Direct activation of PI3K in osteoblasts and osteocytes strengthens murine bone through sex-specific actions on cortical surfaces.

Intracellular phosphoinositide 3-kinase (PI3K) signaling is activated by multiple bone-active receptors. Genetic mutations activating PI3K signaling are associated with clinical syndromes of tissue overgrowth in multiple organs, often including the skeleton. While one formation is increased by removing the PI3K inhibitor (phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)), the effect of direct PI3K activation in the osteoblast lineage has not been reported. We introduced a known gain-of-function mutation in Pik3ca, the gene encoding the p110α catalytic subunit of PI3K, in osteocytes and late osteoblasts using the dentin matrix protein-1 Cre (Dmp1Cre) mouse and assessed the skeletal phenotype. Femur shape was grossly normal, but cortical thickness was significantly greater in both male and female Dmp1Cre.Pik3caH1047R mice, leading to almost doubled bone strength at 12 wk of age. Both sexes had smaller marrow areas from 6 wk of age. Female mice also exhibited greater cross-sectional area, which continued to increase until 24 wk of age, resulting in a further increase in bone strength. Although both male and female mice had increased endocortical mineralizing surface, only female mice had increased periosteal mineralizing surface. The bone formed in the Dmp1Cre.Pik3caH1047R mice showed no increase in intracortical remodeling nor any defect in cortical bone consolidation. In contrast, on both endocortical and periosteal surfaces, there was more lamellar bone formation, including highly organized osteocyte networks extending along the entire surface at a greater thickness than in control mice. In conclusion, direct activation of PI3Kα in cells targeted by Dmp1Cre leads to high cortical bone mass and strength with abundant lamellar cortical bone in female and male mice with no increase in intracortical remodeling. This differs from the effect of PTEN deletion in the same cells, suggesting that activating PI3Kα in osteoblasts and osteocytes may be a more suitable target to promote formation of lamellar bone.

Patients with genetic activation of enzymes called phosphoinositide-3 kinase (PI3K) have tissue overgrowth syndromes, where parts of the body become enlarged, sometimes including the skeleton. There are 2 types of mutations that cause this: one that directly activates the PI3K enzyme, and one that removes the normal brake on PI3K signaling (called PTEN). We tested the effect of directly activating a PI3K enzyme specifically in osteoblasts (the cells that form bone) and osteocytes (osteoblasts that make a network inside the bone tissue itself). We found that mice with these mutations had very strong bones with an outer shell that was thicker than usual. In both male and female mice, it became thicker on the inside of the shell, but in female mice it also became thicker on the outside, making the bones even stronger over time. The new bone was well-organized, which likely helped make the increase in bone strength so profound. This is very different to previous studies of mice with the other type of mutation in their bone-forming cells; they had a shell with many large holes (pores). This indicates that directly stimulating PI3K enzyme is more beneficial for bone than removing the PTEN brake.

