Osteocytes In Mice Research Articles

High-fat and high-carbohydrate diets increase bone fragility through TGF-β-dependent control of osteocyte function.

Obesity can increase the risk of bone fragility, even when bone mass is intact. This fragility stems from poor bone quality, potentially caused by deficiencies in bone matrix material properties. However, cellular and molecular mechanisms leading to obesity-related bone fragility are not fully understood. Using male mouse models of obesity, we discovered TGF-β signaling plays a critical role in mediating the effects of obesity on bone. High-carbohydrate and high-fat diets increase TGF-β signaling in osteocytes, which impairs their mitochondrial function, increases cellular senescence, and compromises perilacunar/canalicular remodeling and bone quality. By specifically inhibiting TGF-β signaling in mouse osteocytes, some of the negative effects of high-fat and high-carbohydrate diets on bones, including the lacunocanalicular network, perilacunar/canalicular remodeling, senescence, and mechanical properties such as yield stress, were mitigated. DMP1-Cre-mediated deletion of TGF-β receptor II also blunted adverse effects of high-fat and high-carbohydrate diets on energy balance and metabolism. These findings suggest osteocytes are key in controlling bone quality in response to high-fat and high-carbohydrate diets. Calibrating osteocyte function could mitigate bone fragility associated with metabolic diseases while reestablishing energy balance.

Eldecalcitol protected osteocytes against ferroptosis of D-gal-induced senescent MLO-Y4 cells and ovariectomized mice

BackgroundActive vitamin D analog eldecalcitol is clinically applied in treatment of postmenopausal osteoporosis. This study aims to determine the role of eldecalcitol in the protection of osteocytes from senescence and the associated ferroptosis. MethodsThe MLO-Y4 osteocytes were exposed to D-gal inducing senescence. The ovariectomized (OVX) mice treated with D-gal using as an aging inducer were intraperitoneally injected with eldecalcitol. The multiplexed confocal imaging, fluorescence in situ hybridization and transmission electron microscopy were applied in assessing osteocytic properties. Immunochemical staining and immunoblotting were carried out to detect abundance and expression of molecules. ResultsThe ablation of vitamin D receptor led to a reduction in amounts of osteocytes, a loss of dendrites, an increase in mRNA expression of SASP factors and in protein expression of senescent factors, as well as changes in mRNA expression of ferroptosis-related genes (PTGS2 & RGS4). Eldecalcitol reversed senescent phenotypes of MLO-Y4 cells shown by improving cell morphology and density, decreasing β-gal-positive cell accumulation, and down-regulating protein expression (P16, P21 & P53). Eldecalcitol reduced intracellular ROS and MDA productions, elevated JC-1 aggregates, and up-regulated expression of Nrf2 and GPX4. Eldecalcitol exhibited osteopreserve effects in D-gal-induced aging OVX mice. The confocal imaging displayed its improvement on osteocytic network organization. Eldecalcitol decreased the numbers of senescent osteocytes at tibial diaphysis by SADS assay and attenuated mRNA expression of SASP factors as well as down-regulated protein expression of senescence-related factors and restored levels of ferroptotic biomarkers in osteocytes-enriched bone fraction. It reduced 4-HNE staining area, stimulated Nrf2-positive staining, and promoted nuclear translocation of Nrf2 in osteocytes of mice as well as inhibited and promoted protein expression of 4-HNE and Nrf2, respectively, in osteocytes-enriched bone fraction. ConclusionsThe present study revealed the ameliorative effects of eldecalcitol on senescence and the associated ferroptosis of osteocytes, contributing to its preservation against osteoporosis of D-gal-induced senescent ovariectomized mice.

Osteocytes contribute to sex-specific differences in osteoarthritic pain.

Osteoarthritic (OA) pain affects 18% of females and 9.6% of males aged over 60 worldwide, with 62% of all OA patients being women. The molecular drivers of sex-based differences in OA are unknown. Bone is intricately coupled with the sensory nervous system and one of the only joint tissues known to show changes that correlate with patient pain in OA. There are fundamental sex differences in pain sensation and bone biology which may be intrinsic to OA disease progression, however these differences are vastly under researched. We have utilised three data sets to investigate the hypothesis that potential mediators responsible for sex dependent pain mechanisms displayed in OA are derived from mechanically stimulated osteocytes. Our published dataset of the in vitro human osteocyte mechanosome was independently compared with published data from, sex-based gene expression differences in human long bone, the sex-based gene expression differences during the skeletal maturation of the mouse osteocyte transcriptome and sex specific OA risk factors and effector genes in a large human GWAS. 80 of the 377 sex-specific genes identified in the mouse osteocyte transcriptome were mechanically regulated in osteocytes with enrichment associated with neural crest migration and axon extension, and DISEASES analysis enrichment for the rheumatoid arthritis pathway. 3861 mechanically regulated osteocytic genes displayed sex-specific differences in human long bone with enrichment for genes associated with the synapse, sensory perception of pain, axon guidance, immune responses, distal peripheral sensory neuropathy, sensory neuropathy, and poor wound healing. 32 of 77 effector genes and 1 of 3 female specific OA risk factor genes identified in the human GWAS were differentially expressed in the osteocyte mechanosome and male and female bone. This analysis lends support to the hypothesis that mechanically regulated genes in osteocytes could influence sex specific differences in osteoarthritic pain and highlights pain pathways with approved drugs that could potentially treat elevated pain susceptibility in females with OA.

