Calcitriol-Dependent and -Independent Regulation of Intestinal Calcium Absorption, Osteoblast Function, and Skeletal Mineralization during Lactation and Recovery in Mice.

Recovery from lactation-induced bone loss appears to be calcitriol-independent, since mice lacking 1-alpha-hydroxylase or vitamin D receptor (VDR) exhibit full skeletal recovery. However, in those studies mice consumed a calcium-, phosphorus-, and lactose-enriched "rescue" diet. Here we assessed whether postweaning skeletal recovery of Vdr null mice required that rescue diet. Wild type (WT) and Vdr null mice were raised on the rescue diet and switched to a normal (1% calcium) diet at Day 21 of lactation until 28 days after weaning. Unmated mice received the same regimen. In WT mice, cortical thickness was significantly reduced by 25% at 21 days of lactation and was completely restored by 28 days after weaning. Three-point bending tests similarly showed a significant reduction during lactation and full recovery of ultimate load and energy absorbed. Although Vdr null mice exhibited a similar lactational reduction in cortical thickness and mechanical strength, neither was even partially restored after weaning. Unmated mice showed no significant changes. In micro-computed tomography scans, diaphyses of Vdr null femora at 28 days after weaning were highly porous and exhibited abundant low-density bone extending into the marrow space from the endocortical surface. To quantify, we segregated bone into low-, mid-, and high-density components. In WT diaphyses, high-density bone was lost during lactation and restored after weaning. Vdr null mice also lost high-density bone during lactation but did not replace it; instead, they demonstrated a threefold increase in low-density bone mass. Histology revealed that intracortical and endocortical surfaces of Vdr null bones after weaning contained very thick (up to 20 micron) osteoid seams, covered with multiple layers of osteoblasts and precursors. We conclude that during the postweaning period, osteoblasts are potently stimulated to produce osteoid despite lacking VDRs, and that either calcitriol or a calcium-enriched diet are needed for this immature bone to become mineralized. © 2022 American Society for Bone and Mineral Research (ASBMR).

Leptin receptor in osteocytes promotes cortical bone consolidation in female mice.

Bone strength is partially determined during cortical bone consolidation, a process comprising coalescence of peripheral trabecular bone and its progressive mineralisation. Mice with genetic deletion of suppressor of cytokine signalling 3 (Socs3), an inhibitor of STAT3 signalling, exhibit delayed cortical bone consolidation, indicated by high cortical porosity, low mineral content, and low bone strength. Since leptin receptor (LepR) is expressed in the osteoblast lineage and is suppressed by SOCS3, we evaluated whether LepR deletion in osteocytes would rectify the Dmp1cre.Socs3fl/fl bone defect. First, we tested LepR deletion in osteocytes by generating Dmp1cre.LepRfl/fl mice and detected no significant bone phenotype. We then generated Dmp1cre.Socs3fl/fl.LepRfl/fl mice and compared them to Dmp1cre.Socs3fl/fl controls. Between 6 and 12 weeks of age, both Dmp1cre.Socs3fl/fl.LepRfl/fl and control (Dmp1cre.Socs3fl/fl) mice showed an increasing proportion of more heavily mineralised bone, indicating some cortical consolidation with time. However, at 12 weeks of age, rather than resolving the phenotype, delayed consolidation was extended in female Dmp1cre.Socs3fl/fl.LepRfl/fl mice. This was indicated in both metaphysis and diaphysis by greater proportions of low-density bone, lower proportions of high-density bone, and greater cortical porosity than Dmp1cre.Socs3fl/fl controls. There was also no change in the proportion of osteocytes staining positive for phospho-STAT3, suggesting the effect of LepR deletion in Dmp1cre.Socs3fl/fl mice is STAT3-independent. This identifies a new role for leptin signalling in bone which opposes our original hypothesis. Although LepR in osteocytes has no irreplaceable physiological role in normal bone maturation, when STAT3 is hyperactive, LepR in Dmp1Cre-expressing cells supports cortical consolidation.

