Nuclear actin structure regulates chromatin accessibility.

Polymerized ß-actin may provide a structural basis for chromatin accessibility and actin transport into the nucleus can guide mesenchymal stem cell (MSC) differentiation. Using MSC, we show that using CK666 to inhibit Arp2/3 directed secondary actin branching results in decreased nuclear actin structure, and significantly alters chromatin access measured with ATACseq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which enhances nuclear actin structure. In addition, nuclear visualization shows Arp2/3 inhibition decreases pericentric H3K9me3 marks. CytoD, alternatively, induces redistribution of H3K27me3 marks centrally. Such alterations in chromatin landscape are consistent with differential gene expression associated with distinctive differentiation patterns. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest increase in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions.

Diet-Stimulated Marrow Adiposity Fails to Worsen Early, Age-Related Bone Loss.

INTRODUCTION: Longitudinal effect of diet-induced obesity on bone is uncertain. Prior work showed both no effect and a decrement in bone density or quality when obesity begins prior to skeletal maturity. We aimed to quantify long-term effects of obesity on bone and bone marrow adipose tissue (BMAT) in adulthood. METHODS: Skeletally mature, female C57BL/6 mice (n = 70) aged 12 weeks were randomly allocated to low-fat diet (LFD; 10% kcal fat; n = 30) or high-fat diet (HFD; 60% kcal fat; n = 30), with analyses at 12, 15, 18, and 24 weeks (n = 10/group). Tibial microarchitecture was analyzed by µCT, and volumetric BMAT was quantified via 9.4T MRI/advanced image analysis. Histomorphometry of adipocytes and osteoclasts, and qPCR were performed. RESULTS: Body weight and visceral white adipose tissue accumulated in response to HFD started in adulthood. Trabecular bone parameters declined with advancing experimental age. BV/TV declined 22% in LFD (p = 0.0001) and 17% in HFD (p = 0.0022) by 24 weeks. HFD failed to appreciably alter BV/TV and had negligible impact on other microarchitecture parameters. Both dietary intervention and age accounted for variance in BMAT, with regional differences: distal femoral BMAT was more responsive to diet, while proximal femoral BMAT was more attenuated by age. BMAT increased 60% in the distal metaphysis in HFD at 18 and 24 weeks (p = 0.0011). BMAT in the proximal femoral diaphysis, unchanged by diet, decreased 45% due to age (p = 0.0002). Marrow adipocyte size via histomorphometry supported MRI quantification. Osteoclast number did not differ between groups. Tibial qPCR showed attenuation of some adipose, metabolism, and bone genes. A regulator of fatty acid ß-oxidation, cytochrome C (CYCS), was 500% more abundant in HFD bone (p < 0.0001; diet effect). CYCS also increased due to age, but to a lesser extent. HFD mildly increased OCN, TRAP, and SOST. CONCLUSIONS: Long-term high fat feeding after skeletal maturity, despite upregulation of visceral adiposity, body weight, and BMAT, failed to attenuate bone microarchitecture. In adulthood, we found aging to be a more potent regulator of microarchitecture than diet-induced obesity.

Extensive Osteonecrosis After Glucocorticoids: Clinical Response to Bisphosphonate.

Osteonecrosis is a devastating complication of long-term glucocorticoid therapy that has been described in both malignant and nonmalignant diseases. Its incidence has been found to greater than 50% using magnetic resonance imaging in asymptomatic patients, thus osteonecrosis is likely underdiagnosed. Recent studies have suggested that treatment with bisphosphonates can improve pain and mobility and decrease bone marrow edema. We describe a patient with acute lymphoblastic leukemia who presented with debilitating osteonecrosis after treatment with prednisone for a total cumulative dose of 5100 mg. Magnetic resonance imaging revealed extensive infarcts of her bilateral tibiae and femora and left humerus, talus, and calcaneus consistent with osteonecrosis that had persisted for more than 2 years. Her severe knee, shoulder, and ankle pain was treated with 1 dose zolendronic acid. Despite a prolonged acute phase reaction, the patient's symptoms improved with near total resolution of pain.

Data driven and cell specific determination of nuclei-associated actin structure.

