Nmp4, a Regulator of Induced Osteoanabolism, Also Influences Insulin Secretion and Sensitivity.

A bidirectional and complex relationship exists between bone and glycemia. Persons with type 2 diabetes (T2D) are at risk for bone loss and fracture, however, heightened osteoanabolism may ameliorate T2D-induced deficits in glycemia as bone-forming osteoblasts contribute to energy metabolism via increased glucose uptake and cellular glycolysis. Mice globally lacking nuclear matrix protein 4 (Nmp4), a transcription factor expressed in all tissues and conserved between humans and rodents, are healthy and exhibit enhanced bone formation in response to anabolic osteoporosis therapies. To test whether loss of Nmp4 similarly impacted bone deficits caused by diet-induced obesity, male wild-type and Nmp4-/- mice (8 weeks) were fed either low-fat diet or high-fat diet (HFD) for 12 weeks. Endpoint parameters included bone architecture, structural and estimated tissue-level mechanical properties, body weight/composition, glucose-stimulated insulin secretion, glucose tolerance, insulin tolerance, and metabolic cage analysis. HFD diminished bone architecture and ultimate force and stiffness equally in both genotypes. Unexpectedly, the Nmp4-/- mice exhibited deficits in pancreatic ß-cell function and were modestly glucose intolerant under normal diet conditions. Despite the ß-cell deficits, the Nmp4-/- mice were less sensitive to HFD-induced weight gain, increases in % fat mass, and decreases in glucose tolerance and insulin sensitivity. We conclude that Nmp4 supports pancreatic ß-cell function but suppresses peripheral glucose utilization, perhaps contributing to its suppression of induced skeletal anabolism. Selective disruption of Nmp4 in peripheral tissues may provide a strategy for improving both induced osteoanabolism and energy metabolism in comorbid patients.

Loss of Nmp4 enhances bone gain from sclerostin antibody administration.

Severe osteoporosis is often treated with one of three Food and Drug Administration (FDA)-approved osteoanabolics. These drugs act by (1) parathyroid hormone (PTH) receptor stimulation using analogues to PTH (teriparatide) or PTH-related peptide (abaloparatide) or by (2) monoclonal antibody neutralization of sclerostin, an innate Wnt inhibitor (Scl-mAb, romosozumab-aqqg). The efficacies of both strategies wane over time. The transcription factor Nmp4 (Nuclear Matrix Protein 4) is expressed in all tissues yet mice lacking this gene are healthy and exhibit enhanced PTH-induced bone formation. Conditional deletion of Nmp4 in mesenchymal stem progenitor cells (MSPCs) phenocopies the elevated response to PTH in global Nmp4-/- mice. However, targeted deletion in later osteoblast stages does not replicate this response. In this study we queried whether loss of Nmp4 improves Scl-mAb potency. Experimental cohorts included global Nmp4-/- and Nmp4+/+ littermates and three conditional knockout models. Nmp4-floxed (Nmp4fl/fl) mice were crossed with mice harboring one of three Cre-drivers (i) Prx1Cre+ targeting MSPCs, (ii) BglapCre+ (mature osteocalcin-expressing osteoblasts), and (iii) Dmp1Cre+ (osteocytes). Female mice were treated with Scl-mAb or 0.9 % saline vehicle for 4 or 7 weeks from 10 weeks of age. Skeletal response was assessed using micro-computed tomography, dual-energy X-ray absorptiometry, bone histomorphometry, and serum analysis. Global Nmp4-/- mice exhibited enhanced Scl-mAb-induced increases in trabecular bone in the femur and spine and a heightened increase in whole body areal bone mineral density compared to global Nmp4+/+ controls. This improved Scl-mAb potency was primarily driven by enhanced increases in bone formation. Nmp4fl/fl;PrxCre+ mice showed an exaggerated Scl-mAb-induced increase in femoral bone but not in the spine since Prrx1 is not expressed in vertebra. The Nmp4fl/fl;BglapCre+ and Nmp4fl/fl;Dmp1Cre+ mice did not exhibit an improved Scl-mAb response. We conclude that Nmp4 expression in MSPCs interferes with the bone anabolic response to anti-sclerostin therapy.

Conditional Loss of Nmp4 in Mesenchymal Stem Progenitor Cells Enhances PTH-Induced Bone Formation.