G-CSF Receptor Deletion Amplifies Cortical Bone Dysfunction in Mice With STAT3 Hyperactivation in Osteocytes.

Bone strength is determined by the structure and composition of its thickened outer shell (cortical bone), yet the mechanisms controlling cortical consolidation are poorly understood. Cortical bone maturation depends on SOCS3-mediated suppression of IL-6 cytokine-induced STAT3 phosphorylation in osteocytes, the cellular network embedded in bone matrix. Because SOCS3 also suppresses granulocyte-colony-stimulating factor receptor (G-CSFR) signaling, we here tested whether global G-CSFR (Csf3r) ablation altereed bone structure in male and female mice lacking SOCS3 in osteocytes, (Dmp1Cre :Socs3f/f mice). Dmp1Cre :Socs3f/f :Csf3r-/- mice were generated by crossing Dmp1Cre :Socs3f/f mice with Csf3r-/- mice. Although G-CSFR is not expressed in osteocytes, Csf3r deletion further delayed cortical consolidation in Dmp1Cre :Socs3f/f mice. Micro-CT images revealed extensive, highly porous low-density bone, with little true cortex in the diaphysis, even at 26 weeks of age; including more low-density bone and less high-density bone in Dmp1Cre :Socs3f/f :Csf3r-/- mice than controls. By histology, the area where cortical bone would normally be found contained immature compressed trabecular bone in Dmp1Cre :Socs3f/f :Csf3r-/- mice and greater than normal levels of intracortical osteoclasts, extensive new woven bone formation, and the presence of more intracortical blood vessels than the already high levels observed in Dmp1Cre :Socs3f/f controls. qRT-PCR of cortical bone from Dmp1Cre :Socs3f/f :Csf3r-/- mice also showed more than a doubling of mRNA levels for osteoclasts, osteoblasts, RANKL, and angiogenesis markers. The further delay in cortical bone maturation was associated with significantly more phospho-STAT1 and phospho-STAT3-positive osteocytes, and a threefold increase in STAT1 and STAT3 target gene mRNA levels, suggesting G-CSFR deletion further increases STAT signaling beyond that of Dmp1Cre :Socs3f/f bone. G-CSFR deficiency therefore promotes STAT1/3 signaling in osteocytes, and when SOCS3 negative feedback is absent, elevated local angiogenesis, bone resorption, and bone formation delays cortical bone consolidation. This points to a critical role of G-CSF in replacing condensed trabecular bone with lamellar bone during cortical bone formation. © 2022 American Society for Bone and Mineral Research (ASBMR).

The effect of carbamazepine on bone structure and strength in control and osteogenesis imperfecta (Col1a2 +/p.G610C ) mice.

The inherited brittle bone disease osteogenesis imperfecta (OI) is commonly caused by COL1A1 and COL1A2 mutations that disrupt the collagen I triple helix. This causes intracellular endoplasmic reticulum (ER) retention of the misfolded collagen and can result in a pathological ER stress response. A therapeutic approach to reduce this toxic mutant load could be to stimulate mutant collagen degradation by manipulating autophagy and/or ER-associated degradation. Since carbamazepine (CBZ) both stimulates autophagy of misfolded collagen X and improves skeletal pathology in a metaphyseal chondrodysplasia model, we tested the effect of CBZ on bone structure and strength in 3-week-old male OI Col1a2 +/p.G610C and control mice. Treatment for 3 or 6 weeks with CBZ, at the dose effective in metaphyseal chondrodysplasia, provided no therapeutic benefit to Col1a2 +/p.G610C mouse bone structure, strength or composition, measured by micro-computed tomography, three point bending tests and Fourier-transform infrared microspectroscopy. In control mice, however, CBZ treatment for 6 weeks impaired femur growth and led to lower femoral cortical and trabecular bone mass. These data, showing the negative impact of CBZ treatment on the developing mouse bones, raise important issues which must be considered in any human clinical applications of CBZ in growing individuals.