Quantitative and volumetric assessment of filamentous actin fibers (F-actin) remains challenging due to their interconnected nature, leading researchers to utilize threshold based or qualitative measurement methods with poor reproducibility. Here we introduce a novel machine learning based methodology for accurate quantification and reconstruction of nuclei-associated F-actin. Utilizing a Convolutional Neural Network (CNN), we segment actin filaments and nuclei from 3D confocal microscopy images and then reconstruct each fiber by connecting intersecting contours on cross-sectional slices. This allowed measurement of the total number of actin filaments and individual actin filament length and volume in a reproducible fashion. Focusing on the role of F-actin in supporting nucleocytoskeletal connectivity, we quantified apical F-actin, basal F-actin, and nuclear architecture in mesenchymal stem cells (MSCs) following the disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) Complexes. Disabling LINC in mesenchymal stem cells (MSCs) generated F-actin disorganization at the nuclear envelope characterized by shorter length and volume of actin fibers contributing a less elongated nuclear shape. Our findings not only present a new tool for mechanobiology but introduce a novel pipeline for developing realistic computational models based on quantitative measures of F-actin.

The skeleton in a physical world.

All organisms exist within a physical space and respond to physical forces as part of daily life. In higher organisms, the skeleton is critical for locomotion in the physical environment, providing a carapace upon which the animal can move to accomplish functions necessary for living. As such, the skeleton has responded evolutionarily, and does in real-time, to physical stresses placed on it to ensure that its structure supports its function in the sea, in the air, and on dry land. In this article, we consider how those cells responsible for remodeling skeletal structure respond to mechanical force including load magnitude, frequency, and cyclicity, and how force rearranges cellular structure in turn. The effects of these forces to balance the mesenchymal stem cell supply of bone-forming osteoblasts and energy storing adipocytes are addressed. That this phenotypic switching is achieved at the level of both gene transactivation and alteration of structural epigenetic controls of gene expression is considered. Finally, as clinicians, we consider this information as it applies to a prescriptive for intelligent exercise.

Mechanically Induced Nuclear Shuttling of ß-Catenin Requires Co-transfer of Actin.

Mesenchymal stem cells (MSCs) respond to environmental forces with both cytoskeletal re-structuring and activation of protein chaperones of mechanical information, ß-catenin, and yes-associated protein 1 (YAP1). To function, MSCs must differentiate between dynamic forces such as cyclic strains of extracellular matrix due to physical activity and static strains due to ECM stiffening. To delineate how MSCs recognize and respond differently to both force types, we compared effects of dynamic (200 cycles × 2%) and static (1 × 2% hold) strain on nuclear translocation of ß-catenin and YAP1 at 3 hours after force application. Dynamic strain induced nuclear accumulation of ß-catenin, and increased cytoskeletal actin structure and cell stiffness, but had no effect on nuclear YAP1 levels. Critically, both nuclear actin and nuclear stiffness increased along with dynamic strain-induced ß-catenin transport. Augmentation of cytoskeletal structure using either static strain or lysophosphatidic acid did not increase nuclear content of ß-catenin or actin, but induced robust nuclear increase in YAP1. As actin binds ß-catenin, we considered whether ß-catenin, which lacks a nuclear localization signal, was dependent on actin to gain entry to the nucleus. Knockdown of cofilin-1 (Cfl1) or importin-9 (Ipo9), which co-mediate nuclear transfer of G-actin, prevented dynamic strain-mediated nuclear transfer of both ß-catenin and actin. In sum, dynamic strain induction of actin re-structuring promotes nuclear transport of G-actin, concurrently supporting nuclear access of ß-catenin via mechanisms used for actin transport. Thus, dynamic and static strain activate alternative mechanoresponses reflected by differences in the cellular distributions of actin, ß-catenin, and YAP1.

Architectural control of mesenchymal stem cell phenotype through nuclear actin.

