AIMS:There is growing evidence supporting paternal contributions to child health. Previous findings from the Vitamin D In Pregnancy study suggest that advanced paternal age is associated with a range of poorer bone measures for offspring at age 11 years. Whether these associations are evident when peak bone mass is attained remains unknown. This study aimed to investigate associations between paternal age and offspring bone health and body composition at age 20 years. METHODS:Data from the Raine study, a multigenerational cohort, were used. Among 1183 offspring with total body DXA data (Norland XR-36 densitometer) at the 20-year follow up, 1142 with information on their father's age were included in this analysis. Linear regression and restricted cubic spline models were developed to examine associations of offspring bone and body composition outcomes at 20 years and paternal age during pregnancy (16-20 weeks' gestation). RESULTS:Fathers' mean age was 31 years (SD 6.6, range 15-58). Restricted cubic spline models adjusted for sex, maternal, paternal and offspring factors revealed complex, nonlinear associations between paternal age and total body bone mineral density (BMD) and content (BMC). Offspring of fathers in the lowest quartile (Q1) of paternal age (median age 25 years) had between 0.018 g/cm2 to 0.023 g/cm2 higher BMD (1.7-2.1%) than those in Q2 (median age 30 years), Q3 (33 years) and Q4 (39 years) and between 61 g and 67 g (2.1-2.3%) higher BMC than those of Q2 and Q3. Paternal age was not significantly associated with offspring total body lean or fat mass in linear regression models. CONCLUSIONS:This study suggests that paternal age in pregnancy is associated with offspring total body bone mass, but not body composition in early adulthood (~20 years). Further confirmatory studies are warranted to better establish the intergenerational effects of paternal age on bone health.
Osteocytes reside within lacunae where they sense mechanical loading and regulate bone formation and resorption. Although lacunae define the osteocyte population, their number and volume are themselves regulated, and evidence regarding lacunar structural properties remains inconsistent. The relationship between lacunar ultrastructure and the bone proteome has not previously been explored. We therefore integrated high-resolution ultrastructural and proteomic analyses of femoral bone from osteoporotic patients undergoing hip arthroplasty after fracture (n = 15) and individuals undergoing surgery for osteoarthritis with normal or near-normal bone mineral density (n = 42). Lacunar morphology was assessed using high-resolution micro-computed tomography, and proteomic profiling was performed by liquid chromatography-tandem mass spectrometry, quantifying approximately three thousand proteins. Lacunar density and total lacunar volume normalized to bone volume were inversely correlated with total hip bone mineral density T-score (r = -0.462 and r = -0.471), indicating increased lacunar number and total lacunar volume in osteoporotic bone. In both sexes, lacunar density correlated strongly with total lacunar volume per bone volume (males: r = 0.69, p = 1.2 × 10-6; females: r = 0.70, p = 1.1 × 10-3), demonstrating tight coupling between lacunar number and volume. Exploratory pathway enrichment analysis revealed enrichment of pathways related to ROBO receptor signaling, translational regulation, and complement-mediated inflammatory processes. These findings link osteocyte lacunar ultrastructure to the bone proteome and identify osteocytes as central regulators integrating immune signaling, mechanosensing, and bone turnover.
Cranial neural crest cell (cNCC)-derived mesenchyme must precisely integrate intrinsic transcriptional programs with localized signaling cues to orchestrate craniofacial skeletogenesis. While the transcription factor Meis2 is an essential regulator of this process, the spatially defined cellular contexts and downstream effector pathways remain poorly characterized. Building upon previous single-cell transcriptomic evidence of Igf2 dysregulation, we employed spatial transcriptomics integrated with single-cell references to map the mesenchymal landscape in Meis2-deficient embryos during early ossification. This spatial mapping identified distinct chondro-osteoprogenitor niches and revealed a significant enrichment of the IGF2 signaling axis within mutant mesenchymal compartments. We validated these spatial expression patterns in vivo, confirming the localized upregulation of Igf2 in Meis2-deficient osteo-chondroprogenitors. Functional in vitro assays demonstrated that IGF signaling is a potent modulator of cNCC-derived mesenchymal differentiation. Exogenous IGF2 enhanced osteogenic potential, whereas inhibition of the IGF1 receptor (IGF1R) significantly attenuated osteogenesis as measured using ALP activity. Furthermore, the impaired bone formation in Meis2 mutants correlated with disrupted cell adhesion dynamics. Collectively, these findings establish the importance of IGF2 signaling during embryonic craniofacial ossification and provide a mechanistic link between Meis2 loss, altered cell adhesion, and dysregulated osteogenic differentiation.