A Fluorescent Intravital Imaging Approach to Study Load-Induced Calcium Signaling Dynamics in Mouse Osteocytes.

Bone tissue is exquisitely sensitive to differences in mechanical load magnitude. Osteocytes, dendritic cells that form a syncytium throughout the bone, are responsible for the mechanosensory function of bone tissue. Studies employing histology, mathematical modeling, cell culture, and ex vivo bone organ cultures have greatly advanced the understanding of osteocyte mechanobiology. However, the fundamental question of how osteocytes respond to and encode mechanical information at the molecular level in vivo is not well understood. Intracellular calcium concentration fluctuations in osteocytes offer a useful target for learning more about acute bone mechanotransduction mechanisms. Here, we report a method for studying osteocyte mechanobiology in vivo, combining a mouse strain with a fluorescently genetically encoded calcium indicator expressed in osteocytes with an in vivo loading and imaging system to directly detect osteocyte calcium levels during loading. This is achieved with a three-point bending device that can deliver well-defined mechanical loads to the third metatarsal of living mice while simultaneously monitoring fluorescently indicated calcium responses of osteocytes using two-photon microscopy. This technique allows for direct in vivo observation of osteocyte calcium signaling events in response to whole bone loading and is useful in the endeavor to reveal mechanisms in osteocyte mechanobiology.

Desferrioxamine alleviates UHMWPE particle-induced osteoclastic osteolysis by inhibiting caspase-1-dependent pyroptosis in osteocytes.

Cell death and inflammation are the two important triggers of wear particle-induced osteolysis. Particles, including cobalt-chromium-molybdenum and tricalcium phosphate, have been reported to induce pyroptosis in macrophages and osteocytes. Although macrophage pyroptosis facilitates osteoclastic bone resorption and osteolysis, whether osteocyte pyroptosis is involved in osteoclastic osteolysis still needs further investigation. Desferrioxamine (DFO), an FDA-approved medication and a powerful iron chelator, has been proven to reduce ultrahigh-molecular-weight polyethylene (UHMWPE) particle-induced osteolysis. However, whether DFO can ameliorate UHMWPE particle-induced osteolysis by decreasing pyroptosis in osteocytes is unknown. A mouse calvarial osteolysis model and the mouse osteocyte cell line MLO-Y4 was used, and we found that pyroptosis in osteocytes was significantly induced by UHMWPE particles. Furthermore, our findings uncovered a role of caspase-1-dependent pyroptosis in osteocytes in facilitating osteoclastic osteolysis induced by UHMWPE particles. In addition, we found that DFO could alleviate UHMWPE particle-induced pyroptosis in osteocytes in vivo and in vitro. We uncovered a role of caspase-1-dependent pyroptosis in osteocytes in facilitating osteoclastic osteolysis induced by UHMWPE particles. Furthermore, we found that DFO alleviated UHMWPE particle-induced osteoclastic osteolysis partly by inhibiting pyroptosis in osteocytes. Schematic of DFO reducing UHMWPE particle-induced osteolysis by inhibiting osteocytic pyroptosis. Wear particles, such as polymers, generated from prosthetic implant materials activate canonical inflammasomes and promote the cleavage and activation of caspase-1. This is followed by caspase-1-dependent IL-β maturation and GSDMD cleavage. The N-terminal fragment of GSDMD binds to phospholipids on the cell membrane and forms holes in the membrane, resulting in the release of mature IL-β and inflammatory intracellular contents. This further facilitates osteoclastic differentiation of BMMs, resulting in excessive bone resorption and ultimately leading to prosthetic osteolysis. DFO reduces UHMWPE particle-induced osteolysis by inhibiting osteocytic pyroptosis.

Accumulation of advanced oxidation protein products contributes to age-related impairment of gap junction intercellular communication in osteocytes of male mice.