STAT3 Hyperactivation Due to SOCS3 Deletion in Murine Osteocytes Accentuates Responses to Exercise- and Load-Induced Bone Formation.

Cortical bone develops and changes in response to mechanical load, which is sensed by bone-embedded osteocytes. The bone formation response to load depends on STAT3 intracellular signals, which are upregulated after loading and are subject to negative feedback from Suppressor of Cytokine Signaling 3 (Socs3). Mice with Dmp1Cre-targeted knockout of Socs3 have elevated STAT3 signaling in osteocytes and display delayed cortical bone maturation characterized by impaired accrual of high-density lamellar bone. This study aimed to determine whether these mice exhibit an altered response to mechanical load. The approach used was to test both treadmill running and tibial compression in female Dmp1Cre.Socs3f/f mice. Treadmill running for 5 days per week from 6 to 11 weeks of age did not change cortical bone mass in control mice, but further delayed cortical bone maturation in Dmp1Cre.Socs3f/f mice; accrual of high-density bone was suppressed, and cortical thickness was less than in genetically-matched sedentary controls. When strain-matched anabolic tibial loading was tested, both control and Dmp1Cre.Socs3f/f mice exhibited a significantly greater cortical thickness and periosteal perimeter in loaded tibia compared with the contralateral non-loaded bone. At the site of greatest compressive strain, the loaded Dmp1Cre.Socs3f/f tibias showed a significantly greater response than controls, indicated by a greater increase in cortical thickness. This was due to a greater bone formation response on both periosteal and endocortical surfaces, including formation of abundant woven bone on the periosteum. This suggests a greater sensitivity to mechanical load in Dmp1Cre.Socs3f/f bone. In summary, mice with targeted SOCS3 deletion and immature cortical bone have an exaggerated response to both physiological and experimental mechanical loads. We conclude that there is an optimal level of osteocytic response to mechanical load required for cortical bone maturation and that load-induced bone formation may be increased by augmenting STAT3 signaling within osteocytes. © 2021 American Society for Bone and Mineral Research (ASBMR).

Dmp1Cre-directed knockdown of parathyroid hormone-related protein (PTHrP) in murine decidua is associated with a life-long increase in bone mass, width, and strength in male progeny.

Parathyroid hormone-related protein (PTHrP, gene name Pthlh) is a pleiotropic regulator of tissue homeostasis. In bone, Dmp1Cre-targeted PTHrP deletion in osteocytes causes osteopenia and impaired cortical strength. We report here that this outcome depends on parental genotype. In contrast to our previous report using mice bred from heterozygous (flox/wild type) Dmp1Cre.Pthlhf/w parents, adult (16-week-old and 26-week-old) flox/flox (f/f) Dmp1Cre.Pthlhf/f mice from homozygous parents (Dmp1Cre.Pthlhf/f(hom) ) have stronger bones, with 40% more trabecular bone mass and 30% greater femoral width than controls. This greater bone size was observed in Dmp1Cre.Pthlhf/f(hom) mice as early as 12 days of age, when greater bone width was also found in male and female Dmp1Cre.Pthlhf/f(hom) mice compared to controls, but not in gene-matched mice from heterozygous parents. This suggested a maternal influence on skeletal size prior to weaning. Although Dmp1Cre has previously been reported to cause gene recombination in mammary gland, milk PTHrP protein levels were normal. The wide-bone phenotype was also noted in utero: Dmp1Cre.Pthlhf/f(hom) embryonic femurs were more mineralized and wider than controls. Closer examination revealed that Dmp1Cre caused PTHrP recombination in placenta, and in the maternal-derived decidual layer that resides between the placenta and the uterus. Decidua from mothers of Dmp1Cre.Pthlhf/f(hom) mice also exhibited lower PTHrP levels by immunohistochemistry and were smaller than controls. We conclude that Dmp1Cre leads to gene recombination in decidua, and that decidual PTHrP might, through an influence on decidual cells, limit embryonic bone radial growth. This suggests a maternal-derived developmental origin of adult bone strength. © 2021 American Society for Bone and Mineral Research (ASBMR).

