Calorie Restriction in Mice Impairs Cortical but not Trabecular Peak Bone Mass by Suppressing Bone Remodeling.

Calorie restriction (CR) can lead to weight loss and decreased substrate availability for bone cells. Ultimately, this can lead to impaired peak bone acquisition in children and adolescence and bone loss in adults. But the mechanisms that drive diet-induced bone loss in humans are not well characterized. To explore those in greater detail, we examined the impact of 30% calorie restriction for 4 and 8 weeks in both male and female 8-week-old C57BL/6 J mice. Body composition, areal bone mineral density (aBMD), skeletal microarchitecture by micro-CT, histomorphometric parameters, and in vitro trajectories of osteoblast and adipocyte differentiation were examined. After both 4 weeks and 8 weeks, CR mice lost weight and exhibited lower femoral and whole-body aBMD vs. ad libitum (AL) mice. By micro-CT, CR mice had lower cortical bone area fraction vs. AL mice, but males had preserved trabecular bone parameters and females showed increased bone volume fraction compared to AL mice after 8 weeks. Histomorphometric analysis revealed that CR mice had a profound suppression in trabecular as well as endocortical and periosteal bone formation in addition to reduced bone resorption compared to AL mice. Bone marrow adipose tissue was significantly increased in CR mice vs. AL mice. In vitro, the pace of adipogenesis in bone marrow stem cells was greatly accelerated with higher markers of adipocyte differentiation and more oil red O staining, whereas osteogenic differentiation was reduced. qRT-PCR and western blotting suggested that the expression of Wnt16 and the canonical ß-catenin pathway were compromised during CR. In sum, CR causes impaired peak cortical bone mass due to a profound suppression in bone remodeling. The increase in marrow adipocytes in vitro and in vivo is related to both progenitor recruitment and adipogenesis in the face of nutrient insufficiency. Long-term calorie restriction may lead to lower bone mass principally in the cortical envelope, possibly due to impaired Wnt signaling.

Calorie restriction led to impaired bone mass and increased accumulation of bone marrow adipose tissue. During the development of bone-fat imbalance due to calorie restriction, bone remodeling was notably inhibited. Calorie restriction may shift the differentiation of bone marrow stem cells towards adipocytes instead of osteoblasts. This process involves a disruption in the canonical Wnt signaling pathway.

Affinity targeting of therapeutic proteins to the bone surface-local delivery of sclerostin-neutralizing antibody enhances efficacy.

Currently available biotherapeutics for the treatment of osteoporosis lack explicit mechanisms for bone localization, potentially limiting efficacy and inducing off-target toxicities. While various strategies have been explored for targeting the bone surface, critical aspects remain poorly understood, including the optimal affinity ligand, the role of binding avidity and circulation time, and, most importantly, whether or not this strategy can enhance the functional activity of clinically relevant protein therapeutics. To investigate, we generated fluorescent proteins (eg, mCherry) with site-specifically attached small molecule (bisphosphonate) or peptide (deca-aspartate, D10) affinity ligands. While both affinity ligands successfully anchored fluorescent protein to the bone surface, quantitative radiotracing revealed only modest femoral and vertebral accumulation and suggested a need for enhanced circulation time. To achieve this, we fused mCherry to the Fc fragment of human IgG1 and attached D10 peptides to each C-terminus. The mCherry-Fc-D10 demonstrated an ~80-fold increase in plasma exposure and marked increases in femoral and vertebral accumulation (13.6% ± 1.4% and 11.4% ± 1.3% of the injected dose/g [%ID/g] at 24 h, respectively). To determine if bone surface targeting could enhance the efficacy of a clinically relevant therapeutic, we generated a bone-targeted sclerostin-neutralizing antibody, anti-sclerostin-D10. The targeted antibody demonstrated marked increases in bone accumulation and retention (20.9 ± 2.5% and 19.5 ± 2.5% ID/g in femur and vertebrae at 7 days) and enhanced effects in a murine model of ovariectomy-induced bone loss (bone volume/total volume, connectivity density, and structure model index all increased [P < .001] vs untargeted anti-sclerostin). Collectively, our results indicate the importance of both bone affinity and circulation time in achieving robust targeting of therapeutic proteins to the bone surface and suggest that this approach may enable lower doses and/or longer dosing intervals without reduction in biotherapeutic efficacy. Future studies will be needed to determine the translational potential of this strategy and its potential impact on off-site toxicities.