Notch signaling plays a fundamental role in skeletal physiology, and activation of NOTCH3 in osteoblasts/osteocytes impairs corticalization, causes cortical osteopenia and suppresses cancellous bone remodeling. To address possible mechanisms responsible, we explored transcriptome profiles in osteocytes and femoral bone from 1-month-old male C57BL/6 mice following activation of NOTCH3 in Dmp1-expressing cells. We used a NOTCH3 gain-of-function mouse line expressing the NOTCH3 intracellular domain (N3ICD) from the Rosa26 locus (R26-N3ICD mice) following the deletion of a loxP flanked STOP cassette, which was accomplished by crossing R26-N3ICD mice with Dmp1-Cre transgenics. Bulk RNA-Sequencing (RNA-Seq) of osteocytes revealed that expression of the N3ICD enhanced pathways associated with rheumatoid arthritis signaling, osteoclast differentiation, collagen degradation and the immune response. Single cell (sc)RNA-Seq of femoral bone from 1-month-old mice revealed clusters of cells related to vascular and osteogenic cells. Trajectory analysis showed a close association among selected clusters in control and N3ICD-expressing femurs. There were modest differences between N3ICD-expressing and control cells in cluster allocation, except for a decrease in the osteogenic cluster in N3ICD-induced cells. Analysis of intron/exon sequences demonstrated that NOTCH3 altered the velocity of the osteogenic cluster. In conclusion, NOTCH3 enhances the activity of selected pathways including osteoclast differentiation and suppresses the transcription of osteogenic cells in femoral bone.
Background Osteosarcoma (OS) represents a common primary malignant bone tumor associated with unfavorable clinical outcomes. Growing evidence underscores the crucial involvement of N6-methyladenosine (m6A) modifications in tumor development, but the specific mechanisms underlying the m6A regulatory network in OS remain to be elucidated. Methods Potential key target genes in OS were identified through bioinformatic analyses, followed by the characterization of m6A-related regulatory proteins, specifically, writer and reader proteins, which showed significant associations with these targets. To elucidate the mechanistic role of m6A methylation in regulating UHRF1 expression, a series of in vitro assays were conducted. These included RNA pull-down, MeRIP-PCR, dot blot, dual-luciferase reporter assays, and RNA stability assays, which collectively confirmed the interaction between m6A regulatory proteins and UHRF1 mRNA. For functional investigations, OS cell lines (U2OS, Saos2, and 143B) with gene silencing or overexpression were established, and the role of UHRF1 in cellular proliferation, migration, and invasion was assessed using CCK-8 assays, Transwell migration and invasion assays, flow cytometry, and wound healing assays. In addition, GSH/GSSG ratio, Fe2+ concentration, and ROS levels were measured using commercial assay kits to explore ferroptosis-related functional mechanisms. To validate the in vivo relevance of our findings, a xenograft mouse model was established. Finally, functional rescue experiments were performed to mechanistically confirm the critical role of the ZCCHC4-UHRF1-CDO1 regulatory axis in OS progression. Results ZCCHC4, functioning as an m6A methyltransferase, enhances the stability of UHRF1 mRNA by catalyzing its m6A modification, thereby promoting increased expression of UHRF1. In parallel, IGF2BP3, an established m6A reader protein, specifically recognizes and binds to the m6A-modified sites on UHRF1 mRNA, further stabilizing the transcript and modulating its downstream biological functions. METTL3/METTL14 knockdown experiments ruled out the contribution of classical m6A methyltransferases, confirming that ZCCHC4 is the primary methyltransferase for UHRF1. Dual-luciferase assays and bisulfite sequencing revealed that UHRF1 suppresses CDO1 transcription by inducing high methylation of its promoter, thereby reducing ROS/Fe2+ levels and increasing GSH, which in turn blocks ferroptosis. In U2OS, Saos2, and 143B cells, silencing UHRF1 or ZCCHC4 inhibited proliferation, migration, and invasion while activating ferroptosis. Overexpression of UHRF1 had the opposite effect. In vivo models confirmed that UHRF1 silencing inhibited tumor growth. Furthermore, UHRF1 overexpression partially reversed the phenotypes induced by ZCCHC4 knockdown. Conclusion In summary, this study reveals for the first time the complete molecular mechanism by which ZCCHC4-mediated UHRF1 m6A methylation promotes OS progression through epigenetic suppression of CDO1 transcription and inhibition of ferroptosis. This regulatory axis (ZCCHC4-IGF2BP3-UHRF1-CDO1-ferroptosis) provides multiple therapeutic targets for OS and lays a solid foundation for the future development of anticancer strategies based on the regulation of ferroptosis, while also offering a promising pathway for clinical translation.
