Inhibition of IL-11 signalling extends mammalian healthspan and lifespan.

For healthspan and lifespan, ERK, AMPK and mTORC1 represent critical pathways and inflammation is a centrally important hallmark1-7. Here we examined whether IL-11, a pro-inflammatory cytokine of the IL-6 family, has a negative effect on age-associated disease and lifespan. As mice age, IL-11 is upregulated across cell types and tissues to regulate an ERK-AMPK-mTORC1 axis to modulate cellular, tissue- and organismal-level ageing pathologies. Deletion of Il11 or Il11ra1 protects against metabolic decline, multi-morbidity and frailty in old age. Administration of anti-IL-11 to 75-week-old mice for 25 weeks improves metabolism and muscle function, and reduces ageing biomarkers and frailty across sexes. In lifespan studies, genetic deletion of Il11 extended the lives of mice of both sexes, by 24.9% on average. Treatment with anti-IL-11 from 75 weeks of age until death extends the median lifespan of male mice by 22.5% and of female mice by 25%. Together, these results demonstrate a role for the pro-inflammatory factor IL-11 in mammalian healthspan and lifespan. We suggest that anti-IL-11 therapy, which is currently in early-stage clinical trials for fibrotic lung disease, may provide a translational opportunity to determine the effects of IL-11 inhibition on ageing pathologies in older people.

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.

Osteoclast-derived coupling factors: origins and state-of-play Louis V Avioli Lecture, ASBMR 2023.

Coupling, the mechanism that controls the sequence of events in bone remodelling, is a fundamental theory for understanding the way the skeleton changes throughout life. This review is an adapted version of the Louis V Avioli lecture, delivered at the Annual Scientific Meeting of the American Society of Bone and Mineral Research. It outlines the history of the coupling concept and details how coupling occurs within trabecular and cortical bone and describes its multiple contexts and the many mechanisms suggested to couple bone forming osteoblasts to the prior action of osteoclasts on the same bone surface. These mechanisms include signals produced at each stage of the remodelling sequence (resorption, reversal, and formation), such as factors released by osteoclasts through their resorptive action and through protein synthesis, molecules deposited in the cement line during the reversal phase, and potentially signals from osteocytes within the local bone environment. The review highlights two examples of coupling factors (Cardiotrophin 1 and EphrinB2:EphB4) to illustrate the limited data available, and the need to integrate both the many functions of these factors within the basic multicellular unit (BMU), and the multiple origins of these factors, including other cell types present during the remodelling sequence (such as osteocytes, macrophages, endothelial cells, and T-cells).

Coupling is a fundamental process by which bone resorbing cells (osteoclasts) are followed by bone forming cells (osteoblasts) on the same surface during the process of bone remodelling. This review outlines the history, basic concepts, and mechanisms proposed, and suggests directions for further research into the way this sequence of events in controlled in bone maintenance, development, and healing.

Fetal growth delay caused by loss of non-canonical imprinting is resolved late in pregnancy and culminates in offspring overgrowth.

Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to growth and developmental changes in mouse offspring. In this study, we show that offspring from mouse oocytes lacking the PRC2 protein Embryonic Ectoderm Development (EED) were initially developmentally delayed, characterised by low blastocyst cell counts and substantial growth delay in mid-gestation embryos. This initial developmental delay was resolved as offspring underwent accelerated fetal development and growth in late gestation resulting in offspring that were similar stage and weight to controls at birth. The accelerated development and growth in offspring from Eed-null oocytes was associated with remodelling of the placenta, which involved an increase in fetal and maternal tissue size, conspicuous expansion of the glycogen-enriched cell population, and delayed parturition. Despite placental remodelling and accelerated offspring fetal growth and development, placental efficiency, and fetal blood glucose levels were low, and the fetal blood metabolome was unchanged. Moreover, while expression of the H3K27me3-imprinted gene and amino acid transporter Slc38a4 was increased, fetal blood levels of individual amino acids were similar to controls, indicating that placental amino acid transport was not enhanced. Genome-wide analyses identified extensive transcriptional dysregulation and DNA methylation changes in affected placentas, including a range of imprinted and non-imprinted genes. Together, while deletion of Eed in growing oocytes resulted in fetal growth and developmental delay and placental hyperplasia, our data indicate a remarkable capacity for offspring fetal growth to be normalised despite inefficient placental function and the loss of H3K27me3-dependent genomic imprinting.