AimsGap junction intercellular communication (GJIC) in osteocytes is impaired by oxidative stress, which is associated with age-related bone loss. Ageing is accompanied by the accumulation of advanced oxidation protein products (AOPPs). However, it is still unknown whether AOPP accumulation is involved in the impairment of osteocytes’ GJIC. This study aims to investigate the effect of AOPP accumulation on osteocytes’ GJIC in aged male mice and its mechanism.MethodsChanges in AOPP levels, expression of connexin43 (Cx43), osteocyte network, and bone mass were detected in 18-month-old and three-month-old male mice. Cx43 expression, GJIC function, mitochondria membrane potential, reactive oxygen species (ROS) levels, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation were detected in murine osteocyte-like cells (MLOY4 cells) treated with AOPPs. The Cx43 expression, osteocyte network, bone mass, and mechanical properties were detected in three-month-old mice treated with AOPPs for 12 weeks.ResultsThe AOPP levels were increased in aged mice and correlated with degeneration of osteocyte network, loss of bone mass, and decreased Cx43 expression. AOPP intervention induced NADPH oxidase activation and mitochondrial dysfunction, triggered ROS generation, reduced Cx43 expression, and ultimately impaired osteocytes’ GJIC, which were ameliorated by NADPH oxidase inhibitor apocynin, mitochondria-targeted superoxide dismutase mimetic (mito-TEMPO), and ROS scavenger N-acetyl cysteine. Chronic AOPP loading accelerated the degradation of osteocyte networks and decreased Cx43 expression, resulting in deterioration of bone mass and mechanical properties in vivo.ConclusionOur study suggests that AOPP accumulation contributes to age-related impairment of GJIC in osteocytes of male mice, which may be part of the pathogenic mechanism responsible for bone loss during ageing.Cite this article: Bone Joint Res 2022;11(7):413–425.

A high-throughput biomimetic bone-on-a-chip platform with artificial intelligence-assisted image analysis for osteoporosis drug testing.

Although numerous organ-on-a-chips have been developed, bone-on-a-chip platforms have rarely been reported because of the high complexity of the bone microenvironment. With an increase in the elderly population, a high-risk group for bone-related diseases such as osteoporosis, it is essential to develop a precise bone-mimicking model for efficient drug screening and accurate evaluation in preclinical studies. Here, we developed a high-throughput biomimetic bone-on-a-chip platform combined with an artificial intelligence (AI)-based image analysis system. To recapitulate the key aspects of natural bone microenvironment, mouse osteocytes (IDG-SW3) and osteoblasts (MC3T3-E1) were cocultured within the osteoblast-derived decellularized extracellular matrix (OB-dECM) built in a well plate-based three-dimensional gel unit. This platform spatiotemporally and configurationally mimics the characteristics of the structural bone unit, known as the osteon. Combinations of native and bioactive ingredients obtained from the OB-dECM and coculture of two types of bone cells synergistically enhanced osteogenic functions such as osteocyte differentiation and osteoblast maturation. This platform provides a uniform and transparent imaging window that facilitates the observation of cell-cell interactions and features high-throughput bone units in a well plate that is compatible with a high-content screening system, enabling fast and easy drug tests. The drug efficacy of anti-SOST antibody, which is a newly developed osteoporosis drug for bone formation, was tested via β-catenin translocation analysis, and the performance of the platform was evaluated using AI-based deep learning analysis. This platform could be a cutting-edge translational tool for bone-related diseases and an efficient alternative to bone models for the development of promising drugs.

Extracellular acidification augments sclerostin and osteoprotegerin production by Ocy454 mouse osteocytes

Osteocytes sense the microenvironmental stimuli, including mechanical stress, and regulate bone resorption by osteoclasts and bone formation by osteoblasts. Diabetes and cancer metastasis to bone raise l-lactic acid in the bone tissue, causing acidification. Here, we investigated the effects of l-lactic acid and extracellular acidification on the function of mouse Ocy454 osteocytes. L- and d-lactic acid with low chiral selectivity and acidification of the medium raised the production of sclerostin and osteoprotegerin by Ocy454 cells. The mRNA expression of their genes increased after either treatment of L- and d-lactic acid or acidification of the medium. Furthermore, the conditioned medium of Ocy454 cells cultured in an acidic environment suppressed the induction of alkaline phosphatase activity in MC3T3-E1 cells, which was recovered by the anti-sclerostin antibody. While it is reported that HDAC5 inhibits the transcription of the sclerostin gene, extracellular acidification reduced the nuclear localization of HDAC5 in Ocy454 cells. While calmodulin kinase II (CaMKII) is known to phosphorylate and induce extranuclear translocation of HDAC5, KN-62, an inhibitor of CaMKII lowered the expression of the sclerostin gene in Ocy454 cells. Collectively, extracellular acidification is a microenvironmental factor that modulates osteocyte functions.