Several biologic therapies have been approved for osteoporosis, but they lack means of localization to bone tissue, potentially limiting their efficacy and leading to off-target toxicities. This manuscript investigates strategies for targeting biotherapeutics to the bone surface and asks the question of whether or not this approach can enhance functional activity and allow for lower or less frequent dosing. To define the key determinants of bone surface targeting, we begin by synthesizing fluorescent model proteins with different bone targeting tags. We show that even 1 tag is enough to make the surface of the femur and vertebrae fluorescent following systemic administration. The results are relatively modest at first, but when we combine the bone targeting tag with a second modification that makes the protein circulate in the body for a longer period of time, we observe a huge increase in bone surface delivery. We then synthesize a bone surface targeted version of a sclerostin-inhibiting antibody and show that it is more effective than the untargeted antibody and provides near complete protection of bone density despite relatively low dose. Our findings could have translational implications for existing bone therapies and help guide design of future strategies for optimized bone surface targeting.

Reduced GS Domain Serine/Threonine Requirements of Fibrodysplasia Ossificans Progressiva Mutant Type I BMP Receptor ACVR1 in the Zebrafish.

Fibrodysplasia ossificans progressiva (FOP) is a rare human genetic condition characterized by altered skeletal development and extraskeletal bone formation. All cases of FOP are caused by mutations in the type I bone morphogenetic protein (BMP) receptor gene ACVR1 that result in overactivation of the BMP signaling pathway. Activation of the wild-type ACVR1 kinase requires assembly of a tetrameric type I and II BMP receptor complex followed by phosphorylation of the ACVR1 GS domain by type II BMP receptors. Previous studies showed that the FOP-mutant ACVR1-R206H required type II BMP receptors and presumptive glycine/serine-rich (GS) domain phosphorylation for overactive signaling. Structural modeling of the ACVR1-R206H mutant kinase domain supports the idea that FOP mutations alter the conformation of the GS domain, but it is unclear how this leads to overactive signaling. Here we show, using a developing zebrafish embryo BMP signaling assay, that the FOP-mutant receptors ACVR1-R206H and -G328R have reduced requirements for GS domain phosphorylatable sites to signal compared to wild-type ACVR1. Further, ligand-independent and ligand-dependent signaling through the FOP-mutant ACVR1 receptors have distinct GS domain phosphorylatable site requirements. ACVR1-G328R showed increased GS domain serine/threonine requirements for ligand-independent signaling compared to ACVR1-R206H, whereas it exhibited reduced serine/threonine requirements for ligand-dependent signaling. Remarkably, while ACVR1-R206H does not require the type I BMP receptor partner, Bmpr1, to signal, a ligand-dependent GS domain mutant of ACVR1-R206H could signal independently of Bmpr1 only when Bmp7 ligand was overexpressed. Of note, unlike human ACVR1-R206H, the zebrafish paralog Acvr1l-R203H does not show increased signaling activity. However, in domain-swapping studies, the human kinase domain, but not the human GS domain, was sufficient to confer overactive signaling to the Acvr1l-R203H receptor. Together these results reflect the importance of GS domain activation and kinase domain functions in regulating ACVR1 signaling and identify mechanisms of reduced regulatory constraints conferred by FOP mutations. © 2023 American Society for Bone and Mineral Research (ASBMR).

High Bone Mass Disorders: New Insights From Connecting the Clinic and the Bench.