Loss of maternal calcitriol reversibly alters early offspring growth and skeletal development in mice.

Ablation of Cyp27b1 eliminates calcitriol but does not disturb fetal mineral homeostasis or skeletal development. However, independent of fetal genotypes, maternal loss of Cyp27b1 altered fetal mineral and hormonal levels compared to offspring of WT dams. We hypothesized that these maternal influences would alter postnatal skeletal development. Cyp27b1 null and WT females were mated to bear only Cyp27b1+/- offspring. Forty-eight hours after birth, pups were cross-fostered to dams of the same or opposite genotype that bore them. Maternal and offspring samples were collected on days 21 (weaning) and 42. Offspring measurements included minerals and hormones, BMC by DXA, ash weight and mineral content, gene expression, 3-point bending tests, and microCT. Maternal lactational behavior was evaluated. Milk was analyzed for nutritional content. At day 21, offspring fostered by nulls, independent of birth dam, had ~20% lower weight, BMC, ash weight, and ash calcium than pups fostered by WT dams. Adjustment for body weight accounted for the lower BMC but not the lower ash weight and ash calcium. Hormones and serum/urine minerals did not differ across offspring groups. Offspring fostered by nulls had shorter femurs and lower cortical thickness, mean polar moment of inertia, cortical area, trabecular bone volume, and trabecular number. Dam lactational behaviors and milk nutritional content did not differ between groups. At day 42, body weight, ash weight, lengths, BMC, and tibial bone strength were no longer different between pups fostered by null vs WT dams. In summary, pups fostered by Cyp27b1 nulls, regardless of birth dam, have proportionately smaller skeletons at 21 d, impaired microstructure, but normal mineral homeostasis. The skeletal effects are largely recovered by day 42 (3 wk after weaning). In conclusion, maternal loss of calcitriol impairs early postnatal cortical bone growth and trabecular bone mass, but affected offspring catch up after weaning.

Severe Osteoporosis With Pathogenic LRP5 Variant.

A 24-year-old female patient was diagnosed with osteoporosis after presenting with numerous fractures throughout her childhood and adolescence. Risk factors included chronic constipation, severe vitamin D deficiency, and long-term high-dose steroid use for severe eczema. Metabolic bone disorder clinical exome screening (limited panel of metabolic bone disorders and gastrointestinal disorders) was undertaken and revealed a class 4 likely pathogenic variant in the LRP5 gene known to cause osteoporosis. Optimal treatment for patients with this variant is not well defined. A literature review of the condition and potential treatment options is discussed.

Craniosynostosis-associated variants in the IL-11R complex: new insights and questions.

Skull growth involves the expansion of both the flat calvarial bones of the skull and the fibrous marginal zones, termed sutures, between them. This process depends on co-ordinated proliferation of mesenchymal-derived progenitor cells within the sutures, and their differentiation to osteoblasts which produce the bone matrix required to expand the size of the bony plates. Defects lead to premature closure of these sutures, termed craniosynostosis, resulting in heterogeneous head shape differences due to restricted growth of one or more sutures. The impact on the individual depends on how many and which sutures are affected and the severity of the effect. Several genetic loci are responsible, including a wide range of variants in the gene for the interleukin 11 receptor (IL11RA, OMIM#600939). Recent work from Kespohl and colleagues provides new insights into how some of these variants influence IL-11R function; we discuss their influences on IL-11R structure and IL-11 function as a stimulus of osteoblast differentiation.

Preclinical Rodent Models for Human Bone Disease, Including a Focus on Cortical Bone.

Preclinical models (typically ovariectomized rats and genetically altered mice) have underpinned much of what we know about skeletal biology. They have been pivotal for developing therapies for osteoporosis and monogenic skeletal conditions, including osteogenesis imperfecta, achondroplasia, hypophosphatasia, and craniodysplasias. Further therapeutic advances, particularly to improve cortical strength, require improved understanding and more rigorous use and reporting. We describe here how trabecular and cortical bone structure develop, are maintained, and degenerate with aging in mice, rats, and humans, and how cortical bone structure is changed in some preclinical models of endocrine conditions (eg, postmenopausal osteoporosis, chronic kidney disease, hyperparathyroidism, diabetes). We provide examples of preclinical models used to identify and test current therapies for osteoporosis, and discuss common concerns raised when comparing rodent preclinical models to the human skeleton. We focus especially on cortical bone, because it differs between small and larger mammals in its organizational structure. We discuss mechanisms common to mouse and human controlling cortical bone strength and structure, including recent examples revealing genetic contributors to cortical porosity and osteocyte network configurations during growth, maturity, and aging. We conclude with guidelines for clear reporting on mouse models with a goal for better consistency in the use and interpretation of these models.