PURPOSE:Photon-counting CT (PCCT) for quantitative microstructural assessments of the central skeleton remains underexplored. This study evaluated the effects of radiation dose, spatial resolution, noise and reconstruction algorithm on bone mineral density (BMD), bone volume fraction (BV/TV), and trabecular separation (Tb.Sp). MATERIALS AND METHODS:Seven excised human vertebral bodies embedded in polymethylmethacrylate were scanned using a clinical PCCT scanner (Naeotom Alpha, Siemens Healthineers): natively at (20 mGy CTDI32 cm) and within a thorax phantom at (6, 9, 12 and 15 mGy). Four repeated scans (15 mGy) served as reference. Filtered back projection (FBP) and iterative (QIR3) reconstructions were used with Br56u, Br76u and Br89u kernels. Bone parameters were estimated from cancellous volumes. Differences were tested with Wilcoxon signed-rank tests. Radiation dose impact was assessed using Bland-Altman analysis and Lin's concordance coefficient (rccc). RESULTS:Median (IQR) ranges for BMD were 177.5 (55.1)-191.9 (57.5) mgHA/cm3, for BV/TV 0.25 (0.20)-0.47 (0.05), and Tb.Sp 0.29 (0.02)-1.50 (1.64) mm. FBP consistently yielded higher BMD and BV/TV than QIR3, albeit non-significant after statistical correction. BMD differences between algorithms were 6.6 mgHA/cm3 at 6.0 mGy Br89u, and 0.1 mgHA/cm3 for Br76u at 20 mGy. Noise was higher in FBP (range: 13-470 HU) vs QIR3 (6-124 HU). The mean absolute (%) differences to the reference across dose levels were best for Br76u/QIR3 (BMD: 0.83 mgHA/cm3 (0.5%); BV/TV: 0.01 (2.6%) and Tb.Sp: 0.79 mm (18%)). Best robustness against radiation dose changes was observed with Br56u/QIR3 and Br76u/QIR3. CONCLUSIONS:Br56u showed enlarged Tb.Sp levels despite excellent agreement, while Br89u showed increased noise levels and instability. Br76u/QIR3 showed the best balance between microstructural accuracy, image noise and stability at clinically diagnostic dose levels, for example 15 mGy CTDIvol.