Monogenic high bone mass (HBM) disorders are characterized by an increased amount of bone in general, or at specific sites in the skeleton. Here, we describe 59 HBM disorders with 50 known disease-causing genes from the literature, and we provide an overview of the signaling pathways and mechanisms involved in the pathogenesis of these disorders. Based on this, we classify the known HBM genes into HBM (sub)groups according to uniform Gene Ontology (GO) terminology. This classification system may aid in hypothesis generation, for both wet lab experimental design and clinical genetic screening strategies. We discuss how functional genomics can shape discovery of novel HBM genes and/or mechanisms in the future, through implementation of omics assessments in existing and future model systems. Finally, we address strategies to improve gene identification in unsolved HBM cases and highlight the importance for cross-laboratory collaborations encompassing multidisciplinary efforts to transfer knowledge generated at the bench to the clinic. © 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).

Inhibition of HMGB1/RAGE Signaling Reduces the Incidence of Medication-Related Osteonecrosis of the Jaw (MRONJ) in Mice.

Medication-related osteonecrosis of the jaw (MRONJ) is a severe complication of antiresorptive or antiangiogenic medications, used in the treatment of bone malignancy or osteoporosis. Bone necrosis, mainly represented by osteocytic death, is always present in MRONJ sites; however, the role of osteocyte death in MRONJ pathogenesis is unknown. High mobility group box 1 (HMGB1) is a non-histone nucleoprotein that in its acetylated form accumulates in the cytoplasm, whereas non-acetylated HMGB1 localizes in the nucleus. SIRT1 deacetylase regulates cellular localization of HMGB1. Interestingly, HMGB1 is released during cell necrosis and promotes inflammation through signaling cascades, including activation of the RAGE receptor. Here, we utilized a well-established mouse MRONJ model that utilizes ligature-induced experimental periodontitis (EP) and treatment with either vehicle or zolendronic acid (ZA). Initially, we evaluated HMGB1-SIRT1 expression in osteocytes at 1, 2, and 4 weeks of treatment. Significantly increased cytoplasmic and perilacunar HMGB1 expression was observed at EP sites of ZA versus vehicle (Veh) animals at all time points. SIRT1 colocalized with cytoplasmic HMGB1 and presented a statistically significant increased expression at the EP sites of ZA animals for all time points. RAGE expression was significantly higher in the submucosal tissues EP sites of ZA animals compared with those in vehicle group. To explore the significance of increased cytoplasmic and extracellular HMGB1 and increased RAGE expression in MRONJ pathogenesis, we used pharmacologic inhibitors of these molecules. Combined HMGB1/RAGE inhibition resulted in lower MRONJ incidence with statistically significant decrease in osteonecrotic areas and bone exposure versus non-inhibitor treated ZA animals. Together, our data point to the role of HMGB1 as a central alarmin, overexpressed at early phase of MRONJ pathogenesis during osteocytic death. Moreover, HMGB1-RAGE pathway may represent a new promising therapeutic target in patients at high risk of MRONJ. © 2022 American Society for Bone and Mineral Research (ASBMR).

Clinical description and genetic analysis of a novel familial skeletal dysplasia characterized by high bone mass and lucent bone lesions.

High bone mass (HBM) disorders are a clinically and genetically heterogeneous subgroup of rare skeletal dysplasias. Here we present a case of a previously unreported familial skeletal dysplasia characterized by HBM and lucent bone lesions that we aimed to clinically characterize and genetically investigate. For phenotyping, we reviewed past clinical records and imaging tests, and performed physical examination (PE), bone densitometry, and mineral panels in affected individuals, including a male proband, his son and daughter, in addition to unaffected controls, including the proband's wife and brother. Affected individuals also underwent impact microindentation (IMI). In an effort to elucidate the disorder's molecular etiology, whole exome sequencing (WES) was performed in all individuals to filter for rare variants present only in affected ones. The cases displayed a unique skeletal phenotype with a mix of sclerotic features and lucent bone lesions, and high IMI values. Bone mineral density was very elevated in the proband and his daughter. The proband's daughter also exhibited idiopathic scoliosis (IS), in addition to mild thrombocytopenia and mild structural thyroid abnormalities, which were the only extra-skeletal abnormalities identified. WES analysis yielded 5 rare putative pathogenic variants in affected members in genes that are associated with bone metabolism including: SEM4AD, TBX18, PTCH1, PTK7, and ADGRE5. The PTK7 variant appeared as possibly implicated in the development of IS while the TBX18 and SEMA4D variants stood out as the strongest candidates for the lucent bone lesions and HBM, respectively, given their high predicted pathogenicity and putative role in bone biology. Variant functionality should be addressed in the future to assess their implication in skeletal metabolism as it is the first time that mutations in TBX18 and SEMA4D have been associated to bone developmental lesions and mineral metabolism in a clinical setting.