Modified enzymatic collagen digestion-mediated isolation of osteocytes

This study established a method for isolating large numbers of high-purity osteocytes from high-density bone. Bone fragments derived from mice tibia and femurs were alternately digested with type I collagenase and EDTA nine times, and the digested cells and bone chips (BC) were cultured, digested, and passaged when cells were fully grown. The types of cells obtained were identified by morphology, viable cell counts, alkaline phosphatase staining, and biochemical activity analyses, and specific osteocyte and osteoblast markers were evaluated by quantitative real-time polymerase chain reaction. Our results showed that among the cells obtained from the third digestion (fractions 7–9) of wild mice tibias and femurs and the remaining BCs, 85%–90% of the cells were osteocytes. Moreover, their morphology was approximately one-tenth to one-fifth the size of osteoblasts, star-shaped or polygonal, with a dendritic structure, negative for alkaline phosphatase staining, and showed a high expression of dmp1 and sclerostin. Ninety percent of the cells in fractions 1–3 were osteoblasts, and were fusiform or polygonal shape. The activity of osteoblast-specific alkaline phosphatase and mRNA expression were high in this fraction, while the expression of osteocyte-specific dmp1 and sclerostin was not detected. In the second portion (fractions 4–6), a large number were osteoblasts, mixed with a small number of osteocytes, and had high alkaline phosphatase activity and osteocyte mRNA levels, a specific level of the osteocyte marker dmp1, and no sclerostin was detected. Osteocytes in daβcatot mice were also successfully isolated by this method, and we found that Wnt signaling increased the proliferation of these osteocytes. The proposed method can be used to culture osteocytes and osteoblasts of high purity and can be used for isolation and culture of these two kinds of cells from high-density bone, which provides an avenue for the study of osteocyte function in vitro.

Estrogen depletion on In vivo osteocyte calcium signaling responses to mechanical loading.

Microstructural adaptation of bone in response to mechanical stimuli is diminished with estrogen deprivation. Here we tested in vivo whether ovariectomy (OVX) alters the acute response of osteocytes, the principal mechanosensory cells of bone, to mechanical loading in mice. We also used super resolution microscopy (Structured Illumination microscopy or SIM) in conjunction with immunohistochemistry to assess changes in the number and organization of "osteocyte mechanosomes" - complexes of Panx1 channels, P2X7 receptors and CaV3 voltage-gated Ca2+ channels clustered around αvβ3 integrin foci on osteocyte processes. Third metatarsals bones of mice expressing an osteocyte-targeted genetically encoded Ca2+ indicator (DMP1-GCaMP3) were cyclically loaded in vivo to strains from 250 to 3000 με and osteocyte intracellular Ca2+ signaling responses were assessed in mid-diaphyses using multiphoton microscopy. The number of Ca2+ signaling osteocytes in control mice increase monotonically with applied strain magnitude for the physiological range of strains. The relationship between the number of Ca2+ signaling osteocytes and loading was unchanged at 2days post-OVX. However, it was altered markedly at 28days post-OVX. At loads up to 1000 με, there was a dramatic reduction in number of responding (i.e. Ca2+ signaling) osteocytes; however, at higher strains the numbers of Ca2+ signaling osteocytes were similar to control mice. OVX significantly altered the abundance, make-up and organization of osteocyte mechanosome complexes on dendritic processes. Numbers of αvβ3 foci also staining with either Panx 1, P2X7R or CaV3 declined by nearly half after OVX, pointing to a loss of osteocyte mechanosomes on the dendritic processes with estrogen depletion. At the same time, the areas of the remaining foci that stained for αvβ3 and channel proteins increased significantly, a redistribution of mechanosome components suggesting a potential compensatory response. These results demonstrate that the deleterious effects of estrogen depletion on skeletal mechanical adaptation appear at the level of mechanosensation; osteocytes lose the ability to sense small (physiological) mechanical stimuli. This decline may result at least partly from changes in the structure and organization of osteocyte mechanosomes, which contribute to the distinctive sensitivity of osteocytes (particularly their dendritic processes) to mechanical stimulation.

Alterations in osteocyte lacunar morphology affect local bone tissue strains

Osteocytes are capable of remodeling their perilacunar bone matrix, which causes considerable variations in the shape and size of their lacunae. If these variations in lacunar morphology cause changes in the mechanical environment of the osteocytes, in particular local strains, they would subsequently affect bone mechanotransduction, since osteocytes are likely able to directly sense these strains. The purpose of this study is to quantify the effect of alterations in osteocyte lacunar morphology on peri-lacunar bone tissue strains. To this end, we related the actual lacunar shape in fibulae of six young-adult (5-month) and six old (23-month) mice, quantified by high-resolution micro-computed tomography, to microscopic strains, analyzed by micro-finite element modeling. We showed that peak effective strain increased by 12.6% in osteocyte cell bodies (OCYs), 9.6% in pericellular matrix (PCM), and 5.3% in extra cellular matrix (ECM) as the lacunae volume increased from 100-200 μm3 to 500–600 μm3. Lacunae with a larger deviation (>8°) in orientation from the longitudinal axis of the bone are exposed to 8% higher strains in OCYs, 6.5% in PCM, 4.2% in ECM than lacunae with a deviation in orientation below 8°. Moreover, increased lacuna sphericity from 0 to 0.5 to 0.7–1 led to 25%, 23%, and 13% decrease in maximum effective strains in OCYs, PCM, and ECM, respectively. We further showed that due to the presence of smaller and more round lacunae in old mice, local bone tissue strains are on average 5% lower in the vicinity of lacunae and their osteocytes of old mice compared to young. Understanding how changes in lacunar morphology affect the micromechanical environment of osteocytes presents a first step in unraveling their potential role in impaired bone mechanoresponsiveness with e.g. aging.