Bone Geometry Is Altered by Follistatin-Induced Muscle Growth in Young Adult Male Mice.

The development of the musculoskeletal system and its maintenance depends on the reciprocal relationship between muscle and bone. The size of skeletal muscles and the forces generated during muscle contraction are potent sources of mechanical stress on the developing skeleton, and they shape bone structure during growth. This is particularly evident in hypermuscular global myostatin (Mstn)-null mice, where larger muscles during development increase bone mass and alter bone shape. However, whether muscle hypertrophy can similarly influence the shape of bones after the embryonic and prepubertal period is unknown. To address this issue, bone structure was assessed after inducing muscle hypertrophy in the lower hindlimbs of young-adult C57BL/6J male mice by administering intramuscular injections of recombinant adeno-associated viral vectors expressing follistatin (FST), a potent antagonist of Mstn. Two FST isoforms were used: the full-length 315 amino acid isoform (FST-315) and a truncated 288 amino acid isoform (FST-288). In both FST-treated cohorts, muscle hypertrophy was observed, and the anterior crest of the tibia, adjacent to the tibialis anterior muscle, was lengthened. Hypertrophy of the muscles surrounding the tibia caused the adjacent cortical shell to recede inward toward the central axis: an event driven by bone resorption adjacent to the hypertrophic muscle. The findings reveal that inducing muscle hypertrophy in mice can confer changes in bone shape in early adulthood. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Cortical bone maturation in mice requires SOCS3 suppression of gp130/STAT3 signalling in osteocytes.

Bone strength is determined by its dense cortical shell, generated by unknown mechanisms. Here we use the Dmp1Cre:Socs3f/f mouse, with delayed cortical bone consolidation, to characterise cortical maturation and identify control signals. We show that cortical maturation requires a reduction in cortical porosity, and a transition from low to high density bone, which continues even after cortical shape is established. Both processes were delayed in Dmp1Cre:Socs3f/f mice. SOCS3 (suppressor of cytokine signalling 3) inhibits signalling by leptin, G-CSF, and IL-6 family cytokines (gp130). In Dmp1Cre:Socs3f/f bone, STAT3 phosphorylation was prolonged in response to gp130-signalling cytokines, but not G-CSF or leptin. Deletion of gp130 in Dmp1Cre:Socs3f/f mice suppressed STAT3 phosphorylation in osteocytes and osteoclastic resorption within cortical bone, leading to rescue of the corticalisation defect, and restoration of compromised bone strength. We conclude that cortical bone development includes both pore closure and accumulation of high density bone, and that these processes require suppression of gp130-STAT3 signalling in osteocytes.

Testing Bone Formation Induction by Calvarial Injection Assay in vivo.

Bone formation occurs during embryogenesis, skeletal growth and during the process of skeletal renewal throughout life. In the process of bone formation, osteoblasts lay down a collagen-containing matrix, termed osteoid, which is gradually hardened by incorporation of mineral crystals. Although osteoblasts can be induced to differentiate and to deposit mineral in culture, this system does not always provide results that reflect the ability of agents to stimulate bone formation in vivo. This protocol describes a rapid and reliable method for testing local administration of agents on bone formation in vivo. In this method, mice are injected with the agent of question for 5 successive days. Fluorochrome labels are injected prior to, and after agents used for testing, and samples are collected and analysed by undecalcified bone histology and histomorphometry. This provides a robust method for assessing the ability of agents to stimulate bone formation, and if a short-term modification is used, can also be used for testing gene responses in bone to the same stimuli.

IL-6 exhibits both cis- and trans-signaling in osteocytes and osteoblasts, but only trans-signaling promotes bone formation and osteoclastogenesis.