Bisphosphonates (BPs) are widely used to treat bone complications, yet their impact on the coupling between resorption and formation during cortical bone remodeling remains incompletely understood. We investigated associations between short-term alendronate treatment and osteoclast morphology, intracortical pore dynamics, and osteoprogenitor organization in a synchronized rat model of endo- and intracortical bone remodeling induced by lactation and dietary calcium restriction. Twenty-four lactating Sprague Dawley rats were randomized to vehicle, alendronate (28 μg/kg, twice weekly), or raloxifene (1 mg/kg, five times weekly) with raloxifene serving as a comparative control, for seven days under a low-calcium diet. Femurs were analyzed by micro-CT, histomorphometry, and multiplex RNA in situ hybridization combined with AI-driven image analysis. Alendronate was associated with a higher proportion of eroded pores (29% vs. 12% in controls) and lower density of formative pores (5.2 vs. 10.0 pores/mm2), indicating a prolonged reversal-resorption phase. Osteoclasts on endocortical surfaces were significantly larger (208 ± 37 μm2vs. 146 ± 46 μm2) and contained more nuclei (2.8 ± 0.40 nuclei vs. 2.2 ± 0.46 nuclei) under alendronate treatment, consistent with enhanced fusion. Osteoprogenitor abundance and spatial organization did not differ between treatment groups. Region-specific structural differences included increased midshaft porosity and larger distal metaphyseal cortical area in alendronate-treated rats. Raloxifene was associated with minimal differences on early remodeling events. Together, these findings support the interpretation that short-term bisphosphonate treatment is associated with early alterations in cortical remodeling dynamics under high-turnover conditions and highlight the importance of site-specific evaluation within defined physiological remodeling contexts.
Intravenous (IV) iron is recommended for the treatment of iron deficiency anemia (IDA) when oral iron is ineffective or poorly tolerated, or when rapid iron replenishment is needed. However, specific IV iron formulations, including saccharated ferric oxide (SFO) and ferric carboxymaltose (FCM), are associated with increased rates of hypophosphatemia and subsequent osteomalacia. Clinical trials of these IV iron formulations were not sufficiently large or long enough to assess whether these effects translate into increased risk of fracture. This observational cohort study used claims data from a Japanese health insurance database between June 2018 and May 2021 to assess the risk of fracture following treatment with SFO in patients with IDA who were propensity score-matched (approximately 4:1) to controls who had anemia but were not treated with IV iron. Since kidney dysfunction partially protects against severe IV iron-induced hypophosphatemia, a secondary analysis was restricted to patients with normal kidney function. Of the patients treated with SFO, 29,037 were matched to 128,311 untreated controls. Treatment with SFO was associated with significantly higher risk of incident fracture compared with controls (hazard ratio [HR]: 1.154; 95% confidence interval [CI]: 1.088, 1.225; p < 0.0001). In patients with normal kidney function, the risk of fracture was increased in those who received higher cumulative doses of SFO. Patients with kidney dysfunction were also at significantly increased risk of fracture following SFO (HR: 1.512; 95% CI: 1.344, 1.702; p < 0.0001). These real-world data suggest that SFO, known to cause significant hypophosphatemia, also increases the risk of fracture.
INTRODUCTION:Dual-energy quantitative computed tomography (DE-QCT) enables the quantitative measurement of bone mineral density (BMD), but the in vivo precision of these measurements remains largely unexplored. Voxel-based morphometry (VBM) provides a framework for assessing BMD precision at the voxel level, offering spatial detail that is not available using conventional region-based methods. This study aimed to evaluate the absolute and relative precision of DE-QCT-based BMD measurements at regional and voxel levels for future studies of knee osteoarthritis. METHODS:Thirty healthy participants (20 female; mean age = 35.7 ± 12.5 years) underwent two same-day bilateral DE-QCT knee scans with repositioning between scans. Scans were calibrated to provide quantitative measures of BMD, and the femur, tibia, and patella were segmented for analysis. Regional precision was assessed by comparing scan-rescan mean BMD values using root-mean-square standard deviation (SDRMS) and root-mean-square percent coefficient of variation (CVRMS), with least significant change (LSC) calculated for both metrics. Voxel-wise precision was evaluated using the same metrics following spatial normalization and Gaussian smoothing with 5 mm and 8 mm full-width-at-half-maximum (FWHM) kernels. RESULTS:Regional precision was high, with pooled precision estimates across the three bones yielding low LSC values, ranging from 7.08-9.97 mg HA/cm3 (LSC SDRMS) and ≤ 3.03% (LSC CVRMS). At the voxel level, LSC SDRMS was generally low and relatively uniform throughout each bone, with mean values ranging 6.02-6.58 mg HA/cm3 at 5 mm FWHM smoothing and 4.83-5.40 mg HA/cm3 at 8 mm FWHM smoothing across the three bones. Voxel-wise CVRMS exhibited greater spatial variability, with the highest values observed in the femoral and tibial metaphyses. CONCLUSION:Using DE-QCT, we provided spatial maps of reproducibility for voxel-wise BMD measurements in the distal femur, proximal tibia, and patella. At the voxel level, SDRMS provides a more stable and interpretable precision metric than CVRMS and is recommended for voxel-based comparisons.