There is growing appreciation that architectural components of the nucleus regulate gene accessibility by altering chromatin organization. While nuclear membrane connector proteins link the mechanosensitive actin cytoskeleton to the nucleoskeleton, actin's contribution to the inner architecture of the nucleus remains enigmatic. Control of actin transport into the nucleus, plus the presence of proteins that control actin structure (the actin tool-box) within the nucleus, suggests that nuclear actin may support biomechanical regulation of gene expression. Cellular actin structure is mechanoresponsive: actin cables generated through forces experienced at the plasma membrane transmit force into the nucleus. We posit that dynamic actin remodeling in response to such biomechanical cues provides a novel level of structural control over the epigenetic landscape. We here propose to bring awareness to the fact that mechanical forces can promote actin transfer into the nucleus and control structural arrangements as illustrated in mesenchymal stem cells, thereby modulating lineage commitment.

G protein-coupled receptor kinase 3 modulates mesenchymal stem cell proliferation and differentiation through sphingosine-1-phosphate receptor regulation.

BACKGROUND: The bone marrow niche supports hematopoietic cell development through intimate contact with multipotent stromal mesenchymal stem cells; however, the intracellular signaling, function, and regulation of such supportive niche cells are still being defined. Our study was designed to understand how G protein receptor kinase 3 (GRK3) affects bone marrow mesenchymal stem cell function by examining primary cells from GRK3-deficient mice, which we have previously published to have a hypercellular bone marrow and leukocytosis through negative regulation of CXCL12/CXCR4 signaling. METHODS: Murine GRK3-deficient bone marrow mesenchymal stromal cells were harvested and cultured to differentiate into three lineages (adipocyte, chondrocyte, and osteoblast) to confirm multipotency and compared to wild type cells. Immunoblotting, modified-TANGO experiments, and flow cytometry were used to further examine the effects of GRK3 deficiency on bone marrow mesenchymal stromal cell receptor signaling. Microcomputed tomography was used to determine trabecular and cortical bone composition of GRK3-deficient mice and standard ELISA to quantitate CXCL12 production from cellular cultures. RESULTS: GRK3-deficient, bone marrow-derived mesenchymal stem cells exhibit enhanced and earlier osteogenic differentiation in vitro. The addition of a sphingosine kinase inhibitor abrogated the osteogenic proliferation and differentiation, suggesting that sphingosine-1-phosphate receptor signaling was a putative G protein-coupled receptor regulated by GRK3. Immunoblotting showed prolonged ERK1/2 signaling after stimulation with sphingosine-1-phosphate in GRK3-deficient cells, and modified-TANGO assays suggested the involvement of ß-arrestin-2 in sphingosine-1-phosphate receptor internalization. CONCLUSIONS: Our work suggests that GRK3 regulates sphingosine-1-phosphate receptor signaling on bone marrow mesenchymal stem cells by recruiting ß-arrestin to the occupied GPCR to promote internalization, and lack of such regulation affects mesenchymal stem cell functionality.

Exercise to Mend Aged-tissue Crosstalk in Bone Targeting Osteoporosis & Osteoarthritis.

Aging induces alterations in bone structure and strength through a multitude of processes, exacerbating common aging- related diseases like osteoporosis and osteoarthritis. Cellular hallmarks of aging are examined, as related to bone and the marrow microenvironment, and ways in which these might contribute to a variety of age-related perturbations in osteoblasts, osteocytes, marrow adipocytes, chondrocytes, osteoclasts, and their respective progenitors. Cellular senescence, stem cell exhaustion, mitochondrial dysfunction, epigenetic and intracellular communication changes are central pathways and recognized as associated and potentially causal in aging. We focus on these in musculoskeletal system and highlight knowledge gaps in the literature regarding cellular and tissue crosstalk in bone, cartilage, and the bone marrow niche. While senolytics have been utilized to target aging pathways, here we propose non-pharmacologic, exercise-based interventions as prospective "senolytics" against aging effects on the skeleton. Increased bone mass and delayed onset or progression of osteoporosis and osteoarthritis are some of the recognized benefits of regular exercise across the lifespan. Further investigation is needed to delineate how cellular indicators of aging manifest in bone and the marrow niche and how altered cellular and tissue crosstalk impact disease progression, as well as consideration of exercise as a therapeutic modality, as a means to enhance discovery of bone-targeted therapies.