The Posterior Part Influences the Anterior Part of the Mouse Cranial Base Development.

The cranial base is a critical structure in the head, which is composed of endoskeletal and dermal skeletal. The braincase floor, part of the cranial base, is a midline structure of the head. Because it is a midline structure connecting the posterior skull with the facial region, braincase floor is critical for the orientation of the facial structure. Shortened braincase floor leads to mid-facial hypoplasia and malocclusions. During embryonic development, elongation of the braincase floor occurs through endochondral ossification in the parachordal cartilage, hypophyseal cartilage, and trabecular cartilage, which leads to formation of basioccipital (BO), basisphenoid (BS), and presphenoid (PS) bones, respectively. Currently, little is known about whether maturation of parachordal cartilage, hypophyseal cartilage, and trabecular cartilage occurs in a simultaneous or sequential manner and if the formation of one impacts the others. Our previous studies demonstrated that loss of function of ciliary protein Evc2 leads to premature fusion in the intersphenoid synchondrosis (ISS). In this study, we take advantage of Evc2 mutant mice to delineate the mechanism governing synchondrosis formation. Our analysis supports a cascade mechanism on the spatiotemporal regulation of the braincase floor development that the hypertrophy of parachordal cartilage (posterior side) impacts the hypertrophy of hypophyseal cartilage (middle) and trabecular cartilage (anterior side) in a sequential manner. The cascade mechanism well explains the premature fusion of the ISS in Evc2 mutant mice and is instructive to understand the specifically shortened anterior end of the braincase floor in various types of genetic syndromes. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Differentiation of Induced Pluripotent Stem Cells Into Chondrocytes: Methods and Applications for Disease Modeling and Drug Discovery.

Induced pluripotent stem cell (iPSC) technology allows pathomechanistic and therapeutic investigation of human heritable disorders affecting tissue types whose collection from patients is difficult or even impossible. Among them are cartilage diseases. Over the past decade, iPSC-chondrocyte disease models have been shown to exhibit several key aspects of known disease mechanisms. Concurrently, an increasing number of protocols to differentiate iPSCs into chondrocytes have been published, each with its respective (dis)advantages. In this review we provide a comprehensive overview of the different differentiation approaches, the hitherto described iPSC-chondrocyte disease models and mechanistic and/or therapeutic insights that have been derived from their investigation, and the current model limitations. Key lessons are that the most appropriate differentiation approach is dependent upon the cartilage disease under investigation and that further optimization is still required to recapitulate the in vivo cartilage. © 2022 American Society for Bone and Mineral Research (ASBMR).

Molecular Diagnoses of X-Linked and Other Genetic Hypophosphatemias: Results From a Sponsored Genetic Testing Program.