Autophagy Prevents Osteocyte Cell Death under Hypoxic Conditions

Hypoxia occurs under important clinical conditions such as cancers, heart disease, and ischemia. However, the relationship between hypoxia and autophagy in osteocytes is still unclear. The objective of the present study was to uncover the regulatory mechanisms that prevent regulated cell death, such as apoptosis, necrosis, and autophagy, under hypoxia. MLO-Y4 cells, a mouse osteocyte cell line, were exposed to various O₂ partial pressures (PO₂). Subsequently, the cells underwent apoptosis, autophagy, autophagic cell death, and/or necrosis, and thereby we designated PO₂ = 2% as a representative hypoxic condition. Immunofluorescence staining showed an increase of LC3 and a decrease of p62 in MLO-Y4 cells exposed to hypoxia, indicating the induction of autophagy. We then hypothesized that β-estradiol (E2) and vitamin D play an important role in apoptosis and autophagy of osteocytes under hypoxia. 1,25α-dihydroxyvitamin D3 (VitD) protected MLO-Y4 cells from cell death and induced autophagy. However, E2 showed little effect. Finally, Western blotting for phosphorylated mTOR and Akt was carried out in order to investigate the altered autophagy signaling pathways affected by the addition of VitD and E2. However, neither E2 nor VitD were capable of recovering the decreased phosphorylation of those factors. Our results indicated that the effects of VitD on autophagy under hypoxia were dependent on the Akt and mTOR pathways. Thus, the results of the present study showed that VitD suppresses osteocyte cell death in an mTOR pathway-dependent manner in hypoxic conditions. This suggests the potential of VitD as a therapeutic intervention for diseases in which the cell death of osteocytes mainly occurs via hypoxia.

Novel Aptamer-Based Small-Molecule Drug Screening Assay to Identify Potential Sclerostin Inhibitors against Osteoporosis.

A novel aptamer-based competitive drug screening platform for osteoporosis was devised in which fluorescence-labeled, sclerostin-specific aptamers compete with compounds from selected chemical libraries for the binding of immobilized recombinant human sclerostin to achieve high-throughput screening for potential small-molecule sclerostin inhibitors and to facilitate drug repurposing and drug discovery. Of the 96 selected inhibitors and FDA-approved drugs, six were shown to result in a significant decrease in the fluorescence intensity of the aptamer, suggesting a higher affinity toward sclerostin compared with that of the aptamer. The targets of these potential sclerostin inhibitors were correlated to lipid or bone metabolism, and several of the compounds have already been shown to be potential osteogenic activators, indicating that the aptamer-based competitive drug screening assay offered a potentially reliable strategy for the discovery of target-specific new drugs. The six potential sclerostin inhibitors suppressed the level of both intracellular and/or extracellular sclerostin in mouse osteocyte IDG-SW3 and increased alkaline phosphatase activity in IDG-SW3 cells, human bone marrow-derived mesenchymal stem cells and human fetal osteoblasts hFOB1.19. Potential small-molecule drug candidates obtained in this study are expected to provide new therapeutics for osteoporosis as well as insights into the structure–activity relationship of sclerostin inhibitors for rational drug design.

Reduced Bone Mass and Increased Osteocyte Tartrate-Resistant Acid Phosphatase (TRAP) Activity, But Not Low Mineralized Matrix Around Osteocyte Lacunae, Are Restored After Recovery From Exogenous Hyperthyroidism in Male Mice.