Interleukin 6 (IL-6) supports development of bone-resorbing osteoclasts by acting early in the osteoblast lineage via membrane-bound (cis) or soluble (trans) receptors. Here, we investigated how IL-6 signals and modifies gene expression in differentiated osteoblasts and osteocytes and determined whether these activities can promote bone formation or support osteoclastogenesis. Moreover, we used a genetically altered mouse with circulating levels of the pharmacological IL-6 trans-signaling inhibitor sgp130-Fc to determine whether IL-6 trans-signaling is required for normal bone growth and remodeling. We found that IL-6 increases suppressor of cytokine signaling 3 (Socs3) and CCAAT enhancer-binding protein δ (Cebpd) mRNA levels and promotes signal transducer and activator of transcription 3 (STAT3) phosphorylation by both cis- and trans-signaling in cultured osteocytes. In contrast, RANKL (Tnfsf11) mRNA levels were elevated only by trans-signaling. Furthermore, we observed soluble IL-6 receptor release and ADAM metallopeptidase domain 17 (ADAM17) sheddase expression by osteocytes. Despite the observation that IL-6 cis-signaling occurs, IL-6 stimulated bone formation in vivo only via trans-signaling. Although IL-6 stimulated RANKL (Tnfsf11) mRNA in osteocytes, these cells did not support osteoclast formation in response to IL-6 alone; binucleated TRAP+ cells formed, and only in response to trans-signaling. Finally, pharmacological, sgp130-Fc-mediated inhibition of IL-6 trans-signaling did not impair bone growth or remodeling unless mice had circulating sgp130-Fc levels > 10 µg/ml. At those levels, osteopenia and impaired bone growth occurred, reducing bone strength. We conclude that high sgp130-Fc levels may have detrimental off-target effects on the skeleton.

IL-6 trans-signalling mediates trabecular, but not cortical, bone loss after ovariectomy.

Bone loss associated with estrogen deficiency occurs due to a high level of bone remodelling, with a greater increase in the level of osteoclast-mediated bone resorption than osteoblast-mediated bone formation. Early studies showed that Interleukin-6 (IL-6) inhibition could prevent the increase in osteoclast numbers associated with ovariectomy. However, IL-6 signals through two possible pathways: classic IL-6 signalling (cis) utilizes a membrane-bound IL-6 receptor (IL-6R), while IL-6 trans-signalling occurs through a soluble IL-6R (sIL-6R). It is not known which of these pathways mediates the bone loss after ovariectomy. We therefore sought to determine whether specific pharmacological inhibition of IL-6 trans-signalling could prevent ovariectomy-induced bone loss in mice. We report that IL-6 trans-signalling inhibition prevented the increase in osteoclasts, and trabecular bone loss, associated with ovariectomy. IL-6 trans-signalling inhibition also reduced bone formation rate, but did not prevent the increase in osteoblast numbers. In contrast, cortical bone loss was not prevented by any IL-6 signalling inhibitor. This suggests that local production of sIL-6R mediates trabecular bone loss in estrogen deficiency, but the increased cortical bone resorption that leads to marrow expansion is independent of IL-6 signalling.

Autocrine and Paracrine Regulation of the Murine Skeleton by Osteocyte-Derived Parathyroid Hormone-Related Protein.

Parathyroid hormone-related protein (PTHrP) and parathyroid hormone (PTH) have N-terminal domains that bind a common receptor, PTHR1. N-terminal PTH (teriparatide) and now a modified N-terminal PTHrP (abaloparatide) are US Food and Drug Administration (FDA)-approved therapies for osteoporosis. In physiology, PTHrP does not normally circulate at significant levels, but acts locally, and osteocytes, cells residing within the bone matrix, express both PTHrP and the PTHR1. Because PTHR1 in osteocytes is required for normal bone resorption, we determined how osteocyte-derived PTHrP influences the skeleton. We observed that adult mice with low PTHrP in osteocytes (targeted with the Dmp1(10kb)-Cre) have low trabecular bone volume and osteoblast numbers, but osteoclast numbers were unaffected. In addition, bone size was normal, but cortical bone strength was impaired. Osteocyte-derived PTHrP therefore stimulates bone formation and bone matrix strength, but is not required for normal osteoclastogenesis. PTHrP knockdown and overexpression studies in cultured osteocytes indicate that osteocyte-secreted PTHrP regulates their expression of genes involved in matrix mineralization. We determined that osteocytes secrete full-length PTHrP with no evidence for secretion of lower molecular weight forms containing the N-terminus. We conclude that osteocyte-derived full-length PTHrP acts through both PTHR1 receptor-mediated and receptor-independent actions in a paracrine/autocrine manner to stimulate bone formation and to modify adult cortical bone strength. © 2017 American Society for Bone and Mineral Research.