Lamin A/C Is Dispensable to Mechanical Repression of Adipogenesis.

Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C.

Lumbar Scoliosis in Postmenopausal Women Increases with Age but is not Associated with Osteoporosis.

CONTEXT: The contribution of lumbar scoliosis to osteoporosis is unknown. OBJECTIVE: This work aimed to determine the prevalence and relationship of lumbar scoliosis to osteoporosis in aging women. METHODS: A cross-sectional analysis used dual-energy x-ray absorptiometry (DXA) scans of randomly selected groups of postmenopausal women (64-68, 74-78, and 84-88 years; N = 300 each) in a university teaching hospital from 2014 to 2019. Lumbar Cobb angle was tested for an association to femoral neck (FN), total hip (TH), and spine T score, age, weight, and ethnicity. Logistic regression tested an association between scoliosis (Cobb angle > 10°) and osteoporosis (T score ≤ -2.5). Available sequential DXA scans (N = 51) were analyzed for changes in Cobb angle using a linear mixed model of these longitudinal data. RESULTS: Osteoporosis and Cobb angle both increased with age: from 22% and 4.4 (SD = 7.8) respectively in 64- to 68-year-olds to 32.9% and to 9.7 (SD = 9.2) in women age 84 to 88 years. The prevalence of clinically significant scoliosis rose from 11.5% in the youngest group, to 27.3% and 39.4% in the age 74 to 78 and 84 to 88 cohorts, respectively. Cobb angle increased 0.7° per year of follow-up. After adjusting for covariates, there was no significant association between T scores at any site (TH, FN, or spine) and Cobb angle. CONCLUSION: Based on screening DXAs, the incidence and degree of lumbar scoliosis increases significantly in women between age 65 and 85 years. There was no association between the incidence of lumbar scoliosis and FN bone density.

Low-Dose Tamoxifen Induces Significant Bone Formation in Mice.

Use of the selective estrogen receptor modulator Tamoxifen (TAM) is a mainstay to induce conditional expression of Cre recombinase in transgenic laboratory mice. To excise ß-catenin fl/fl in 28-day-old male and female Prrx1-CreER/ß-catenin fl/fl mice (C57BL/6), we utilized TAM at 150 mg/kg; despite ß-catenin knockout in MSC, we found a significant increase in trabecular and cortical bone volume in all genders. Because TAM was similarly anabolic in KO and control mice, we investigated a dose effect on bone formation by treating wild-type mice (WT C57BL/6, 4 weeks) with TAM (total dose 0, 20, 40, 200 mg/kg via four injections). TAM increased bone in a dose-dependent manner analyzed by micro-computed tomography (µCT), which showed that, compared to control, 20 mg/kg TAM increased femoral bone volume fraction (bone volume/total volume [BV/TV]) (21.6% ± 1.5% to 33% ± 2.5%; 153%, p < 0.005). With TAM 40 mg/kg and 200 mg/kg, BV/TV increased to 48.1% ± 4.4% (223%, p < 0.0005) and 58% ± 3.8% (269%, p < 0.0001) respectively, compared to control. Osteoblast markers increased with 200 mg/kg TAM: Dlx5 (224%, p < 0.0001), Alp (166%, p < 0.0001), Bglap (223%, p < 0.0001), and Sp7 (228%, p < 0.0001). Osteoclasts per bone surface (Oc#/BS) nearly doubled at the lowest TAM dose (20 mg/kg), but decreased to <20% control with 200 mg/kg TAM. Our data establish that use of TAM at even very low doses to excise a floxed target in postnatal mice has profound effects on trabecular and cortical bone formation. As such, TAM treatment is a major confounder in the interpretation of bone phenotypes in conditional gene knockout mouse models. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Exercise Increases Bone in SEIPIN Deficient Lipodystrophy, Despite Low Marrow Adiposity.