Hyperthyroidism causes secondary osteoporosis through favoring bone resorption over bone formation, leading to bone loss with elevated bone fragility. Osteocytes that reside within lacunae inside the mineralized bone matrix orchestrate the process of bone remodeling and can themselves actively resorb bone upon certain stimuli. Nevertheless, the interaction between thyroid hormones and osteocytes and the impact of hyperthyroidism on osteocyte cell function are still unknown. In a preliminary study, we analyzed bones from male C57BL/6 mice with drug-induced hyperthyroidism, which led to mild osteocytic osteolysis with 1.14-fold larger osteocyte lacunae and by 108.33% higher tartrate-resistant acid phosphatase (TRAP) activity in osteocytes of hyperthyroid mice compared to euthyroid mice. To test whether hyperthyroidism-induced bone changes are reversible, we rendered male mice hyperthyroid by adding levothyroxine into their drinking water for 4 weeks, followed by a weaning period of 4 weeks with access to normal drinking water. Hyperthyroid mice displayed cortical and trabecular bone loss due to high bone turnover, which recovered with weaning. Although canalicular number and osteocyte lacunar area were similar in euthyroid, hyperthyroid and weaned mice, the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive osteocytes was 100% lower in the weaning group compared to euthyroid mice and the osteocytic TRAP activity was eightfold higher in hyperthyroid animals. The latter, along with a 3.75% lower average mineralization around the osteocyte lacunae in trabecular bone, suggests osteocytic osteolysis activity that, however, did not result in significantly enlarged osteocyte lacunae. In conclusion, we show a recovery of bone microarchitecture and turnover after reversal of hyperthyroidism to a euthyroid state. In contrast, osteocytic osteolysis was initiated in hyperthyroidism, but its effects were not reversed after 4 weeks of weaning. Due to the vast number of osteocytes in bone, we speculate that even minor individual cell functions might contribute to altered bone quality and mineral homeostasis in the setting of hyperthyroidism-induced bone disease. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Skeletal Effects of Inducible ERα Deletion in Osteocytes in Adult Mice.

Estrogen is known to regulate bone metabolism in both women and men, but substantial gaps remain in our knowledge of estrogen and estrogen receptor alpha (ERα) regulation of adult bone metabolism. Studies using global ERα-knockout mice were confounded by high circulating sex-steroid levels, and osteocyte/osteoblast-specific ERα deletion may be confounded by ERα effects on growth versus the adult skeleton. Thus, we developed mice expressing the tamoxifen-inducible CreERT2 in osteocytes using the 8-kilobase (kb) Dmp1 promoter (Dmp1CreERT2 ). These mice were crossed with ERαfl//fl mice to create ERαΔOcy mice, permitting inducible osteocyte-specific ERα deletion in adulthood. After intermittent tamoxifen treatment of adult 4-month-old mice for 1 month, female, but not male, ERαΔOcy mice exhibited reduced spine bone volume fraction (BV/TV (-20.1%, p=0.004) accompanied by decreased trabecular bone formation rate (-18.9%, p=0.0496) and serum P1NP levels (-38.9%, p=0.014). Periosteal (+65.6%, p=0.004) and endocortical (+64.1%, p=0.003) expansion were higher in ERαΔOcy mice compared to control (Dmp1CreERT2 ) mice at the tibial diaphysis, reflecting the known effects of estrogen to inhibit periosteal apposition and promote endocortical formation. Increases in Sost (2.1-fold, p=0.001) messenger RNA (mRNA) levels were observed in trabecular bone at the spine in ERαΔOcy mice, consistent with previous reports that estrogen deficiency is associated with increased circulating sclerostin as well as bone SOST mRNA levels in humans. Further, the biological consequences of increased Sost expression were reflected in significant overall downregulation in panels of osteoblast and Wnt target genes in osteocyte-enriched bones from ERαΔOcy mice. These findings thus establish that osteocytic ERα is critical for estrogen action in female, but not male, adult bone metabolism. Moreover, the reduction in bone formation accompanied by increased Sost, decreased osteoblast, and decreased Wnt target gene expression in ERαΔOcy mice provides a direct link in vivo between ERα and Wnt signaling. © 2022 American Society for Bone and Mineral Research (ASBMR).

Implicación de la Cx43 y el cilio primario en la actividad de los osteocitos

The bone tissue has the ability to adapt to the stimuli of the environment by altering its morphology and metabolism. The development and maintenance of this tissue are dynamic processes, regulated by the different bone cells communicated with each other through Gap Junctions (GJs). Connexin 43 (Cx43), which is the most abundant protein in GJs, has key roles in the signal transduction of bone tissue and in the response to hormonal and mechanical stimuli. Other mechanosensing elements in the osteocytes are: the primary cilium, mainly formed of microtubules and developed when the cells are in the G0 phase of the cell cycle; and the integrins, a family of glycoproteins forming heterodimers composed of two subunits (the alpha and beta chain) and located in the cell membrane. The involvement of Cx43, the primary cilium and integrins in the mouse osteocyte cell line MLO-Y4 control and deficient in Cx43 will be studied in this project. The response of this cell line to hormonal stimuli (ligand of the parathyroid hormone receptor type 1, the protein related to parathyroid hormone, PTHrP) and mechanical stimuli (by fluid flow of culture medium) will also be analyzed. In addition, the possible functional interaction between these three mechanical elements will be studied. For protein expression analyses of IFT88 (homolog of the intraflagelar transport protein 88), Cx43, P-ERK (Kinases regulated by extracellular signal phosphorylated) and P-GSK3 (Glycogen synthase kinase 3 beta phosphorylated), Western blotting was performed. The expression of the integrins was determined by PCR (Polymerase Chain Reaction) and, to characterize the possible colocalization between the primary cilium and Cx43 an immunofluorescence was performed. The results obtained show that Cx43 and the primary cilium do not collocate, and that the presence of Cx43 is not necessary for ciliogenesis. On the other hand, it was determined that fluid flow and PTHrP increase the phosphorylation of ERK and GSK3. Therefore, these stimuli regulate cell survival pathways in osteocytes. Finally, it was observed that integrin α2 increased its expression in control cells (Cx43 +/+). However, integrins α6, β1 and β3 increased in Cx43 deficient cells (Cx43 -/-), suggesting an interaction between these two protein families.