Oncostatin M: Dual Regulator of the Skeletal and Hematopoietic Systems.

PURPOSE OF THE REVIEW: The bone and hematopoietic tissues coemerge during development and are functionally intertwined throughout mammalian life. Oncostatin M (OSM) is an inflammatory cytokine of the interleukin-6 family produced by osteoblasts, bone marrow macrophages, and neutrophils. OSM acts via two heterodimeric receptors comprising GP130 with either an OSM receptor (OSMR) or a leukemia inhibitory factor receptor (LIFR). OSMR is expressed on osteoblasts, mesenchymal, and endothelial cells and mice deficient for the Osm or Osmr genes have both bone and blood phenotypes illustrating the importance of OSM and OSMR in regulating these two intertwined tissues. RECENT FINDINGS: OSM regulates bone mass through signaling via OSMR, adaptor protein SHC1, and transducer STAT3 to both stimulate osteoclast formation and promote osteoblast commitment; the effect on bone formation is also supported by action through LIFR. OSM produced by macrophages is an important inducer of neurogenic heterotopic ossifications in peri-articular muscles following spinal cord injury. OSM produced by neutrophils in the bone marrow induces hematopoietic stem and progenitor cell proliferation in an indirect manner via OSMR expressed by bone marrow stromal and endothelial cells that form hematopoietic stem cell niches. OSM acts as a brake to therapeutic hematopoietic stem cell mobilization in response to G-CSF and CXCR4 antagonist plerixafor. Excessive OSM production by macrophages in the bone marrow is a key contributor to poor hematopoietic stem cell mobilization (mobilopathy) in people with diabetes. OSM and OSMR may also play important roles in the progression of several cancers. It is increasingly clear that OSM plays unique roles in regulating the maintenance and regeneration of bone, hematopoietic stem and progenitor cells, inflammation, and skeletal muscles. Dysregulated OSM production can lead to bone pathologies, defective muscle repair and formation of heterotopic ossifications in injured muscles, suboptimal mobilization of hematopoietic stem cells, exacerbated inflammatory responses, and anti-tumoral immunity. Ongoing research will establish whether neutralizing antibodies or cytokine traps may be useful to correct pathologies associated with excessive OSM production.

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.

Sexing Bones: Improving Transparency of Sex Reporting to Address Bias Within Preclinical Studies.

Despite knowledge that sexually dimorphic mechanisms regulate bone homeostasis, sex often remains unreported and unconsidered in preclinical experimental design. Failure to report sex could lead to inappropriate generalizations of research findings and less effective translation into clinical practice. Preclinical sex bias (preferential selection of one sex) is present across other fields, including neuroscience and immunology, but remains uninvestigated in skeletal research. For context, we first summarized key literature describing sexually dimorphic bone phenotypes in mice. We then investigated sex reporting practices in skeletal research, specifically how customary it is for murine sex to be included in journal article titles or abstracts and then determined whether any bias in sex reporting exists. Because sex hormones are important regulators of bone health (gonadectomy procedures, ie, ovariectomy [OVX] and orchidectomy [ORX], are common yet typically not reported with sex), we incorporated reporting of OVX and ORX terms, representing female and male mice, respectively, into our investigations around sex bias. Between 1999 and 2020, inclusion of sex in titles or abstracts was low in murine skeletal studies (2.6%-4.06%). Reporting of OVX and ORX terms was low (1.44%-2.64%) and reporting of OVX and ORX with sex uncommon (0.4%-0.3%). When studies were combined to include both sexes and OVX (representing female) and ORX terms (representing male), a bias toward reporting of female mice was evident. However, when the terms OVX and ORX were removed, a bias toward the use of male mice was identified. Thus, studies focusing on sex hormones are biased toward female reporting with all other studies biased in reporting of male mice. We now call upon journal editors to introduce consistent guidance for transparent and accessible reporting of murine sex in skeletal research to better monitor preclinical sex bias, to diversify development of treatments for bone health, and to enable global skeletal health equity. © 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).

Transient Polycomb activity represses developmental genes in growing oocytes.

