genes implicated in bone resorption. From this list of candidate genes, we investigated the role of Rho/Rnd3. Using primary RhoE-deficient OCs, we demonstrated that RhoE is indispensable for OC migration and bone resorption by maintaining fast actin turnover in podosomes. We further showed that RhoE activates podosome component cofilin by inhibiting its Rock-mediated phosphorylation. We conclude that the RhoE-Rock-cofilin pathway, by promoting podosome dynamics and patterning, is central for OC migration, SZ formation, and, ultimately, bone resorption.

Bone metastasis affects70% of patients with advanced breast cancer. However, the molecular mechanisms underlying this process remain unclear. On the basis of analysis of clinical datasets, and in vitro and in vivo experiments, we report that the ZNF217 oncogene is a crucial mediator and indicator of bone metastasis. Patients with high ZNF217 mRNA expression levels in primary breast tumours had a higher risk of developing bone metastases. MDA-MB-231 breast cancer cells stably transfected with ZNF217 (MDA-MB-231-ZNF217) showed the dysregulated expression of a set of genes with bone-homing and metastasis characteristics, which overlapped with two previously described 'osteolytic bone metastasis' gene signatures, while also highlighting the bone morphogenetic protein (BMP) pathway. The latter was activated in MDA-MB-231-ZNF217 cells, and its silencing by inhibitors (Noggin and LDN-193189) was sufficient to rescue ZNF217-dependent cell migration, invasion or chemotaxis towards the bone environment. Finally, by using non-invasive multimodal in vivo imaging, we found that ZNF217 increases the metastatic growth rate in the bone and accelerates the development of severe osteolytic lesions. Altogether, the findings of this study highlight ZNF217 as an indicator of the emergence of breast cancer bone metastasis; future therapies targeting ZNF217 and/or the BMP signalling pathway may be beneficial by preventing the development of bone metastases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

Microbiota and infant development Malnutrition in children is a persistent challenge that is not always remedied by improvements in nutrition. This is because a characteristic community of gut microbes seems to mediate some of the pathology. Human gut microbes can be transplanted effectively into germ-free mice to recapitulate their associated phenotypes. Using this model, Blanton et al. found that the microbiota of healthy children relieved the harmful effects on growth caused by the microbiota of malnourished children. In infant mammals, chronic undernutrition results in growth hormone resistance and stunting. In mice, Schwarzer et al. showed that strains of Lactobacillus plantarum in the gut microbiota sustained growth hormone activity via signaling pathways in the liver, thus overcoming growth hormone resistance. Together these studies reveal that specific beneficial microbes could potentially be exploited to resolve undernutrition syndromes. Science , this issue p. 10.1126/science.aad3311 , p. 854

11 pages, 8 figures.-- PMID: 19197242 [PubMed].-- Supporting information available (Suppl. figures S1-S7 + Suppl. table I)., Bone undergoes continuous remodelling throughout adult life, and the equilibrium between bone formation by osteoblasts and bone resorption by osteoclasts defines the final bone mass. Here we show that Snail1 regulates this balance by controlling osteoblast differentiation. Snail1 is necessary for the early steps of osteoblast development, and it must be downregulated for their final differentiation. At the molecular level, Snail1 controls bone mass by repressing the transcription of both the osteoblast differentiation factor Runx2 and the vitamin D receptor (VDR) genes in osteoblasts. Sustained activation of Snail1 in transgenic mice provokes deficient osteoblast differentiation, which, together with the loss of vitamin D signalling in the bone, also impairs osteoclastogenesis. Indeed, the mineralisation of the bone matrix is severely affected, leading to hypocalcemia-independent osteomalacia. Our data show that the impact of Snail1 activity on the osteoblast population regulates the course of bone cells differentiation and ensures normal bone remodelling., This work has been supported by the Spanish Ministry of Education and Science (Grants BFU2005-05772, BFU2008-01042, NAN2004-09230-C04-04 and CONSOLIDER-INGENIO 2010 CSD2007-00017) and from the Generalitat Valenciana (Prometeo 2008/049) to MAN. RD is recipient of an INSERM grant.