Observation of dendrite osteocytes of mice at different developmental stages using Ploton silver staining and phalloidin staining

To assess the value of Ploton silver staining and phalloidin-iFlour 488 staining in observation of the morphology of osteocyte dendrites of mice at different developmental stages. The humerus and femurs were harvested from mice at 0 (P0), 5 (P5), 15 (P15), 21 (P21), 28 (P28), and 35 days (P35) after birth to prepare cryo-sections and paraffin sections. HE staining of P35 mouse femur sections served as a reference for observing osteocytes in the trabecular bone and cortical bone. The humeral sections at different developmental stages were stained with Ploton silver staining to observe the morphology of osteocytes and canaliculi, and the canalicular lengths in the cortical and trabecular bones of the humerus of the mice in each developmental stage were recorded. The cryo-sections of the humerus from P10 and P15 mice were stained with phalloidin iFlour-488 to observe the morphology of osteocytes and measurement of the length of osteocyte dendrites in the cortical bone. In the trabecular bone of the humerus of P0-P15 mice, Ploton silver staining only visualized the outline of the osteocytes, and the morphology of the canaliculi was poorly defined. In P21 or older mice, Ploton silver staining revealed the morphology of the trabecular bone osteocytes and the canaliculi, which were neatly arranged and whose lengths increased significantly with age (P21 vs P28, P < 0.05; P21 vs P35, P < 0.05). In the humeral cortical bone of P15 mice, the morphology of the osteocytes and canalicular could be observed with Ploton silver staining, and the length of the regularly arranged canaliculi of the osteocytes increased significantly with age (P15 vs P21, P < 0.005; P15 vs P28, P < 0.0001; P15 vs P35, P < 0.0001). Phalloidin iFlour-488 staining was capable of visualizing the complete morphology of the osteocytes at P10 and P15; the osteocyte dendrites elongated progressively with age (P10 vs P15, P < 0.01) to form connections with the surrounding osteocytes. Mouse osteocyte dendrites elongate progressively and their arrangement gradually becomes regular with age. Ploton silver staining can clearly visualize the morphology of the osteocytes and the canaliculi in adult mice but not in mice in early stages of development. Phalloidin iFlour-488 staining for labeling the cytoskeleton can be applied for mouse osteocytes at all developmental stages and allows morphological observation of mouse osteocytes in early developmental stages.

BMP-induced Atoh8 attenuates osteoclastogenesis by suppressing Runx2 transcriptional activity and reducing the Rankl/Opg expression ratio in osteoblasts

Adult bone structural integrity is maintained by remodeling via the coupling of osteoclastic bone resorption and osteoblastic bone formation. Osteocytes or osteoblasts express receptor activator of nuclear factor κ-B ligand (Rankl) or osteoprotegerin (Opg) to promote or inhibit osteoclastogenesis, respectively. Bone morphogenetic protein (BMP) is a potent bone inducer, but its major role in adult bone is to induce osteocytes to upregulate sclerostin (Sost) and increase the Rankl/Opg expression ratio, resulting in promotion of osteoclastogenesis. However, the precise effect of BMP-target gene(s) in osteoblasts on the Rankl/Opg expression ratio remains unclear. In the present study, we identified atonal homolog 8 (Atoh8), which is directly upregulated by the BMP-Smad1 axis in osteoblasts. In vivo, Atoh8 was detected in osteoblasts but not osteocytes in adult mice. Although global Atoh8-knockout mice showed only a mild phenotype in the neonate skeleton, the bone volume was decreased and osteoclasts were increased in the adult phase. Atoh8-null marrow stroma cells were more potent than wild-type cells in inducing osteoclastogenesis in marrow cells. Atoh8 loss in osteoblasts increased Runx2 expression and the Rankl/Opg expression ratio, while Runx2 knockdown normalized the Rankl/Opg expression ratio. Moreover, Atoh8 formed a protein complex with Runx2 to inhibit Runx2 transcriptional activity and decrease the Rankl/Opg expression ratio. These results suggest that bone remodeling is regulated elaborately by BMP signaling; while BMP primarily promotes bone resorption, it simultaneously induces Atoh8 to inhibit Runx2 and reduce the Rankl/Opg expression ratio in osteoblasts, suppressing osteoclastogenesis and preventing excessive BMP-mediated bone resorption.