Exercise, typically beneficial for skeletal health, has not yet been studied in lipodystrophy, a condition characterized by paucity of white adipose tissue, with eventual diabetes, and steatosis. We applied a mouse model of global deficiency of Bscl2 (SEIPIN), required for lipid droplet formation. Male twelve-week-old B6 knockouts (KO) and wild type (WT) littermates were assigned six-weeks of voluntary, running exercise (E) versus non-exercise (N=5-8). KO weighed 14% less than WT (p=0.01) and exhibited an absence of epididymal adipose tissue; KO liver Plin1 via qPCR was 9-fold that of WT (p=0.04), consistent with steatosis. Bone marrow adipose tissue (BMAT), unlike white adipose, was measurable, although 40.5% lower in KO vs WT (p=0.0003) via 9.4T MRI/advanced image analysis. SEIPIN ablation's most notable effect marrow adiposity was in the proximal femoral diaphysis (-56% KO vs WT, p=0.005), with relative preservation in KO-distal-femur. Bone via µCT was preserved in SEIPIN KO, though some quality parameters were attenuated. Running distance, speed, and time were comparable in KO and WT. Exercise reduced weight (-24% WT-E vs WT p<0.001) but not in KO. Notably, exercise increased trabecular BV/TV in both (+31%, KO-E vs KO, p=0.004; +14%, WT-E vs WT, p=0.006). The presence and distribution of BMAT in SEIPIN KO, though lower than WT, is unexpected and points to a uniqueness of this depot. That trabecular bone increases were achievable in both KO and WT, despite a difference in BMAT quantity/distribution, points to potential metabolic flexibility during exercise-induced skeletal anabolism.

Evaluation of zoledronate, cytochalasin-D, and desferrioxamine on osseointegration in an intra-medullary femoral implant model.

OBJECTIVE: The rise in primary and revision surgeries utilizing joint replacement implants suggest the need for more reliable means of promoting implant fixation. Zoledronate-(Zol), cytochalasin-D-(cytoD), and desferrioxamine-(DFO) have been shown to enhance mesenchymal stem cell (MSC) differentiation into osteoblasts promoting bone formation. The objective was to determine whether Zol, cytoD, and DFO can improve fixation strength and enhance peri-implant bone volume about intra-medullary femoral implants. METHODS: 48 Sprague-Dawley female rats were randomized into four treatments, saline-control or experimental: Zol-(0.8 µg/µL), cytoD-(0.05 µg/µL), DFO-(0.4 µg/µL). Implants were placed bilaterally in the femoral canals following injection of treatment solution and followed for 28 days. Mechanical push-out testing and micro-CT were our primary evaluations, measuring load to failure and bone volume. Qualitative evaluation included histological assessment. Data was analyzed with a one-way ANOVA with Holm-Sidak mean comparison testing. RESULTS: Significant results included pushout tests showing an increase in maximum energy for Zol (124%) and cytoD (82%); Zol showed an increase in maximum load by 48%; Zol micro-CT showed increase in BV/TV by 35%. CONCLUSIONS: Our findings suggest that locally applied Zol and cytoD enhance implant mechanical stability. Bisphosphonates and actin regulators, like cytoD, might be further investigated as a new strategy for improving osseointegration.

ß-Catenin Preserves the Stem State of Murine Bone Marrow Stromal Cells Through Activation of EZH2.

During bone marrow stromal cell (BMSC) differentiation, both Wnt signaling and the development of a rigid cytoskeleton promote commitment to the osteoblastic over adipogenic lineage. ß-catenin plays a critical role in the Wnt signaling pathway to facilitate downstream effects on gene expression. We show that ß-catenin was additive with cytoskeletal signals to prevent adipogenesis, and ß-catenin knockdown promoted adipogenesis even when the actin cytoskeleton was depolymerized. ß-catenin also prevented osteoblast commitment in a cytoskeletal-independent manner, with ß-catenin knockdown enhancing lineage commitment. Chromatin immunoprecipitation (ChIP)-sequencing demonstrated binding of ß-catenin to the promoter of enhancer of zeste homolog 2 (EZH2), a key component of the polycomb repressive complex 2 (PRC2) complex that catalyzes histone methylation. Knockdown of ß-catenin reduced EZH2 protein levels and decreased methylated histone 3 (H3K27me3) at osteogenic loci. Further, when EZH2 was inhibited, ß-catenin's anti-differentiation effects were lost. These results indicate that regulating EZH2 activity is key to ß-catenin's effects on BMSCs to preserve multipotentiality. © 2020 American Society for Bone and Mineral Research.