The semaphorin-signaling transducer collapsin response mediator protein 2 (CRMP2) has been identified in the nervous system where it mediates Sema3A-induced growth cone navigation. In the present study, we provide first evidence that CRMP2 is present in the immune system and plays a critical role in T lymphocyte function. CRMP2 redistribution at the uropod in polarized T cells, a structural support of lymphocyte motility, suggests that it may regulate T cell migration. This was evidenced in primary T cells by small-interfering RNA-mediated CRMP2 gene silencing and blocking Ab, as well as CRMP2 overexpression in Jurkat T cells tested in a chemokine- and semaphorin-mediated transmigration assay. Expression analysis in PBMC from healthy donors showed that CRMP2 is enhanced in cell subsets bearing the activation markers CD69+ and HLA-DR+. Heightened expression in T lymphocytes of patients suffering from neuroinflammatory disease with enhanced T cell-transmigrating activity points to a role for CRMP2 in pathogenesis. The elucidation of the signals and mechanisms that control this pathway will lead to a better understanding of T cell trafficking in physiological and pathological situations.

Background information. Sema-7A is a glycosylphosphatidylinositol-anchored semaphorin that was first identified in the immune system. It is a member of a large family of proteins involved in axon guidance signalling. Sema-7A is expressed in the myeloid and the lymphoid lineage and seems to be involved in cytokine expression and chemotaxy through its receptor Plexin C1. However, it can promote axon outgrowth, acting through a β1 subunit-containing integrin receptor. Results and conclusions. In the present study, we have investigated its regulation and function in bone cells. Semiquantitative reverse transcriptase—PCR demonstrated that Sema-7A mRNA is present during all stages of osteoblast differentiation and maturation in mouse calvaria cells and in MC3T3 cell line in vitro. Its expression is also regulated during primary osteoclast differentiation in vitro. We report that Sema-7A is capable of increasing the migration of MC3T3 cells and that this process is mediated by the mitogen-activated protein kinase pathway in osteoblasts, probably through the integrin subunit β1. Moreover, the addition of recombinant soluble Sema-7A to the culture enhances osteoclast fusion. These findings indicate for the first time the possible involvement of Sema-7A in bone cell differentiation.

Semaphorin 7A (SEMA 7A) is a membrane-anchored member of the semaphorin family of guidance proteins, previously identified in the immune system. Expressed in central and peripheral nervous system during embryonic and post-natal stages, it can mediate neuronal functions by promoting axonal growth. We show here that SEMA 7A is expressed in human odontoblasts in vivo and in vitro and that its expression is correlated with the establishment of dentin-pulp complex terminal innervation . Co-cultures of trigeminal ganglion (TG) with COS cells overexpressing SEMA 7A demonstrate that SEMA 7A can promote the growth of trigeminal nerve fibers. Finally, by RT-PCR and immunochemistry, we show that beta1-integrin, a SEMA 7A putative receptor, is expressed in pulpal nerve fibers but we failed to detect a co-localization between nerves and odontoblasts through these molecules. On the basis of these data, we suggest that SEMA 7A might be a molecule involved in the terminal innervation of the dentin-pulp complex.