X-linked hypophosphataemia (XLH) is a rare, lifelong heritable metabolic bone disorder characterized by fibroblast growth factor 23 (FGF23) excess, chronic hypophosphataemia, renal phosphate wasting, progressive skeletal deformities and impaired quality of life. Burosumab, targeted anti-FGF-23 therapy, is approved for use in XLH across the lifespan. Clinical trials have demonstrated benefit, although distinct outcomes in response to treatment vary depending on whether initiated during childhood, adolescence or adulthood. We uniquely describe three patients with XLH across three generations within a single family, each initiated on burosumab at different stages of life. This familial case study highlights the broad phenotypic spectrum of XLH, and differential efficacy profile of burosumab across various stages, such as pre- and post-growth plate closure or in the presence of established musculoskeletal morbidity. We also demonstrate the unique inter-generational impact of burosumab in XLH, as a targeted novel treatment available for patients with a dominantly inherited disorder across the lifespan.
Longitudinal HR-pQCT scans can identify mineralised-tissue gains and losses after image registration, but whether baseline trabecular architecture can rank the locations of subsequent image-derived transitions remains unclear. This study tested whether surface-conditioned local trabecular support can rank subsequent formation-labelled transitions directly from baseline images and whether a longitudinal axial-prolate neighbourhood provides additional information beyond the isotropic local support. Baseline and 6-month distal-tibia HR-pQCT scans of nine postmenopausal women are analysed, giving 18 side-level image pairs. The analysis is restricted to baseline surface-marrow voxels, defined as marrow voxels sharing a face with baseline bone. A spherical support count provides an isotropic local-BV/TV-equivalent baseline. The proposed axial-prolate operator counts baseline bone voxels within an ellipsoidal neighbourhood, with its long axis aligned with the scanner's z-axis. Ranking performance is evaluated using leave-one-subject-out ROC-AUC. The obtained results indicate that the spherical local support ranks subsequent formation-labelled transitions above chance, showing that an isotropic local bone amount already contains prospective surface-conditioned information. The axial-prolate operator further improves this process. Matched-volume comparisons show that both anisotropy and longitudinal orientation contribute to this gain, while local-BV/TV-stratified analyses indicate that the directional signal is not fully reducible to the isotropic local bone amount. These findings establish an image-derived framework for ranking trabecular formation-labelled transitions from the baseline architecture alone. The distal-tibia application indicates that site-informed directional kernels can refine the general local-support framework. External validation is required to determine whether the kernel scale and alignment should be recalibrated for other anatomical sites and populations.
Bone loss is an underestimated consequence of spinal cord injury (SCI), that manifests rapidly and is resistant to currently available treatments including exercise, functional electrical stimulation, anabolic agents, and bisphosphonates. While changes in adrenergic signaling have been linked to bone loss, it remains unclear whether maladaptive sympathetic signaling drives SCI-induced bone loss. We evaluated the effects of sympathetic blockade on SCI-induced bone loss using primary bone marrow osteoclast cultures and an in vivo rat model of a moderate T11 spinal contusion injury using young (300-350 g) male Sprague Dawley rats. In vitro, pre-osteoclast formation was significantly elevated in SCI cultures compared to shams, and exogenous norepinephrine (NE) robustly enhanced this effect. Concomitant administration of adrenergic receptor (AR) antagonists, with varying α- and β-AR selectivity, completely attenuated this NE-driven osteoclastogenesis. Moving in vivo, we utilized a novel intraosseous catheter for targeted delivery of labetalol (mixed α- and β-AR antagonist) and butoxamine (selective β-AR antagonist) directly into the sublesional (below the level of injury) bone marrow space for 28 consecutive day, beginning 24 h post-injury. Analysis of the femur microarchitecture from both legs revealed that blocking local sympathetic signaling failed to protect trabecular bone after SCI. Furthermore, while both labetalol and butoxamine had no effect on locomotor recovery, post-injury weight loss was increased compared to saline-treated SCI controls. These findings demonstrate that while blocking β2-ARs is sufficient to reduce NE-driven osteoclastogenesis, excessive NE does not fully explain SCI-induced bone loss. Local AR antagonists do not appear to be a viable therapeutic strategy to mitigate SCI-induced osteoporosis.