Effects of Iron Isomaltoside vs Ferric Carboxymaltose on Hypophosphatemia in Iron-Deficiency Anemia: Two Randomized Clinical Trials.

Importance: Intravenous iron enables rapid correction of iron-deficiency anemia, but certain formulations induce fibroblast growth factor 23-mediated hypophosphatemia. Objective: To compare risks of hypophosphatemia and effects on biomarkers of mineral and bone homeostasis of intravenous iron isomaltoside (now known as ferric derisomaltose) vs ferric carboxymaltose. Design, Setting, and Participants: Between October 2017 and June 2018, 245 patients aged 18 years and older with iron-deficiency anemia (hemoglobin level ≤11 g/dL; serum ferritin level ≤100 ng/mL) and intolerance or unresponsiveness to 1 month or more of oral iron were recruited from 30 outpatient clinic sites in the United States into 2 identically designed, open-label, randomized clinical trials. Patients with reduced kidney function were excluded. Serum phosphate and 12 additional biomarkers of mineral and bone homeostasis were measured on days 0, 1, 7, 8, 14, 21, and 35. The date of final follow-up was June 19, 2018, for trial A and May 29, 2018, for trial B. Interventions: Intravenous administration of iron isomaltoside, 1000 mg, on day 0 or ferric carboxymaltose, 750 mg, infused on days 0 and 7. Main Outcomes and Measures: The primary end point was the incidence of hypophosphatemia (serum phosphate level <2.0 mg/dL) between baseline and day 35. Results: In trial A, 123 patients were randomized (mean [SD] age, 45.1 [11.0] years; 95.9% women), including 62 to iron isomaltoside and 61 to ferric carboxymaltose; 95.1% completed the trial. In trial B, 122 patients were randomized (mean [SD] age, 42.6 [12.2] years; 94.1% women), including 61 to iron isomaltoside and 61 to ferric carboxymaltose; 93.4% completed the trial. The incidence of hypophosphatemia was significantly lower following iron isomaltoside vs ferric carboxymaltose (trial A: 7.9% vs 75.0% [adjusted rate difference, -67.0% {95% CI, -77.4% to -51.5%}], P < .001; trial B: 8.1% vs 73.7% [adjusted rate difference, -65.8% {95% CI, -76.6% to -49.8%}], P < .001). Beyond hypophosphatemia and increased parathyroid hormone, the most common adverse drug reactions (No./total No.) were nausea (iron isomaltoside: 1/125; ferric carboxymaltose: 8/117) and headache (iron isomaltoside: 4/125; ferric carboxymaltose: 5/117). Conclusions and Relevance: In 2 randomized trials of patients with iron-deficiency anemia who were intolerant of or unresponsive to oral iron, iron isomaltoside (now called ferric derisomaltose), compared with ferric carboxymaltose, resulted in lower incidence of hypophosphatemia over 35 days. However, further research is needed to determine the clinical importance of this difference. Trial Registration: ClinicalTrials.gov Identifiers: NCT03238911 and NCT03237065.

Exercise Degrades Bone in Caloric Restriction, Despite Suppression of Marrow Adipose Tissue (MAT).