The function of osteoclasts is to digest the calcified bone matrix. Osteoclasts, together with myotubes, are among the rare examples of multinucleated cells found in higher vertebrates, resulting from the fusion of mononucleated progenitors belonging to the monocyte/macrophage lineage. So far, no information is available about function and transcriptional activity of multiple nuclei in osteoclasts. We have used a run-on technique to visualize RNA synthesis in individual nucleus. We provide the first evidence that nuclei of resorbing osteoclasts, isolated from chick embryo long bones, or differentiated in vitro from murine spleen cells in presence of RANKL and macrophage-colony stimulating factor, are all transcriptionally active. Nevertheless, if transcriptional activity is the same for all the nuclei within a cell, its level varies between osteoclasts: osteoclasts with highly active nuclei are always associated with resorption pits. We found that global transcription activity of resorbing osteoclasts seeded on calcified matrix is down-regulated after 5-h treatment with calcitonin, which transiently blocks resorption. This effect is reversible because calcitonin removal led to nuclear transcription activation. These results indicate a strong correlation between transcription and resorption. Finally, our data indicate that the resorption pit surface is linearly related to the nuclei number per osteoclast, strongly suggesting that functional advantage of osteoclast multinucleation is to improve resorption efficiency.

International audience; Lysophosphatidic acid (LPA) is a natural bioactive lipid that acts through six different G protein-coupled receptors (LPA1–6) with pleiotropic activities on multiple cell types. We have previously demonstrated that LPA is necessary for successful in vitro osteoclastogenesis of bone marrow cells. Bone cells controlling bone remodeling (i.e. osteoblasts, osteoclasts, and osteocytes) express LPA1, but delineating the role of this receptor in bone remodeling is still pending. Despite Lpar1−/− mice displaying a low bone mass phenotype, we demonstrated that bone marrow cell-induced osteoclastogenesis was reduced in Lpar1−/− mice but not in Lpar2−/− and Lpar3−/− animals. Expression of LPA1 was up-regulated during osteoclastogenesis, and LPA1 antagonists (Ki16425, Debio0719, and VPC12249) inhibited osteoclast differentiation. Blocking LPA1 activity with Ki16425 inhibited expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and dendritic cell-specific transmembrane protein and interfered with the fusion but not the proliferation of osteoclast precursors. Similar to wild type osteoclasts treated with Ki16425, mature Lpar1−/− osteoclasts had reduced podosome belt and sealing zone resulting in reduced mineralized matrix resorption. Additionally, LPA1 expression markedly increased in the bone of ovariectomized mice, which was blocked by bisphosphonate treatment. Conversely, systemic treatment with Debio0719 prevented ovariectomy-induced cancellous bone loss. Moreover, intravital multiphoton microscopy revealed that Debio0719 reduced the retention of CX3CR1-EGFP+ osteoclast precursors in bone by increasing their mobility in the bone marrow cavity. Overall, our results demonstrate that LPA1 is essential for in vitro and in vivo osteoclast activities. Therefore, LPA1 emerges as a new target for the treatment of diseases associated with excess bone loss.

Background Autotaxin (ATX) is a secreted protein produced by various tissues in the body including the liver, adipose tissue and bone. Autotaxin (ATX) is an enzyme with a phospholipase D activity responsible for cleavage of lysophosphatidyl-choline (LPC) in lysophosphatidic acid (LPA). LPA is a bio phospholipid, which acts as a growth factor, affecting proliferation, differentiation, and migration. It has been shown that the biological effect of LPA could be the direct consequence of local production of Autotaxin (ATX) in a given tissue or cell type (1). Recently, we have shown that LPA controls two steps of osteoclastogenesis: the fusion and bone resorption capacity of osteoclasts (2). Objectives The aim of this study is to test if ATX produced by osteoclasts could play a direct role in osteoclastogenesis and in bone mass control. Results First we observed that Enpp2 (ATX gene) was a target gene of RANK-L and as a consequence was up regulated during the course of osteoclastogenesis from bone marrow (BM) wild type (WT) cells. Next we generated Ctsk- Cre+;ATXfl/fl mice and use of these animals as a source of osteoclasts-ATX deficient progenitors. In vitro experiments showed a major impact of ATX on osteoclastogenesis and osteoclast mediated bone resorption. Because LPA is massively present in sera, to explore either LPA or ATX putative effect in culture, the use of delipidated serum was mandatory. Using such conditions, we observed a drastic reduction in the number of mature osteoclasts after 5 days of differentiation from BM-WT progenitors, but osteoclasts number were restored by the use of LPA, recombinant ATX plus LPC, or LPC. These results were confirmed by the use of LPA and ATX specific inhibitors (KI16425 and PF8380). Osteocorning bone resorption assays showed that ATX is required for osteoclasts activity, that decreased of 50% when delipidated sera is used in the assays and that was fully restored by the addition of either LPC or recombinant ATX plus LPC. Conclusions All together, and more specifically the results obtained in presence of LPC alone suggest that (i) ATX is secreted by osteoclasts and (ii) is functionally involved in osteoclast differentiation and function in vitro. Current studies are conducted on Ctsk-Cre+;ATXfl/fl mice to fully characterize the role of ATX in the bone mass control in physiological and pathological conditions due to ageing (osteoporosis) and inflammation (rheumatoid arthritis). References Nishimura S. et al Diabetes. 2014 Dec;63(12):4154–64) David M.et al J Biol Chem. 2014 Mar 7;289(10):6551–64 Disclosure of Interest None declared