Activation of bone anabolic pathways is a fruitful approach for treating severe osteoporosis, yet FDA-approved osteoanabolics, eg, parathyroid hormone (PTH), have limited efficacy. Improving their potency is a promising strategy for maximizing bone anabolic output. Nmp4 (Nuclear Matrix Protein 4) global knockout mice exhibit enhanced PTH-induced increases in trabecular bone but display no overt baseline skeletal phenotype. Nmp4 is expressed in all tissues; therefore, to determine which cell type is responsible for driving the beneficial effects of Nmp4 inhibition, we conditionally removed this gene from cells at distinct stages of osteogenic differentiation. Nmp4-floxed (Nmp4fl/fl ) mice were crossed with mice bearing one of three Cre drivers including (i) Prx1Cre+  to remove Nmp4 from mesenchymal stem/progenitor cells (MSPCs) in long bones; (ii) BglapCre+  targeting mature osteoblasts, and (iii) Dmp1Cre+  to disable Nmp4 in osteocytes. Virgin female Cre+  and Cre- mice (10 weeks of age) were sorted into cohorts by weight and genotype. Mice were administered daily injections of either human PTH 1-34 at 30 µg/kg or vehicle for 4 weeks or 7 weeks. Skeletal response was assessed using dual-energy X-ray absorptiometry, micro-computed tomography, bone histomorphometry, and serum analysis for remodeling markers. Nmp4fl/fl ;Prx1Cre+  mice virtually phenocopied the global Nmp4-/- skeleton in the femur, ie, a mild baseline phenotype but significantly enhanced PTH-induced increase in femur trabecular bone volume/total volume (BV/TV) compared with their Nmp4fl/fl ;Prx1Cre- controls. This was not observed in the spine, where Prrx1 is not expressed. Heightened response to PTH was coincident with enhanced bone formation. Conditional loss of Nmp4 from the mature osteoblasts (Nmp4fl/fl ;BglapCre+ ) failed to increase BV/TV or enhance PTH response. However, conditional disabling of Nmp4 in osteocytes (Nmp4fl/fl ;Dmp1Cre+ ) increased BV/TV without boosting response to hormone under our experimental regimen. We conclude that Nmp4-/- Prx1-expressing MSPCs drive the improved response to PTH therapy and that this gene has stage-specific effects on osteoanabolism. © 2022 American Society for Bone and Mineral Research (ASBMR).

Osteocytic miR21 deficiency improves bone strength independent of sex despite having sex divergent effects on osteocyte viability and bone turnover.

Osteocytes play a critical role in mediating cell-cell communication and regulating bone homeostasis, and osteocyte apoptosis is associated with increased bone resorption. miR21, an oncogenic microRNA, regulates bone metabolism by acting directly on osteoblasts and osteoclasts, but its role in osteocytes is not clear. Here, we show that osteocytic miR21 deletion has sex-divergent effects in bone. In females, miR21 deletion reduces osteocyte viability, but suppresses bone turnover. Conversely, in males, miR21 deletion increases osteocyte viability, but stimulates bone turnover and enhances bone structure. Further, miR21 deletion differentially alters osteocyte cytokine production in the two sexes. Interestingly, despite these changes, miR21 deletion increases bone mechanical properties in both sexes, albeit to a greater extent in males. Collectively, our findings suggest that miR21 exerts both sex-divergent and sex-equivalent roles in osteocytes, regulating osteocyte viability and altering bone metabolism through paracrine actions on osteoblasts and osteoclasts differentially in males vs females, whereas, influencing bone mechanical properties independent of sex.

The Emerging Role of Osteocytes in Cancer in Bone.

Advances in the last decade have established the osteocyte, the most abundant cell in bone, as a dynamic and multifunctional cell capable of controlling bone homeostasis by regulating the function of both osteoblasts and osteoclasts. In addition, accumulating evidence demonstrates that osteocyte function is altered in several skeletal disorders, and targeting osteocytes and their derived factors improves skeletal health. Despite the remarkable progress in our understanding of osteocyte biology, there has been a paucity of information regarding the role of osteocytes in the progression of cancer in bone. Exciting, recent discoveries suggest that tumor cells communicate with osteocytes to generate a microenvironment that supports the growth and survival of cancer cells and stimulates bone destruction. This review features these novel findings and discussions regarding the impact of chemotherapy on osteocyte function and the potential of targeting osteocytes for the treatment of cancer in bone. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Aplidin (plitidepsin) is a novel anti-myeloma agent with potent anti-resorptive activity mediated by direct effects on osteoclasts.