X-linked hypophosphatemia (XLH), a dominant disorder caused by pathogenic variants in the PHEX gene, affects both sexes of all ages and results in elevated serum fibroblast growth factor 23 (FGF23) and below-normal serum phosphate. In XLH, rickets, osteomalacia, short stature, and lower limb deformity may be present with muscle pain and/or weakness/fatigue, bone pain, joint pain/stiffness, hearing difficulty, enthesopathy, osteoarthritis, and dental abscesses. Invitae and Ultragenyx collaborated to provide a no-charge sponsored testing program using a 13-gene next-generation sequencing panel to confirm clinical XLH or aid diagnosis of suspected XLH/other genetic hypophosphatemia. Individuals aged ≥6 months with clinical XLH or suspected genetic hypophosphatemia were eligible. Of 831 unrelated individuals tested between February 2019 and June 2020 in this cross-sectional study, 519 (62.5%) individuals had a pathogenic or likely pathogenic variant in PHEX (PHEX-positive). Among the 312 PHEX-negative individuals, 38 received molecular diagnoses in other genes, including ALPL, CYP27B1, ENPP1, and FGF23; the remaining 274 did not have a molecular diagnosis. Among 319 patients with a provider-reported clinical diagnosis of XLH, 88.7% (n = 283) had a reportable PHEX variant; 81.5% (n = 260) were PHEX-positive. The most common variant among PHEX-positive individuals was an allele with both the gain of exons 13-15 and c.*231A>G (3'UTR variant) (n = 66/519). Importantly, over 80% of copy number variants would have been missed by traditional microarray analysis. A positive molecular diagnosis in 41 probands (4.9%; 29 PHEX positive, 12 non-PHEX positive) resulted in at least one family member receiving family testing. Additional clinical or family member information resulted in variant(s) of uncertain significance (VUS) reclassification to pathogenic/likely pathogenic (P/LP) in 48 individuals, highlighting the importance of segregation and clinical data. In one of the largest XLH genetic studies to date, 65 novel PHEX variants were identified and a high XLH diagnostic yield demonstrated broad insight into the genetic basis of XLH. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Osteoblast-Specific Wnt Secretion Is Required for Skeletal Homeostasis and Loading-Induced Bone Formation in Adult Mice.

Wnt signaling is critical to many aspects of skeletal regulation, but the importance of Wnt ligands in the bone anabolic response to mechanical loading is not well established. Recent transcriptome profiling studies by our laboratory and others show that mechanical loading potently induces genes encoding Wnt ligands, including Wnt1 and Wnt7b. Based on these findings, we hypothesized that mechanical loading stimulates adult bone formation by inducing Wnt ligand expression. To test this hypothesis, we inhibited Wnt ligand secretion in adult (5 months old) mice using a systemic (drug) and a bone-targeted (conditional gene knockout) approach, and subjected them to axial tibial loading to induce lamellar bone formation. Mice treated with the Wnt secretion inhibitor WNT974 exhibited a decrease in bone formation in non-loaded bones as well as a 54% decline in the periosteal bone formation response to tibial loading. Next, osteoblast-specific Wnt secretion was inhibited by dosing 5-month-old Osx-CreERT2; WlsF/F mice with tamoxifen. Within 1 to 2 weeks of Wls deletion, skeletal homeostasis was altered with decreased bone formation and increased resorption, and the anabolic response to loading was reduced 65% compared to control (WlsF/F ). Together, these findings show that Wnt ligand secretion is required for adult bone homeostasis, and furthermore establish a role for osteoblast-derived Wnts in mediating the bone anabolic response to tibial loading. © 2021 American Society for Bone and Mineral Research (ASBMR).

gp130 Cytokines Activate Novel Signaling Pathways and Alter Bone Dissemination in ER+ Breast Cancer Cells.

Breast cancer cells frequently home to the bone marrow, where they encounter signals that promote survival and quiescence or stimulate their proliferation. The interleukin-6 (IL-6) cytokines signal through the co-receptor glycoprotein130 (gp130) and are abundantly secreted within the bone microenvironment. Breast cancer cell expression of leukemia inhibitory factor (LIF) receptor (LIFR)/STAT3 signaling promotes tumor dormancy in the bone, but it is unclear which, if any of the cytokines that signal through LIFR, including LIF, oncostatin M (OSM), and ciliary neurotrophic factor (CNTF), promote tumor dormancy and which signaling pathways are induced. We first confirmed that LIF, OSM, and CNTF and their receptor components were expressed across a panel of breast cancer cell lines, although expression was lower in estrogen receptor-negative (ER- ) bone metastatic clones compared with parental cell lines. In estrogen receptor-positive (ER+ ) cells, OSM robustly stimulated phosphorylation of known gp130 signaling targets STAT3, ERK, and AKT, while CNTF activated STAT3 signaling. In ER- breast cancer cells, OSM alone stimulated AKT and ERK signaling. Overexpression of OSM, but not CNTF, reduced dormancy gene expression and increased ER+ breast cancer bone dissemination. Reverse-phase protein array revealed distinct and overlapping pathways stimulated by OSM, LIF, and CNTF with known roles in breast cancer progression and metastasis. In breast cancer patients, downregulation of the cytokines or receptors was associated with reduced relapse-free survival, but OSM was significantly elevated in patients with invasive disease and distant metastasis. Together these data indicate that the gp130 cytokines induce multiple signaling cascades in breast cancer cells, with a potential pro-tumorigenic role for OSM and pro-dormancy role for CNTF. © 2021 American Society for Bone and Mineral Research (ASBMR).