Absence of Calcitriol Causes Increased Lactational Bone Loss and Lower Milk Calcium but Does Not Impair Post-lactation Bone Recovery in Cyp27b1 Null Mice.

We hypothesized that adaptation to calcium supply demands of pregnancy and lactation do not require calcitriol. Adult Cyp27b1 null mice lack calcitriol and have hypocalcemia, hypophosphatemia, and rickets. We studied wild-type (WT) and null sister pairs raised on a calcium-, phosphorus-, and lactose-enriched "rescue" diet that prevents hypocalcemia and rickets. Bone mineral content (BMC) increased >30% in pregnant nulls, declined 30% during lactation, and increased 30% by 4 weeks post-weaning. WT showed less marked changes. Micro-CT revealed loss of trabecular bone and recovery in both genotypes. In lactating nulls, femoral cortical thickness declined >30%, whereas endocortical perimeter increased; both recovered to baseline after weaning; there were no such changes in WT. Histomorphometry revealed a profound increase in osteoid surface and thickness in lactating nulls, which recovered after weaning. By three-point bend test, nulls had a >50% decline in ultimate load to failure that recovered after weaning. Although nulls showed bone loss during lactation, their milk calcium content was 30% lower compared with WT. Serum parathyroid hormone (PTH) was markedly elevated in nulls at baseline, reduced substantially in pregnancy, but increased again during lactation and remained high post-weaning. In summary, pregnant Cyp27b1 nulls gained BMC with reduced secondary hyperparathyroidism, implying increased intestinal calcium delivery. Lactating nulls lost more bone mass and strength than WT, accompanied by increased osteoid, reduced milk calcium, and worsened secondary hyperparathyroidism. This implies suboptimal intestinal calcium absorption. Post-weaning, bone mass and strength recovered to baseline, whereas BMC exceeded baseline by 40%. In conclusion, calcitriol-independent mechanisms regulate intestinal calcium absorption and trabecular bone metabolism during pregnancy and post-weaning but not during lactation; calcitriol may protect cortical bone during lactation. © 2017 American Society for Bone and Mineral Research.

Osteoblasts Are Rapidly Ablated by Virus-Induced Systemic Inflammation following Lymphocytic Choriomeningitis Virus or Pneumonia Virus of Mice Infection in Mice.

A link between inflammatory disease and bone loss is now recognized. However, limited data exist on the impact of virus infection on bone loss and regeneration. Bone loss results from an imbalance in remodeling, the physiological process whereby the skeleton undergoes continual cycles of formation and resorption. The specific molecular and cellular mechanisms linking virus-induced inflammation to bone loss remain unclear. In the current study, we provide evidence that infection of mice with either lymphocytic choriomeningitis virus (LCMV) or pneumonia virus of mice (PVM) resulted in rapid and substantial loss of osteoblasts from the bone surface. Osteoblast ablation was associated with elevated levels of circulating inflammatory cytokines, including TNF-α, IFN-γ, IL-6, and CCL2. Both LCMV and PVM infections resulted in reduced osteoblast-specific gene expression in bone, loss of osteoblasts, and reduced serum markers of bone formation, including osteocalcin and procollagen type 1 N propeptide. Infection of Rag-1-deficient mice (which lack adaptive immune cells) or specific depletion of CD8+ T lymphocytes limited osteoblast loss associated with LCMV infection. By contrast, CD8+ T cell depletion had no apparent impact on osteoblast ablation in association with PVM infection. In summary, our data demonstrate dramatic loss of osteoblasts in response to virus infection and associated systemic inflammation. Further, the inflammatory mechanisms mediating viral infection-induced bone loss depend on the specific inflammatory condition.