International audience; Multinucleated muscle fibres arise by fusion of precursor cells called myoblasts. We previously showed that CKIP-1 ectopic expression in C2C12 myoblasts increased cell fusion. In this work, we report that CKIP-1 depletion drastically impairs C2C12 myoblast fusion in vitro and in vivo during zebrafish muscle development. Within developing fast-twich myotome, Ckip-1 localises at the periphery of fast precursor cells, closed to the plasma membrane. Unlike wild-type myoblasts that form spatially arrayed multinucleated fast myofibres, Ckip-1-deficient myoblasts show a drastic reduction in fusion capacity. A search for CKIP-1 binding partners identified the ARPC1 subunit of Arp2/3 actin nucleation complex essential for myoblast fusion. We demonstrate that CKIP-1, through binding to plasma membrane phosphoinositides via its PH domain, regulates cell morphology and lamellipodia formation by recruiting the Arp2/3 complex at the plasma membrane. These results establish CKIP-1 as a regulator of cortical actin that recruits the Arp2/3 complex at the plasma membrane essential for muscle precursor elongation and fusion.

International audience; Osteoporosis is one of the most common bone pathologies, which are characterized by a decrease in bone mass. It is well established that bone mass, which results from a balanced bone formation and bone resorption, is regulated by many hormonal, environmental and genetic factors. Here we report that the immune semaphorin 4D (Sema4D) is a novel factor controlling bone resorption. Sema4D-deficient primary osteoclasts showed impaired spreading, adhesion, migration and resorption due to altered ß3 integrin sub-unit downstream signaling. In apparent accordance with these in vitro results, Sema4D deletion in sexually mature female mice led to a high bone mass phenotype due to defective bone resorption by osteoclasts. Mutant males, however, displayed normal bone mass and the female osteopetrotic phenotype was only detected at the onset of sexual maturity, indicating that, in vivo, this intrinsic osteoclast defect might be overcome in these mice. Using bone marrow cross transplantation, we confirmed that Sema4D controls bone resorption through an indirect mechanism. In addition, we show that Sema4D -/- mice were less fertile than their WT littermates. A decrease in Gnrh1 hypothalamic expression and a reduced number of ovarian follicles can explain this attenuated fertility. Interestingly, ovariectomy abrogated the bone resorption phenotype in Sema4D -/- mice, providing the evidence that the observed high bone mass phenotype is strictly dependent on ovarian function. Altogether, this study reveals that, in vivo, Sema4D is an indirect regulator of bone resorption, which acts via its effect on reproductive function.