RNA-seq Analysis of Peri-Implant Tissue Shows Differences in Immune, Notch, Wnt, and Angiogenesis Pathways in Aged Versus Young Mice.

The number of total joint replacements (TJRs) in the United States is increasing annually. Cementless implants are intended to improve upon traditional cemented implants by allowing bone growth directly on the surface to improve implant longevity. One major complication of TJR is implant loosening, which is related to deficient osseointegration in cementless TJRs. Although poor osseointegration in aged patients is typically attributed to decreased basal bone mass, little is known about the molecular pathways that compromise the growth of bone onto porous titanium implants. To identify the pathways important for osseointegration that are compromised by aging, we developed an approach for transcriptomic profiling of peri-implant tissue in young and aged mice using our murine model of osseointegration. Based on previous findings of changes of bone quality associated with aging, we hypothesized that aged mice have impaired activation of bone anabolic pathways at the bone-implant interface. We found that pathways most significantly downregulated in aged mice relative to young mice are related to angiogenic, Notch, and Wnt signaling. Downregulation of these pathways is associated with markedly increased expression of inflammatory and immune genes at the bone-implant interface in aged mice. These results identify osseointegration pathways affected by aging and suggest that an increased inflammatory response in aged mice may compromise peri-implant bone healing. Targeting the Notch and Wnt pathways, promoting angiogenesis, or modulating the immune response at the peri-implant site may enhance osseointegration and improve the outcome of joint replacement in older patients. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Mesenchymal Stem Cells: Potential Therapeutic Prospect of Paracrine Pathways in Neonatal Infection.

Infection is the leading cause of admission and mortality in neonatal intensive care units. Immature immune function and antibiotic resistance make the treatment more difficult. However, there is no effective prevention for it. Recently, more and more researches are focusing on stem cell therapy, especially mesenchymal stem cells (MSCs); their potential paracrine effect confer MSCs with a major advantage to treat the immune and inflammatory disorders associated with neonatal infection. In this review, we summarize the basal properties and preclinical evidence of MSCs and explore the potential mechanisms of paracrine factors of MSCs for neonatal infection.

Notum Deletion From Late-Stage Skeletal Cells Increases Cortical Bone Formation and Potentiates Skeletal Effects of Sclerostin Inhibition.

Wnt signaling plays a vital role in the cell biology of skeletal patterning, differentiation, and maintenance. Notum is a secreted member of the α/ß-hydrolase superfamily that hydrolyzes the palmitoleoylate modification on Wnt proteins, thereby disrupting Wnt signaling. As a secreted inhibitor of Wnt, Notum presents an attractive molecular target for improving skeletal health. To determine the cell type of action for Notum's effect on the skeleton, we generated mice with Notum deficiency globally (Notum-/- ) and selectively (Notumf/f ) in limb bud mesenchyme (Prx1-Cre) and late osteoblasts/osteocytes (Dmp1-Cre). Late-stage deletion induced increased cortical bone properties, similar to global mutants. Notum expression was enhanced in response to sclerostin inhibition, so dual inhibition (Notum/sclerostin) was also investigated using a combined genetic and pharmacologic approach. Co-suppression increased cortical properties beyond either factor alone. Notum suppressed Wnt signaling in cell reporter assays, but surprisingly also enhanced Shh signaling independent of effects on Wnt. Notum is an osteocyte-active suppressor of cortical bone formation that is likely involved in multiple signaling pathways important for bone homeostasis © 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.

Mike Wakelam: an appreciation.