BACKGROUND:Osteoporosis burden is underdiagnosed and undertreated. While increasing community awareness and education are recognised priorities, research on how older adults view osteoporosis is limited. The aim was to assess osteoporosis-related knowledge, intentions and attitudes in relation to other health conditions in Australian adults aged 50 years and older. METHODS:A validated online questionnaire, developed with an expert panel, was administered to 1432 healthy volunteers, (64.3 ± 9.5 years). The questionnaire assessed knowledge of osteoporosis risk factors, attitudes and intentions. Multivariable logistic regression identified factors associated with low knowledge. RESULTS:Knowledge score across six topics was 29.5 ± 14.5 (100 possible). Lowest scores were for 'exercises to improve balance' (11.1 ± 11.5) and highest for 'sources of calcium' (35.0 ± 26.4). The likelihood of being in the lowest knowledge tertile was associated with age (OR: 1.02, CI: 1.00-1.03), lower education level (OR: 1.38, CI: 1.00-1.89), being male (OR: 2.40, CI: 1.87-3.09) and those without a family history of osteoporosis (OR: 1.79, CI: 1.30-2.45). CONCLUSION:The study identifies a concerning lack of knowledge about osteoporosis among older adults. Health promotion strategies to target discrete behaviours and address barriers and promoters for osteoporosis prevention are needed.
Chronic kidney disease (CKD) induces a progressive decline in kidney function, leading to increased inorganic phosphate (Pi) load resulting in higher fibroblast growth factor 23 (FGF23) and cardiovascular complications including vascular calcifications (VC). Parallel rise of parathyroid hormone (PTH) increases bone resorption and release of calcium and Pi. These mineral and bone disorders (MBD) are central to the abnormalities observed in CKD. CKD has higher prevalence in women but higher cardiovascular impact on men, with increased risk of progressing to late stages. Here we characterize CKD-induced MBD in CD1 mice, and investigate sex differences. Twenty-week old male (M) and female (F) mice underwent 5/6 nephrectomy (Nx) then were fed either a normal (NP, 0.55% Pi) or a high phosphate diet (HP, 1.65% Pi) for 10 weeks. Nx mice showed increased markers of mineral metabolism, particularly under HP, including FGF23 (especially in F; HP vs NP: M +184%, F +770%), PTH (M +118%, F +201%) and osteopontin (M +92%, F +74%). Phosphatemia remained stable indicating that the phosphaturic effects of FGF23 were still sufficient, similar to early-stage CKD. Under HP cardiomyocyte hypertrophy was significant in M (+28% cardiomyocyte area vs NP) while bone disorders were more pronounced in F, with increased cortical porosity (+28% vs NP) and turnover. No VC was detected. Thus, HP diet induced a more severe mineral and bone phenotype in Nx CD1 female mice, making this strain a sensitive model to investigate female-predominant skeletal vulnerability in CKD.