Bone corticalization requires local SOCS3 activity and is promoted by androgen action via interleukin-6.

Long bone strength is determined by its outer shell (cortical bone), which forms by coalescence of thin trabeculae at the metaphysis (corticalization), but the factors that control this process are unknown. Here we show that SOCS3-dependent cytokine expression regulates bone corticalization. Young male and female Dmp1Cre.Socs3 f/f mice, in which SOCS3 has been ablated in osteocytes, have high trabecular bone volume and poorly defined metaphyseal cortices. After puberty, male mice recover, but female corticalization is still impaired, leading to a lasting defect in bone strength. The phenotype depends on sex-steroid hormones: dihydrotestosterone treatment of gonadectomized female Dmp1Cre.Socs3 f/f mice restores normal cortical morphology, whereas in males, estradiol treatment, or IL-6 deletion, recapitulates the female phenotype. This suggests that androgen action promotes metaphyseal corticalization, at least in part, via IL-6 signaling.The strength of long bones is determined by coalescence of trabeculae during corticalization. Here the authors show that this process is regulated by SOCS3 via a mechanism dependent on IL-6 and expression of sex hormones.

Murine Oncostatin M Acts via Leukemia Inhibitory Factor Receptor to Phosphorylate Signal Transducer and Activator of Transcription 3 (STAT3) but Not STAT1, an Effect That Protects Bone Mass.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) are IL-6 family members with a wide range of biological functions. Human OSM (hOSM) and murine LIF (mLIF) act in mouse cells via a LIF receptor (LIFR)-glycoprotein 130 (gp130) heterodimer. In contrast, murine OSM (mOSM) signals mainly via an OSM receptor (OSMR)-gp130 heterodimer and binds with only very low affinity to mLIFR. hOSM and mLIF stimulate bone remodeling by both reducing osteocytic sclerostin and up-regulating the pro-osteoclastic factor receptor activator of NF-κB ligand (RANKL) in osteoblasts. In the absence of OSMR, mOSM still strongly suppressed sclerostin and stimulated bone formation but did not induce RANKL, suggesting that intracellular signaling activated by the low affinity interaction of mOSM with mLIFR is different from the downstream effects when mLIF or hOSM interacts with the same receptor. Both STAT1 and STAT3 were activated by mOSM in wild type cells or by mLIF/hOSM in wild type and Osmr-/- cells. In contrast, in Osmr-/- primary osteocyte-like cells stimulated with mOSM (therefore acting through mLIFR), microarray expression profiling and Western blotting analysis identified preferential phosphorylation of STAT3 and induction of its target genes but not of STAT1 and its target genes; this correlated with reduced phosphorylation of both gp130 and LIFR. In a mouse model of spontaneous osteopenia caused by hyperactivation of STAT1/3 signaling downstream of gp130 (gp130Y757F/Y757F), STAT1 deletion rescued the osteopenic phenotype, indicating a beneficial effect of promoting STAT3 signaling over STAT1 downstream of gp130 in this low bone mass condition, and this may have therapeutic value.

Prepubertal Di-n-Butyl Phthalate Exposure Alters Sertoli and Leydig Cell Function and Lowers Bone Density in Adult Male Mice.