Increasing muscle contractility through low-frequency stimulation alters tibial bone geometry and reduces bone strength in mdx and dko dystrophic mice.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by mutations or deletions in the dystrophin gene, for which there remains no cure. As DMD patients also develop bone fragility because of muscle weakness and immobilization, better understanding of the pathophysiological mechanisms of dystrophin deficiency will help develop therapies to improve musculoskeletal health. Since alterations in muscle phenotype can influence bone structure, we investigated whether modifying muscle contractile activity through low-frequency stimulation (LFS) could alter bone architecture in mouse models of DMD. We tested the hypothesis that increasing muscle contractile activity could influence bone mass and structure in dystrophin-deficient (mdx) and dystrophin- and utrophin-deficient (dko) dystrophic mice. Tibial bone structure in dko mice was significantly different from that in mdx and wild-type (C57BL/10) control mice. Effects of LFS on bone architecture differed between dystrophic and healthy mice, with LFS thinning cortical bone in both dystrophic models. Bone mass was maintained in LFS-treated healthy mice, with a reduced proportion of high-density bone and concomitant increase in low-density bone. LFS-treated dko mice exhibited a net deficit in cortical thickness and reduced high-density bone but no equivalent increase in low-density bone. These alterations in bone structure and mineral density reduced mechanical strength in mdx and dko mice. The findings reveal that muscle activity can regulate bone mass, structure, mineral accrual, and strength, especially in the context of dystrophin and/or utrophin deficiency. The results provide unique insights into the development of bone fragility in DMD and for devising interventions to improve musculoskeletal health.NEW & NOTEWORTHY Patients with Duchenne muscular dystrophy (DMD) develop bone fragility because of muscle weakness and immobilization. We investigated whether increasing muscle contractile activity through low-frequency stimulation (LFS) could alter bone architecture in dystrophin-deficient (mdx) or dystrophin- and utrophin-deficient (dko) mouse models of DMD. Chronic LFS reduced tibial diaphysis cross sections in mdx and dko mice, without affecting bone shape in healthy mice. LFS affected the distribution of bone mineral density across all phenotypes, with the magnitude of effect being dependent on disease severity.

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).

Similarities and differences between IL11 and IL11RA1 knockout mice for lung fibro-inflammation, fertility and craniosynostosis.

Loss of function (LOF) in IL11RA infers IL11 signaling as important for fertility, fibrosis, inflammation and incompletely penetrant craniosynostosis. The impact of LOF in IL11 has not been characterized. We generated IL11 knockout (Il11-/-) mice that are born in expected ratios and have normal hematological profiles. Lung fibroblasts from Il11-/- mice are resistant to pro-fibrotic stimulation with TGFß1. Following bleomycin-induced lung injury, Il11-/- mice are protected from pulmonary fibrosis and exhibit lesser ERK, STAT3 and NF-kB activation, reduced Il1b, Timp1, Ccl2 and diminished IL6 expression, both at baseline and after injury: placing Il11 activity upstream of IL6 in this model. Il11-/- female mice are infertile. Unlike Il11ra1-/- mice, Il11-/- mice do not have craniosynostosis, have normal long bone mass and reduced body weights. These data further establish the role of IL11 signaling in lung fibrosis while suggesting that bone development abnormalities can be associated with mutation of IL11RA but not IL11, which may have implications for therapeutic targeting of IL11 signaling.

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.

Measuring Bone Volume at Multiple Densities by Micro-computed Tomography.

Bone strength is controlled by both bone mass, and the organization and quality of the bone material. The current standard method for measuring bone mass in mouse and rat studies is micro-computed tomography. This method typically uses a single threshold to identify bone material in the cortical and trabecular regions. However, this single threshold method obscures information about the mineral content of the bone material and depends on normal morphology to separately analyze cortical and trabecular structures. To extend this method to identify bone mass at multiple density levels, we have established a protocol for unbiased selection and application of multiple thresholds using a standard laboratory-based micro-computed tomography instrument. This non-invasive method can be applied to longitudinal studies and archived samples and provides additional information about bone structure and strength.

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.

Osteopontin is An Important Regulative Component of the Fetal Bone Marrow Hematopoietic Stem Cell Niche.