Despite recent progress in its treatment, Multiple Myeloma (MM) remains incurable and its associated bone disease persists even after complete remission. Thus, identification of new therapeutic agents that simultaneously suppress MM growth and protect bone is an unmet need. Herein, we examined the effects of Aplidin, a novel anti-cancer marine-derived compound, on MM and bone cells. In vitro, Aplidin potently inhibited MM cell growth and induced apoptosis, effects that were enhanced by dexamethasone (Dex) and bortezomib (Btz). Aplidin modestly reduced osteocyte/osteoblast viability and decreased osteoblast mineralization, effects that were enhanced by Dex and partially prevented by Btz. Further, Aplidin markedly decreased osteoclast precursor numbers and differentiation, and reduced mature osteoclast number and resorption activity. Moreover, Aplidin reduced Dex-induced osteoclast differentiation and further decreased osteoclast number when combined with Btz. Lastly, Aplidin alone, or suboptimal doses of Aplidin combined with Dex or Btz, decreased tumor growth and bone resorption in ex vivo bone organ cultures that reproduce the 3D-organization and the cellular diversity of the MM/bone marrow niche. These results demonstrate that Aplidin has potent anti-myeloma and anti-resorptive properties, and enhances proteasome inhibitors blockade of MM growth and bone destruction.

Age- and sex-dependent role of osteocytic pannexin1 on bone and muscle mass and strength.

Pannexins (Panxs), glycoproteins that oligomerize to form hemichannels on the cell membrane, are topologically similar to connexins, but do not form cell-to-cell gap junction channels. There are 3 members of the family, 1-3, with Panx1 being the most abundant. All Panxs are expressed in bone, but their role in bone cell biology is not completely understood. We now report that osteocytic Panx1 deletion (Panx1Δot) alters bone mass and strength in female mice. Bone mineral density after reaching skeletal maturity is higher in female Panx1Δot mice than in control Panx1fl/fl mice. Further, osteocytic Panx1 deletion partially prevented aging effects on cortical bone structure and mechanical properties. Young 4-month-old female Panx1Δot mice exhibited increased lean body mass, even though pannexin levels in skeletal muscle were not affected; whereas no difference in lean body mass was detected in male mice. Furthermore, female Panx1-deficient mice exhibited increased muscle mass without changes in strength, whereas Panx1Δot males showed unchanged muscle mass and decreased in vivo maximum plantarflexion torque, indicating reduced muscle strength. Our results suggest that osteocytic Panx1 deletion increases bone mass in young and old female mice and muscle mass in young female mice, but has deleterious effects on muscle strength only in males.

Reversal of loss of bone mass in old mice treated with mefloquine.

Aging is accompanied by imbalanced bone remodeling, elevated osteocyte apoptosis, and decreased bone mass and mechanical properties; and improved pharmacologic approaches to counteract bone deterioration with aging are needed. We examined herein the effect of mefloquine, a drug used to treat malaria and systemic lupus erythematosus and shown to ameliorate bone loss in glucocorticoid-treated patients, on bone mass and mechanical properties in young and old mice. Young 3.5-month-old and old 21-month-old female C57BL/6 mice received daily injections of 5 mg/kg/day mefloquine for 14 days. Aging resulted in the expected changes in bone volume and mechanical properties. In old mice mefloquine administration reversed the lower vertebral cancellous bone volume and bone formation; and had modest effects on cortical bone volume, thickness, and moment of inertia. Mefloquine administration did not change the levels of the circulating bone formation markers P1NP or alkaline phosphatase, whereas levels of the resorption marker CTX showed trends towards increase with mefloquine treatment. In addition, and as expected, aging bones exhibited an accumulation of active caspase3-expressing osteocytes and higher expression of apoptosis-related genes compared to young mice, which were not altered by mefloquine administration at either age. In young animals, mefloquine induced higher periosteal bone formation, but lower endocortical bone formation. Further, osteoclast numbers were higher on the endocortical bone surface and circulating CTX levels were increased, in mefloquine- compared to vehicle-treated young mice. Consistent with this, addition of mefloquine to bone marrow cells isolated from young mice led to increased osteoclastic gene expression and a tendency towards increased osteoclast numbers in vitro. Taken together our findings identify the age and bone-site specific skeletal effects of mefloquine. Further, our results highlight a beneficial effect of mefloquine administration on vertebral cancellous bone mass in old animals, raising the possibility of using this pharmacologic inhibitor to preserve skeletal health with aging.