Diffuse idiopathic skeletal hyperostosis (DISH) and ossification of the posterior longitudinal ligament (OPLL) are prototypical ectopic spinal ossification disorders, but objective methods for early activity detection and prognostication remain limited. We evaluated ossification trajectories, blood biomarkers, circulating microRNAs, and genetic susceptibility for progression stratification. We analyzed 694 health-screening participants who underwent CT at baseline and follow-up (≥5 years); DISH was scored level-by-level and OPLL was assessed across all levels, using age- and sex-matched controls. Baseline plasma small-RNA sequencing in 40 DISH participants with extreme progression phenotypes (top versus bottom 20 by score change) identified candidate miRNAs via nested cross-validation, repeating feature selection within each fold; genetic plausibility was assessed by two-sample Mendelian randomization (MR) using GTEx miRNA eQTLs and UK Biobank DISH GWAS. DISH showed thoracic-predominant progression; OPLL clustered in the cervical spine, predominantly at C4-C6. OPLL had substantially higher BMI (SMD 0.85); its only FDR-surviving biomarker difference, lower HDL, lost significance after BMI adjustment (adiposity-confounded). CRP/hs-CRP (DISH) and HbA1c (OPLL) were nominally higher but did not survive FDR adjustment. A four-miRNA plasma panel discriminated DISH progression status (nested AUC 0.953), unimproved by adding age, sex, and BMI (AUC 0.897). MR gave nominal genetic evidence linking the miR-15a locus (a single shared -3p/-5p signal) to the DISH flow score, unlike the progression-panel miRNAs. These findings distinguish DISH and OPLL ossification trajectories and biomarker profiles, yield a discovery-stage miRNA panel, and provide genetic evidence for miR-15a in susceptibility; independent cohort validation and orthogonal assay confirmation are required for clinical translation.
Osteoprotegerin (OPG), encoded by TNFRSF11B, plays a critical role in suppressing osteoclastogenesis as a secreted decoy receptor for RANKL. Loss-of-function mutations in TNFRSF11B cause juvenile Paget's disease and related bone metabolic disorders; however, the mechanisms by which individual mutations impair OPG secretion and function remain incompletely understood. Here, we quantitatively analyzed the effects of disease-associated TNFRSF11B variants on OPG secretion and osteoclastogenesis-inhibitory activity. Wild-type and variant OPG proteins were expressed as Gaussia luciferase fusion constructs to measure extracellular secretion, and functional activity was evaluated using a mouse osteoclastogenesis assay. The cysteine-substituted variants C65F and C87Y exhibited severe secretion defects, whereas the T76P and D182del variants showed reductions in both secretion and inhibitory activity. The F117L variant showed normal secretion but markedly reduced inhibitory activity, indicating a secretion-independent functional defect. In contrast, the truncation variants D323fs and R333Ter exhibited reduced secretion while retaining substantial anti-RANKL activity. Further analysis revealed that endoplasmic reticulum-associated degradation (ERAD) contributes to the regulation of OPG secretion. Proteasome inhibition and knockdown of the ERAD components HRD1 and SEL1L increased extracellular levels of wild-type OPG as well as those of D323fs and R333Ter variants. Collectively, our results define mutation-specific mechanisms underlying OPG dysfunction and identify ERAD as a regulator of OPG secretion.
Alcohol use disorder (AUD) is the most commonly diagnosed substance use disorder in the United States, with one in ten people (12 years of age and older) diagnosed as of 2023. AUD can lead to a variety of deleterious outcomes, including increased fracture risk. The reduced bone mineral density (BMD) observed in this patient population is often blamed for this increased fracture risk. However, no other routine skeletal measures are made in the clinic, suggesting AUD could induce detrimental skeletal effects outside of reduced bone mass. Consequently, animal models of AUD have been developed to assess additional measures, but many do not achieve the magnitude of blood ethanol concentrations (BECs) observed in patients with AUD. The crossed high alcohol preferring (cHAP) mouse line achieves these clinically relevant BECs through voluntary alcohol consumption, avoiding the potentially confounding effects of stress associated with forced exposure procedures. Until recently, there has been relatively little toxicological work with this line. Here, the skeletal effects of alcohol consumption in male and female cHAP mice were explored for the first time. We hypothesized 8 weeks of ethanol consumption would cause a loss of skeletal mass, architecture, and mechanical integrity. Ethanol drinking had a significant main effect on body composition measures resulting in lower total BMD in both sexes. Cortical and whole-bone mechanical measures were also lower in both sexes with ethanol drinking. While osteoclast number was not different, adipocyte numbers were higher, particularly in ethanol-drinking females. These findings indicate the cHAP mouse is a useful model of the skeletal outcomes of AUD.