This issue of Essays in Biochemistry explores lipid mediators - biologically active metabolites formed by enzymic and non-enzymic oxidation of polyunsaturated fatty acids. These can be exported across the cell membrane into the extracellular space, where they activate cell surface receptors to stimulate the cells of origin (autocrine) or nearby cells (paracrine). Lipid mediators are involved in many physiological processes, which may become dysregulated during ageing and in lipid-related diseases such as diabetes, atherosclerosis, arthritis, cancer, Alzheimer's disease and metabolic syndrome. Following the death in March 2020 of Professor Mike Wakelam, with the loss of his major input into the lipid signalling field, Portland Press and Guest Editors John Harwood and Emyr Lloyd-Evans decided to dedicate this issue to his memory. This Editorial briefly recalls his work and influence.

Unique, Gender-Dependent Serum microRNA Profile in PLS3 Gene-Related Osteoporosis.

Plastin 3 (PLS3), encoded by PLS3, is a newly recognized regulator of bone metabolism, and mutations in the encoding gene result in severe childhood-onset osteoporosis. Because it is an X chromosomal gene, PLS3 mutation-positive males are typically more severely affected whereas females portray normal to increased skeletal fragility. Despite the severe skeletal pathology, conventional metabolic bone markers tend to be normal and are thus insufficient for diagnosing or monitoring patients. Our study aimed to explore serum microRNA (miRNA) concentrations in subjects with defective PLS3 function to identify novel markers that could differentiate subjects according to mutation status and give insight into the molecular mechanisms by which PLS3 regulates skeletal health. We analyzed fasting serum samples for a custom-designed panel comprising 192 miRNAs in 15 mutation-positive (five males, age range 8-76 years, median 41 years) and 14 mutation-negative (six males, age range 8-69 years, median 40 years) subjects from four Finnish families with different PLS3 mutations. We identified a unique miRNA expression profile in the mutation-positive subjects with seven significantly upregulated or downregulated miRNAs (miR-93-3p, miR-532-3p, miR-133a-3p, miR-301b-3p, miR-181c-5p, miR-203a-3p, and miR-590-3p; p values, range .004-.044). Surprisingly, gender subgroup analysis revealed the difference to be even more distinct in female mutation-positive subjects (congruent p values, range .007-.086) than in males (p values, range .127-.843) in comparison to corresponding mutation-negative subjects. Although the seven identified miRNAs have all been linked to bone metabolism and two of them (miR-181c-5p and miR-203a-3p) have bioinformatically predicted targets in the PLS3 3' untranslated region (3'-UTR), none have previously been reported to associate with PLS3. Our results indicate that PLS3 mutations are reflected in altered serum miRNA levels and suggest there is crosstalk between PLS3 and these miRNAs in bone metabolism. These provide new understanding of the pathomechanisms by which mutations in PLS3 lead to skeletal disease and may provide novel avenues for exploring miRNAs as biomarkers in PLS3 osteoporosis or as target molecules in future therapeutic applications. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

TGFß Regulation of Perilacunar/Canalicular Remodeling Is Sexually Dimorphic.

Bone fragility is the product of defects in bone mass and bone quality, both of which show sex-specific differences. Despite this, the cellular and molecular mechanisms underpinning the sexually dimorphic control of bone quality remain unclear, limiting our ability to effectively prevent fractures, especially in postmenopausal osteoporosis. Recently, using male mice, we found that systemic or osteocyte-intrinsic inhibition of TGFß signaling, achieved using the 9.6-kb DMP1 promoter-driven Cre recombinase (TßRIIocy-/- mice), suppresses osteocyte perilacunar/canalicular remodeling (PLR) and compromises bone quality. Because systemic TGFß inhibition more robustly increases bone mass in female than male mice, we postulated that sex-specific differences in bone quality could likewise result, in part, from dimorphic regulation of PLR by TGFß. Moreover, because lactation induces PLR, we examined the effect of TGFß inhibition on the female skeleton during lactation. In contrast to males, female mice that possess an osteocyte-intrinsic defect in TGFß signaling were protected from TGFß-dependent defects in PLR and bone quality. The expression of requisite PLR enzymes, the lacunocanalicular network (LCN), and the flexural strength of female TßRIIocy-/- bone was intact. With lactation, however, bone loss and induction in PLR and osteocytic parathyroid hormone type I receptor (PTHR1) expression, were suppressed in TßRIIocy-/- bone, relative to the control littermates. Indeed, differential control of PTHR1 expression, by TGFß and other factors, may contribute to dimorphism in PLR regulation in male and female TßRIIocy-/- mice. These findings provide key insights into the sex-based differences in osteocyte PLR that underlie bone quality and highlight TGFß signaling as a crucial regulator of lactation-induced PLR. © 2020 American Society for Bone and Mineral Research.