Phthalate exposure impairs testis development and function; however, whether phthalates affect nonreproductive functions is not well understood. To investigate this, C57BL/6J mice were fed 1-500 mg di-n-butyl phthalate (DBP) in corn oil, or vehicle only, daily from 4 to 14 days, after which tissues were collected (prepubertal study). Another group was fed 1-500 mg/kg·d DBP from 4 to 21 days and then maintained untreated until 8 weeks for determination of adult consequences of prepubertal exposure. Bones were assessed by microcomputed tomography and dual-energy X-ray absorptiometry and T by RIA. DBP exposure decreased prepubertal femur length, marrow volume, and mean moment of inertia. Adult animals exposed prepubertally to low DBP doses had lower bone mineral content and bone mineral density and less lean tissue mass than vehicle-treated animals. Altered dynamics of the emerging Leydig population were found in 14-day-old animals fed 100-500 mg/kg·d DBP. Adult mice had variable testicular T and serum T and LH concentrations after prepubertal exposure and a dose-dependent reduction in cytochrome p450, family 11, subfamily A, polypeptide 1. Insulin-like 3 was detected in Sertoli cells of adult mice administered the highest dose of 500 mg/kg·d DBP prepubertally, a finding supported by the induction of insulin-like 3 expression in TM4 cells exposed to 50 µM, but not 5 µM, DBP. We propose that low-dose DBP exposure is detrimental to bone but that normal bone mineral density/bone mineral content after high-dose DBP exposure reflects changes in testicular somatic cells that confer protection to bones. These findings will fuel concerns that low-dose DBP exposure impacts health beyond the reproductive axis.

Chondrocytic ephrin B2 promotes cartilage destruction by osteoclasts in endochondral ossification.

The majority of the skeleton arises by endochondral ossification, whereby cartilaginous templates expand and are resorbed by osteoclasts then replaced by osteoblastic bone formation. Ephrin B2 is a receptor tyrosine kinase expressed by osteoblasts and growth plate chondrocytes that promotes osteoblast differentiation and inhibits osteoclast formation. We investigated the role of ephrin B2 in endochondral ossification using Osx1Cre-targeted gene deletion. Neonatal Osx1Cre.Efnb2(Δ/Δ) mice exhibited a transient osteopetrosis demonstrated by increased trabecular bone volume with a high content of growth plate cartilage remnants and increased cortical thickness, but normal osteoclast numbers within the primary spongiosa. Osteoclasts at the growth plate had an abnormal morphology and expressed low levels of tartrate-resistant acid phosphatase; this was not observed in more mature bone. Electron microscopy revealed a lack of sealing zones and poor attachment of Osx1Cre.Efnb2(Δ/Δ) osteoclasts to growth plate cartilage. Osteoblasts at the growth plate were also poorly attached and impaired in their ability to deposit osteoid. By 6 months of age, trabecular bone mass, osteoclast morphology and osteoid deposition by Osx1Cre.Efnb2(Δ/Δ) osteoblasts were normal. Cultured chondrocytes from Osx1Cre.Efnb2(Δ/Δ) neonates showed impaired support of osteoclastogenesis but no significant change in Rankl (Tnfsf11) levels, whereas Adamts4 levels were significantly reduced. A population of ADAMTS4(+) early hypertrophic chondrocytes seen in controls was absent from Osx1Cre.Efnb2(Δ/Δ) neonates. This suggests that Osx1Cre-expressing cells, including hypertrophic chondrocytes, are dependent on ephrin B2 for their production of cartilage-degrading enzymes, including ADAMTS4, and this might be required for attachment of osteoclasts and osteoblasts to the cartilage surface during endochondral ossification.

Loss of Gsα in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy.

Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including Gsα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of Gsα in the osteoblast lineage. Postnatal removal of Gsα in the osteoblast lineage (P-Gsα(OsxKO) mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1-34) (80 µg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-Gsα(OsxKO) mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-Gsα(OsxKO) mice. In mice that express a mutated PTH1R that activates adenylyl cyclase and protein kinase A (PKA) via Gsα but not phospholipase C via Gq/11 (D/D mice), PTH significantly enhanced bone formation, indicating that phospholipase C activation is not required for increased bone turnover in response to PTH. Therefore, although the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of Gsα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond Gsα and Gq/11 act downstream of PTH on osteoblast differentiation.