Cx43 overexpression in osteocytes prevents osteocyte apoptosis and preserves cortical bone quality in aging mice.

Young, skeletally mature mice lacking Cx43 in osteocytes exhibit increased osteocyte apoptosis and decreased bone strength, resembling the phenotype of old mice. Further, the expression of Cx43 in bone decreases with age, suggesting a contribution of reduced Cx43 levels to the age-related changes in the skeleton. We report herein that Cx43 overexpression in osteocytes achieved by using the DMP1-8kb promoter (Cx43OT mice) attenuates the skeletal cortical, but not trabecular bone phenotype of aged, 14-month-old mice. The percentage of Cx43-expressing osteocytes was higher in Cx43OT mice, whereas the percentage of Cx43 positive osteoblasts remained similar to wild type (WT) littermate control mice. The percentage of apoptotic osteocytes and osteoblasts was increased in aged WT mice compared to skeletally mature, 6-month-old WT mice, and the percentage of apoptotic osteocytes, but not osteoblasts, was decreased in age-matched Cx43OT mice. Aged WT mice exhibited decreased bone formation and increased bone resorption as quantified by histomorphometric analysis and circulating markers, compared to skeletally mature mice. Further, aged WT mice exhibited the expected decrease in bone biomechanical structural and material properties compared to young mice. Cx43 overexpression prevented the increase in osteoclasts and decrease in bone formation on the endocortical surfaces, and the changes in circulating markers in the aged mice. Moreover, the ability of bone to resist damage was preserved in aged Cx43OT mice both at the structural and material level. All together, these findings suggest that increased Cx43 expression in osteocytes ameliorates age-induced cortical bone changes by preserving osteocyte viability and maintaining bone formation, leading to improved bone strength.

Connexin37 deficiency alters organic bone matrix, cortical bone geometry, and increases Wnt/ß-catenin signaling.

Deletion of connexin (Cx) 37 in mice leads to increased cancellous bone mass due to defective osteoclast differentiation. Paradoxically; however, Cx37-deficient mice exhibit reduced cortical thickness accompanied by higher bone strength, suggesting a contribution of Cx37 to bone matrix composition. Thus, we investigated whether global deletion of Cx37 alters the composition of organic bone extracellular matrix. Five-month-old Cx37-/- mice exhibited increased marrow cavity area, and periosteal and endocortical bone surface resulting in higher total area in tibia compared to Cx37+/+ control mice. Deletion of Cx37 increased genes involved in collagen maturation (loxl3 and loxl4) and glycosaminoglycans- (chsy1, chpf and has3) proteoglycans- associated genes (biglycan and decorin). In addition, expression of type II collagen assessed by immunostaining was increased by 82% whereas collagen maturity by picrosirius-polarizarion tended to be reduced (p=0.071). Expression of glycosaminoglycans by histochemistry was decreased, whereas immunostaining revealed that biglycan was unchanged and decorin was slightly increased in Cx37-/- bone sections. Consistent with these in vivo findings, MLO-Y4 osteocytic cells silenced for Cx37 gene exhibited increased mRNA levels for collagen synthesis (col1a1 and col3a1) and collagen maturation (lox, loxl1 and loxl2 genes). Furthermore, mechanistic studies showed Wnt/ß-catenin activation in MLO-Y4 osteocytic cells, L5 vertebra, and authentic calvaria-derived osteocytes isolated by fluorescent-activated cell sorter. Our findings demonstrate that altered profile of the bone matrix components in Cx37-deficient mice acts in favor of higher resistance to fracture in long bones.

Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging.