CAMP regulates 11β-hydroxysteroid dehydrogenase-2 and Sp1 expression in MLO-Y4/MC3T3-E1 cells.

11β-hydroxysteroid dehydrogenase-2 (11β-HSD2) is one of the key enzymes in glucocorticoid metabolism, which can inactivate local corticosterone and regulate the level of active glucocorticoid in tissues. The expression of 11β-HSD2 and its regulatory pathway serve an important role in the apoptosis of steroid induced osteonecrosis of the femoral head (SANFH). The present study aimed to identify the regulatory effects of cAMP on the expression of Sp1 transcription factor (Sp1) and 11β-HSD2 in osteocytes at the cellular level. Murine long bone osteocyte Y4 (MLO-Y4) clone cells and mouse embryo osteoblast-like (MC3T3-E1) cells were cultured in vitro with adenylate cyclase activator or inhibitor (forskolin and SQ22536, respectively) to investigate the effects of alterations to intracellular cAMP levels. mRNA and protein expression levels of Sp1 and 11β-HSD2 were detected by reverse transcription-quantitative PCR and western blotting, respectively. Compared with the negative control group, the mRNA and protein expression levels of Sp1 were significantly increased in the activation group, whereas Sp1 expression levels were significantly decreased in the inhibition group. Similarly, compared with the negative control group, the mRNA and protein expression levels of 11β-HSD2 were significantly increased in the activator group, but significantly decreased in the inhibitor group. The aforementioned results indicated that intracellular cAMP levels significantly regulated the expression of Sp1 and 11β-HSD2 in mouse osteocytes and osteoblasts. Therefore, the present study suggested a potential therapeutic strategy for the prevention of osteonecrosis of the femoral head.

Abstract P5-05-15: Activation of osteocytes connexin43 hemichannels suppresses breast cancer bone metastasis, a potential therapeutic target

Abstract Bone is a fertile place for metastasized breast cancer cell (BCC), however, mechanical loading is reported to inhibit BCC growth. Osteocytes, representing over 95% of bone cells, are mechanosensitive; they coordinate bone remodeling and modulate the extracellular environment. Mechanical loading activates osteocityc connexin 43 (Cx43) hemichannels, which mediates communication between the intracellular and extracellular space with message of molecules below 1 kDa. We have shown that ATP released by osteocytic hemichannels through the activation of BCC P2X7 receptor, inhibits BCC growth and migration. Here, we evaluate if activation of Cx43 hemichannels in osteocytes plays a critical role in inhibiting breast cancer bone metastasis. Our lab develops a monoclonal antibody, MHC2, which binds to extracellular domain of Cx43 and increases the activity of osteocytic hemichannels. This antibody was immune-reactive in HeLa cells transfected with Cx43 and osteocytes cell line MLO-Y4. It also labeled mouse osteocytes in situ. MHC2 antibody enhanced the activity of Cx43 hemichannels in cells and in vivo osteocytes and promoted the ATP release from osteocytic MLO-Y4 cells. Moreover, conditioned media collected from MHC2-treated osteocytes reduced BCC migration in vitro. The weekly injection of MHC2 reduced the breast cancer growth in bone in WT mice, but not in specific osteocyte Cx43 knockout mice. MHC2 antibody increase levels of lymphocytes T cytotoxic (CD3+/CD8+) and helper (CD3+/CD4+) in tumor and draining lymph nodes. Moreover, the weekly injection of MHC2, in intracardiac injected tumor cells model, in both immunocompetent and immune-compromised mice increased survival rate and reduced tumor spreading. As is known, osteocytic Cx43 hemichannels are permeable to ATP. We showed that P2X7 specific agonist benzoyl ATP reduced in vitro breast cancer cell migration, increased cytoplasmic free Ca2+ and induced formation of tubulin bundles, an indicator of cytoskeleton reorganization. The effect of benzoyl ATP required activation of CamKII in breast cancer cells. Our results suggest that Cx43 hemichannels are likely a potential therapeutic target in treating breast cancer growth and metastasizing to the bone, probably through the enhancement of the extracellular ATP level and the activation of P2X7/CamKII axis. Citation Format: Manuel A. Riquelme, Sumin Gu, Jeffery B. Chavez, Zhiqiang An, Jean X. Jiang. Activation of osteocytes connexin43 hemichannels suppresses breast cancer bone metastasis, a potential therapeutic target [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-15.