BACKGROUND: Non-genetic disease inheritance and offspring phenotype are substantially influenced by germline epigenetic programming, including genomic imprinting. Loss of Polycomb Repressive Complex 2 (PRC2) function in oocytes causes non-genetically inherited effects on offspring, including embryonic growth restriction followed by post-natal offspring overgrowth. While PRC2-dependent non-canonical imprinting is likely to contribute, less is known about germline epigenetic programming of non-imprinted genes during oocyte growth. In addition, de novo germline mutations in genes encoding PRC2 lead to overgrowth syndromes in human patients, but the extent to which PRC2 activity is conserved in human oocytes is poorly understood. RESULTS: In this study, we identify a discrete period of early oocyte growth during which PRC2 is expressed in mouse growing oocytes. Deletion of Eed during this window led to the de-repression of 343 genes. A high proportion of these were developmental regulators, and the vast majority were not imprinted genes. Many of the de-repressed genes were also marked by the PRC2-dependent epigenetic modification histone 3 lysine 27 trimethylation (H3K27me3) in primary-secondary mouse oocytes, at a time concurrent with PRC2 expression. In addition, we found H3K27me3 was also enriched on many of these genes by the germinal vesicle (GV) stage in human oocytes, strongly indicating that this PRC2 function is conserved in the human germline. However, while the 343 genes were de-repressed in mouse oocytes lacking EED, they were not de-repressed in pre-implantation embryos and lost H3K27me3 during pre-implantation development. This implies that H3K27me3 is a transient feature that represses a wide range of genes in oocytes. CONCLUSIONS: Together, these data indicate that EED has spatially and temporally distinct functions in the female germline to repress a wide range of developmentally important genes and that this activity is conserved in the mouse and human germlines.

Localized, time-dependent responses of rat cranial bone to repeated mild traumatic brain injuries.

While it is well-established that bone responds dynamically to mechanical loading, the effects of mild traumatic brain injury (mTBI) on cranial bone composition are unclear. We hypothesized that repeated mTBI (rmTBI) would change the microstructure of cranial bones, without gross skull fractures. To address this, young adult female Piebald Viral Glaxo rats received sham, 1×, 2× or 3× closed-head mTBIs delivered at 24 h intervals, using a weight-drop device custom-built for reproducible impact. Skull bones were collected at 2 or 10 weeks after the final injury/sham procedure, imaged by micro computed tomography and analyzed at predetermined regions of interest. In the interparietal bone, proximal to the injury site, modest increases in bone thickness were observed at 2 weeks, particularly following 2× and 3× mTBI. By 10 weeks, 2× mTBI induced a robust increase in the volume and thickness of the interparietal bone, alongside a corresponding decrease in the volume of marrow cavities in the diploë region. In contrast, neither parietal nor frontal skull samples were affected by rmTBI. Our findings demonstrate time- and location-dependent effects of rmTBI on cranial bone structure, highlighting a need to consider microstructural alterations to cranial bone when assessing the consequences of rmTBI.

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.

Oncostatin M regulates hematopoietic stem cell (HSC) niches in the bone marrow to restrict HSC mobilization.

We show that pro-inflammatory oncostatin M (OSM) is an important regulator of hematopoietic stem cell (HSC) niches in the bone marrow (BM). Treatment of healthy humans and mice with granulocyte colony-stimulating factor (G-CSF) dramatically increases OSM release in blood and BM. Using mice null for the OSM receptor (OSMR) gene, we demonstrate that OSM provides a negative feed-back acting as a brake on HSPC mobilization in response to clinically relevant mobilizing molecules G-CSF and CXCR4 antagonist. Likewise, injection of a recombinant OSM molecular trap made of OSMR complex extracellular domains enhances HSC mobilization in poor mobilizing C57BL/6 and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. Mechanistically, OSM attenuates HSC chemotactic response to CXCL12 and increases HSC homing to the BM signaling indirectly via BM endothelial and mesenchymal cells which are the only cells expressing OSMR in the BM. OSM up-regulates E-selectin expression on BM endothelial cells indirectly increasing HSC proliferation. RNA sequencing of HSCs from Osmr-/- and wild-type mice suggest that HSCs have altered cytoskeleton reorganization, energy usage and cycling in the absence of OSM signaling in niches. Therefore OSM is an important regulator of HSC niche function restraining HSC mobilization and anti-OSM therapy combined with current mobilizing regimens may improve HSPC mobilization for transplantation.