Osteopontin (OPN) is an important component in both bone and blood regulation, functioning as a bridge between the two. Previously, thrombin-cleaved osteopontin (trOPN), the dominant form of OPN in adult bone marrow (BM), was demonstrated to be a critical negative regulator of adult hematopoietic stem cells (HSC) via interactions with α4ß1 and α9ß1 integrins. We now demonstrate OPN is also required for fetal hematopoiesis in maintaining the HSC and progenitor pool in fetal BM. Specifically, we showed that trOPN is highly expressed in fetal BM and its receptors, α4ß1 and α9ß1 integrins, are both highly expressed and endogenously activated on fetal BM HSC and progenitors. Notably, the endogenous activation of integrins expressed by HSC was attributed to high concentrations of three divalent metal cations, Ca2+, Mg2+ and Mn2+, which were highly prevalent in developing fetal BM. In contrast, minimal levels of OPN were detected in fetal liver, and α4ß1 and α9ß1 integrins expressed by fetal liver HSC were not in the activated state, thereby permitting the massive expansion of HSC and progenitors required during early fetal hematopoiesis. Consistent with these results, no differences in the number or composition of hematopoietic cells in the liver of fetal OPN-/- mice were detected, but significant increases in the hematopoietic progenitor pool in fetal BM as well as an increase in the BM HSC pool following birth and into adulthood were observed. Together, the data demonstrates OPN is a necessary negative regulator of fetal and neonatal BM progenitors and HSC, and it exhibits preserved regulatory roles during early development, adulthood and ageing.

Dynll1 is essential for development and promotes endochondral bone formation by regulating intraflagellar dynein function in primary cilia.

Mutations in subunits of the cilia-specific cytoplasmic dynein-2 (CD2) complex cause short-rib thoracic dystrophy syndromes (SRTDs), characterized by impaired bone growth and life-threatening perinatal respiratory complications. Different SRTD mutations result in varying disease severities. It remains unresolved whether this reflects the extent of retained hypomorphic protein functions or relative importance of the affected subunits for the activity of the CD2 holoenzyme. To define the contribution of the LC8-type dynein light chain subunit to the CD2 complex, we have generated Dynll1-deficient mouse strains, including the first-ever conditional knockout (KO) mutant for any CD2 subunit. Germline Dynll1 KO mice exhibit a severe ciliopathy-like phenotype similar to mice lacking another CD2 subunit, Dync2li1. Limb mesoderm-specific loss of Dynll1 results in severe bone shortening similar to human SRTD patients. Mechanistically, loss of Dynll1 leads to a partial depletion of other SRTD-related CD2 subunits, severely impaired retrograde intra-flagellar transport, significant thickening of primary cilia and cilia signaling defects. Interestingly, phenotypes of Dynll1-deficient mice are very similar to entirely cilia-deficient Kif3a/Ift88-null mice, except that they never present with polydactyly and retain relatively higher signaling outputs in parts of the hedgehog pathway. Compared to complete loss of Dynll1, maintaining very low DYNLL1 levels in mice lacking the Dynll1-transcription factor ASCIZ (ATMIN) results in significantly attenuated phenotypes and improved CD2 protein levels. The results suggest that primary cilia can maintain some functionality in the absence of intact CD2 complexes and provide a viable animal model for the analysis of the underlying bone development defects of SRTDs.

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.

Granulocyte-CSF links destructive inflammation and comorbidities in obstructive lung disease.

Chronic obstructive pulmonary disease (COPD) is an incurable inflammatory lung disease that afflicts millions of people worldwide, and it is the fourth leading cause of death. Systemic comorbidities affecting the heart, skeletal muscle, bone, and metabolism are major contributors to morbidity and mortality. Given the surprising finding in large prospective clinical biomarker studies that peripheral white blood cell count is more closely associated with disease than inflammatory biomarkers, we probed the role of blood growth factors. Using the SHIP-1-deficient COPD mouse model, which manifests a syndrome of destructive lung disease and a complex of comorbid pathologies, we have identified a critical and unexpected role for granulocyte-CSF (G-CSF) in linking these conditions. Deletion of G-CSF greatly reduced airway inflammation and lung tissue destruction, and attenuated systemic inflammation, right heart hypertrophy, loss of fat reserves, and bone osteoporosis. In human clinical translational studies, bronchoalveolar lavage fluid of patients with COPD demonstrated elevated G-CSF levels. These studies suggest that G-CSF may play a central and unforeseen pathogenic role in COPD and its complex comorbidities, and identify G-CSF and its regulators as potential therapeutic targets.

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.

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.