Skeletal aging results in apoptosis of osteocytes, cells embedded in bone that control the generation/function of bone forming and resorbing cells. Aging also decreases connexin43 (Cx43) expression in bone; and osteocytic Cx43 deletion partially mimics the skeletal phenotype of old mice. Particularly, aging and Cx43 deletion increase osteocyte apoptosis, and osteoclast number and bone resorption on endocortical bone surfaces. We examined herein the molecular signaling events responsible for osteocyte apoptosis and osteoclast recruitment triggered by aging and Cx43 deficiency. Cx43-silenced MLO-Y4 osteocytic (Cx43def ) cells undergo spontaneous cell death in culture through caspase-3 activation and exhibit increased levels of apoptosis-related genes, and only transfection of Cx43 constructs able to form gap junction channels reverses Cx43def cell death. Cx43def cells and bones from old mice exhibit reduced levels of the pro-survival microRNA miR21 and, consistently, increased levels of the miR21 target phosphatase and tensin homolog (PTEN) and reduced phosphorylated Akt, whereas PTEN inhibition reduces Cx43def cell apoptosis. miR21 reduction is sufficient to induce apoptosis of Cx43-expressing cells and miR21 deletion in miR21fl/fl bones increases apoptosis-related gene expression, whereas a miR21 mimic prevents Cx43def cell apoptosis, demonstrating that miR21 lies downstream of Cx43. Cx43def cells release more osteoclastogenic cytokines [receptor activator of NFκB ligand (RANKL)/high-mobility group box-1 (HMGB1)], and caspase-3 inhibition prevents RANKL/HMGB1 release and the increased osteoclastogenesis induced by conditioned media from Cx43def cells, which is blocked by antagonizing HMGB1-RAGE interaction. These findings identify a novel Cx43/miR21/HMGB1/RANKL pathway involved in preventing osteocyte apoptosis that also controls osteoclast formation/recruitment and is impaired with aging.

MLO-Y4 osteocytic cell clones express distinct gene expression patterns characteristic of different stages of osteocyte differentiation / Clones de las células osteocíticas MLO-Y4 tienen diferentes patrones de expresión génica característicos de diferentes estadíos de diferenciación osteocítica

Osteocytes are the most abundant bone cell and are formed when osteoblasts become embedded in the bone matrix. Through changes in gene expression and paracrine effects, osteocytes regulate the number of osteoblasts, bone forming cells, and osteoclasts, bone resorbing cells, which are needed to maintain bone mass. MLO-Y4 is the better characterized osteocytic cell line; however, lacks expression of sclerostin, the product of the SOST gene, which is fundamental for osteocyte function and blocks bone formation. With the objective to isolate MLO-Y4 clones with different gene expression profiles, we performed cultures at very low density of MLO-Y4 cells stably transfected with nuclear green fluorescent protein (MLOnGFP). Cell morphology was visualized under a fluorescence microscope. Once the cells reached 80% confluency, RNA was extracted and quantitative real time PCR was performed. Clones exhibit different sizes and morphology, with some cells showing a spindle-like shape and others with abundant projections and a star-like shape. Gene expression also differed among clones. However, none of the clones examined expressed SOST. We conclude that the MLO-nGFP clones constitute a useful tool to study osteocyte differentiation and the role of osteocytes in the control of bone formation and resorption in vitro. (AU)

Los osteocitos son las células más abundantes del hueso y se forman cuando los osteoblastos se encuentran rodeados de matriz ósea. A través de cambios en la expresión génica y efectos paracrinos, los osteocitos controlan el número de osteoblastos que forman el hueso, y osteoclastos que resorben el hueso, células necesarias para mantener la masa ósea. Las células MLO-Y4 son la línea celular osteocítica más investigada; sin embargo, no expresan esclerostina, el pro esclerostina, el producto del gen SOST que bloquea la formación ósea y es indispensable para la función de los osteocitos. Con el objetivo de aislar clones de las células MLO-Y4 con diferentes perfiles de expresión génica, realizamos cultivos a muy baja densidad de las células transfectadas en forma estable con proteína verde fluorescente nuclear (MLO-nGFP). La morfología celular fue evaluada utilizando un microscopio de fluorescencia. Una vez que las células alcanzaron el 80% de confluencia, el ARN fue extraído y analizado por PCR cuantitativa en tiempo real. Las células de los diferentes clones tienen diferentes tamaños y morfología, algunas células son fusiformes y otras con proyecciones citoplasmáticas abundantes y en forma de estrella. La expresión de los genes también varió en los distintos clones. Sin embargo, ninguno de ellos expresó SOST. En conclusión, los clones de las células MLO-nGFP constituyen una herramienta útil para estudiar la diferenciación de los osteocitos y el rol de estas células en el control de la formación y resorción ósea in vitro. (AU)