Extracellular Vesicles From Osteotropic Breast Cancer Cells Affect Bone Resident Cells.

Extracellular vesicles (EVs) are emerging as mediators of a range of pathological processes, including cancer. However, their role in bone metastases has been poorly explored. We investigated EV-mediated effects of osteotropic breast cancer cells (MDA-MB-231) on bone resident cells and endothelial cells. Pretreatment of osteoblasts with conditioned medium (CM) of MDA-MB-231 (MDA) cells promoted pro-osteoclastogenic and pro-angiogenic effects by osteoblast EVs (OB-EVs), as well as an increase of RANKL-positive OB-EVs. Moreover, when treating osteoblasts with MDA-EVs, we observed a reduction of their number, metabolic activity, and alkaline phosphatase (Alp) activity. MDA-EVs also reduced transcription of Cyclin D1 and of the osteoblast-differentiating genes, while enhancing the expression of the pro-osteoclastogenic factors Rankl, Lcn2, Il1b, and Il6. Interestingly, a cytokine array on CM from osteoblasts treated with MDA-EVs showed an increase of the cytokines CCL3, CXCL2, Reg3G, and VEGF, while OPG and WISP1 were downregulated. MDA-EVs contained mRNAs of genes involved in bone metabolism, as well as cytokines, including PDGF-BB, CCL3, CCL27, VEGF, and Angiopoietin 2. In line with this profile, MDA-EVs increased osteoclastogenesis and in vivo angiogenesis. Finally, intraperitoneal injection of MDA-EVs in mice revealed their ability to reach the bone microenvironment and be integrated by osteoblasts and osteoclasts. In conclusion, we showed a role for osteoblast-derived EVs and tumor cell-derived EVs in the deregulation of bone and endothelial cell physiology, thus fueling the vicious cycle induced by bone tumors. © 2019 American Society for Bone and Mineral Research.

Observations on the Natural History of Camurati-Engelmann Disease.

Camurati-Engelmann disease (OMIM 31300) is a rare cranio-tubular bone dysplasia characterized by osteosclerosis of the long bones and skull caused by dominantly-inherited mutations in the transforming growth factor beta 1 (TGFB1) gene. A wide variation in phenotype has been recognized, even within families carrying the same mutation. In addition, aspects of the natural history of the disorder, in particular whether it is always progressive or can remit spontaneously, remain uncertain. In a large kindred carrying a TGFB1 gene mutation (c.653G > A; p.R218H) we have attempted to clarify the extent of phenotypic variability and the natural history of the disease through detailed individual histories of symptoms, and skeletal imaging by both radiography and scintigraphy. Only one subject had the classical childhood onset with bone pain in the legs and gait disturbance. Eight subjects reported the onset of leg pain in their teenage years that, by their early 20s, had either resolved or persisted at a low level. Two of these eight later developed cranial nerve palsies. There was a wide variation in the radiographic appearance in adults, but disease extent and activity in long bones, as assessed by scintigraphy, was inversely correlated with age (p < 0.025). In younger subjects the radiographic and scintigraphic appearances were concordant, but in older subjects the scintigram could be quiescent despite florid radiographic changes. Sequential scintigrams in two subjects showed reduced activity in the later scan. One subject had suffered meningoencephalitis in early childhood that resulted in paresis of one arm. The affected arm showed markedly less disease involvement, implicating mechanical or growth factors in its etiology. Our data suggest that the natural history of Camurati-Engelmann disease can be benign, and that disease activity commonly attenuates in adulthood. Severe cases of childhood onset and/or with cranial nerve involvement, may occur only in a minority of mutation carriers. © 2019 American Society for Bone and Mineral Research.