Marrow adipose tissue (MAT) and its relevance to skeletal health during caloric restriction (CR) is unknown: It remains unclear whether exercise, which is anabolic to bone in a calorie-replete state, alters bone or MAT in CR. We hypothesized that response of bone and MAT to exercise in CR differs from the calorie-replete state. Ten-week-old female B6 mice fed a regular diet (RD) or 30% CR diet were allocated to sedentary (RD, CR, n = 10/group) or running exercise (RD-E, CR-E, n = 7/group). After 6 weeks, CR mice weighed 20% less than RD, p < 0.001; exercise did not affect weight. Femoral bone volume (BV) via 3D MRI was 20% lower in CR versus RD (p < 0.0001). CR was associated with decreased bone by µCT: Tb.Th was 16% less in CR versus RD, p < 0.003, Ct.Th was 5% less, p < 0.07. In CR-E, Tb.Th was 40% less than RD-E, p < 0.0001. Exercise increased Tb.Th in RD (+23% RD-E versus RD, p < 0.003) but failed to do so in CR. Cortical porosity increased after exercise in CR (+28%, p = 0.04), suggesting exercise during CR is deleterious to bone. In terms of bone fat, metaphyseal MAT/ BV rose 159% in CR versus RD, p = 0.003 via 3D MRI. Exercise decreased MAT/BV by 52% in RD, p < 0.05, and also suppressed MAT in CR (-121%, p = 0.047). Histomorphometric analysis of adipocyte area correlated with MAT by MRI (R2 = 0.6233, p < 0.0001). With respect to bone, TRAP and Sost mRNA were reduced in CR. Intriguingly, the repressed Sost in CR rose with exercise and may underlie the failure of CR-bone quantity to increase in response to exercise. Notably, CD36, a marker of fatty acid uptake, rose 4088% in CR (p < 0.01 versus RD), suggesting that basal increases in MAT during calorie restriction serve to supply local energy needs and are depleted during exercise with a negative impact on bone. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Knockdown of formin mDia2 alters lamin B1 levels and increases osteogenesis in stem cells.

Nuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In marrow-derived MSCs, the principal diaphanous-related formin Diaph3 (mDia2) is present in the nucleus and regulates intranuclear actin polymerization, whereas Diaph1 (mDia1) is localized to the cytoplasm and controls cytoplasmic actin polymerization. We here show that mDia2 can be used as a tool to query actin-lamin nucleoskeletal structure. Silencing mDia2 affected the nucleoskeletal lamin scaffold, altering nuclear morphology without affecting cytoplasmic actin cytoskeleton, and promoted MSC differentiation. Attempting to target intranuclear actin polymerization by silencing mDia2 led to a profound loss in lamin B1 nuclear envelope structure and integrity, increased nuclear height, and reduced nuclear stiffness without compensatory changes in other actin nucleation factors. Loss of mDia2 with the associated loss in lamin B1 promoted Runx2 transcription and robust osteogenic differentiation and suppressed adipogenic differentiation. Hence, mDia2 is a potent tool to query intranuclear actin-lamin nucleoskeletal structure, and its presence serves to retain multipotent stromal cells in an undifferentiated state.

Persistently Elevated PTH After Parathyroidectomy at One Year: Experience in a Tertiary Referral Center.

CONTEXT/OBJECTIVE: Increased PTH after successful parathyroid surgery represents a clinical conundrum. We aimed to determine the prevalence of persistently elevated PTH (PePTH) postsurgery, along with predisposing factors. DESIGN: and Setting: Patients ≥ age 18 with parathyroidectomy performed at University of North Carolina Hospitals for primary hyperparathyroidism (PHPT) over a 12-year period were identified from the Carolina Data Warehouse. Clinical and demographic characteristics were collected, transformed, and analyzed. RESULTS: Five hundred seventy patients met initial criteria for PHPT, and of those 407 had postoperative values. One hundred forty-four had laboratory results within 3 to 18 months post operatively. There was no clinical difference between those with and without long-term laboratory follow-up. Presurgery, patients had average calcium of 11 mg/dL and PTH 125.4 pg/mL. Ninety-seven percent of patients had normalized calcium after surgery, but 30% had PePTH, which can be predicted at 3 months. Patients with PePTH (persistent elevation of PTH) after surgery did not differ from those with normalized PTH in terms of sex, age, body mass index, or excised gland weight; presurgery 25-vitamin D was slightly lower, but not abnormal (26 ± 15 vs 36 ± 11). The presurgical PTH was significantly higher (P < 0.001) in those with PePTH (156.5 pg/mL compared with presurgical level of 102.5 in those whose PTH normalized). CONCLUSIONS: Nearly one-third of PHPT patients have elevated PTH levels postsurgery in a tertiary hospital setting. At presentation, patients with PePTH tend to have higher PTH relative to calcium levels. Whether PePTH after surgical treatment of PHPT has pathological consequences is unknown.