Aim The cysteine protease cathepsin K (CatK), abundantly expressed in osteoclasts, is responsible for the degradation of bone matrix proteins, including collagen type 1. Thus, CatK is an attractive target for new anti-resorptive osteoporosis therapies, but the wider effects of CatK inhibitors on bone cells also need to be evaluated to assess their effects on bone. Therefore, we selected, among a series of synthetized isothiosemicarbazides, two molecules which are highly selective CatK inhibitors (CKIs) to test their effects on osteoblasts and osteoclasts. Research Design and Methods Cell viability upon treatment of CKIs were was assayed on human osteoblast-like Saos-2, mouse monocyte cell line RAW 264.7 and mature mouse osteoclasts differentiated from bone marrow. Osteoblast-induced mineralization in Saos-2 cells and in mouse primary osteoblasts from calvaria, with or without CKIs,; were was monitored by Alizarin Red staining and alkaline phosphatase activity, while osteoclast-induced bone resorption was performed on bovine slices. Results Treatments with two CKIs, CKI-8 and CKI-13 in human osteoblast-like Saos-2, murine RAW 264.7 macrophages stimulated with RANKL and mouse osteoclasts differentiated from bone marrow stimulated with RANKL and MCSF were found not to be toxic at doses of up to 100 nM. As probed by Alizarin Red staining, CKI-8 did not inhibit osteoblast-induced mineralization in mouse primary osteoblasts as well as in osteoblast-like Saos-2 cells. However, CKI-13 led to a reduction in mineralization of around 40% at 10–100 nM concentrations in osteoblast-like Saos-2 cells while it did not in primary cells. After a 48-hour incubation, both CKI-8 and CKI-13 decreased bone resorption on bovine bone slices. CKI-13 was more efficient than the commercial inhibitor E-64 in inhibiting bone resorption induced by osteoclasts on bovine bone slices. Both CKI-8 and CKI-13 created smaller bone resorption pits on bovine bone slices, suggesting that the mobility of osteoclasts was slowed down by the addition of CKI-8 and CKI-13. Conclusion CKI-8 and CKI-13 screened here show promise as antiresorptive osteoporosis therapeutics but some off target effects on osteoblasts were found with CKI-13.

pathway by RANKL is critical for osteoclast formation and activity and RANK knockout mice are osteopetrotic as a result of lack of osteoclast formation. A number of mutations in RANK have been associated with osteoclast-poor osteopetrosis. The seven mutations identified to date are located throughout the whole protein and all prevent osteoclast formation in patients. We are exploiting these different single base-pair mutations to elucidate the precise roles of different domains of RANK on downstream signalling. Here, we investigate a mutation that occurs within the C-terminal, intracellular domain, of RANK, that contains the binding site for the essential signalling cofactor TNF receptor associated factor 6 (TRAF6), as well as the oligomerisation domain. The truncating W434X RANK mutation encodes a RANK protein that lacks this oligomerisation motif (amino acids 534-539), which is necessary for trimer formation, but leaves the TRAF6 binding site (amino acids 340-358) intact. HEK293 cells that had been transfected with W434X-RANK were immunostained and analysed by flow cytometry and confocal microscopy. Like wildtype RANK, W434X-RANK was detected at the plasma membrane, suggesting that it could interact with RANKL. Immunoprecipitation and western blot analysis showed that FLAGtagged W434X-RANK interacted with TRAF6 but did not interact with myc-tagged wildtype RANK (which could, however, interact with FLAG-tagged wildtype RANK). These observations demonstrate that this truncating mutation prevents normal RANK trimer formation. However, by contrast to the predicted inactivating nature of this mutation, immunostaining and fluorescence microscopy demonstrated that nuclear translocation of NFkB occurred as a result of RANKL stimulation in W434X-RANK-transfected cells. Taken together, these results suggest that lack of RANK trimer formation is not sufficient to prevent signalling downstream of the RANK receptor, which is surprising given the absence of osteoclast formation in the patient carrying this truncating mutation. It is therefore likely, due to the nature of the mutation, that this protein is not expressed in the patient, probably as a result of nonsensemediated RNA decay, which would explain the phenotype associated with this case of osteoclast-poor osteopetrosis. This mutant RANK protein provides a useful tool to investigate the role of RANK trimerisation on downstream signalling. Disclosure of Interest: None declared