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.

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.

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.

Exercise and Diet: Uncovering Prospective Mediators of Skeletal Fragility in Bone and Marrow Adipose Tissue.

PURPOSE OF REVIEW: To highlight recent basic, translational, and clinical works demonstrating exercise and diet regulation of marrow adipose tissue (MAT) and bone and how this informs current understanding of the relationship between marrow adiposity and musculoskeletal health. RECENT FINDINGS: Marrow adipocytes accumulate in the bone in the setting of not only hypercaloric intake (calorie excess; e.g., diet-induced obesity) but also with hypocaloric intake (calorie restriction; e.g., anorexia), despite the fact that these states affect bone differently. With hypercaloric intake, bone quantity is largely unaffected, whereas with hypocaloric intake, bone quantity and quality are greatly diminished. Voluntary running exercise in rodents was found to lower MAT and promote bone in eucaloric and hypercaloric states, while degrading bone in hypocaloric states, suggesting differential modulation of MAT and bone, dependent upon whole-body energy status. Energy status alters bone metabolism and bioenergetics via substrate availability or excess, which plays a key role in the response of bone and MAT to mechanical stimuli. Marrow adipose tissue (MAT) is a fat depot with a potential role in-as well as responsivity to-whole-body energy metabolism. Understanding the localized function of this depot in bone cell bioenergetics and substrate storage, principally in the exercised state, will aid to uncover putative therapeutic targets for skeletal fragility.

Intranuclear Actin Structure Modulates Mesenchymal Stem Cell Differentiation.

Actin structure contributes to physiologic events within the nucleus to control mesenchymal stromal cell (MSC) differentiation. Continuous cytochalasin D (Cyto D) disruption of the MSC actin cytoskeleton leads to osteogenic or adipogenic differentiation, both requiring mass transfer of actin into the nucleus. Cyto D remains extranuclear, thus intranuclear actin polymerization is potentiated by actin transfer: we asked whether actin structure affects differentiation. We show that secondary actin filament branching via the Arp2/3 complex is required for osteogenesis and that preventing actin branching stimulates adipogenesis, as shown by expression profiling of osteogenic and adipogenic biomarkers and unbiased RNA-seq analysis. Mechanistically, Cyto D activates osteoblast master regulators (e.g., Runx2, Sp7, Dlx5) and novel coregulated genes (e.g., Atoh8, Nr4a3, Slfn5). Formin-induced primary actin filament formation is critical for Arp2/3 complex recruitment: osteogenesis is prevented by silencing of the formin mDia1, but not its paralog mDia2. Furthermore, while inhibition of actin, branching is a potent adipogenic stimulus, silencing of either mDia1 or mDia2 blocks adipogenic gene expression. We propose that mDia1, which localizes in the cytoplasm of multipotential MSCs and traffics into the nucleus after cytoskeletal disruption, joins intranuclear mDia2 to facilitate primary filament formation before mediating subsequent branching via Arp2/3 complex recruitment. The resulting intranuclear branched actin network specifies osteogenic differentiation, while actin polymerization in the absence of Arp2/3 complex-mediated secondary branching causes adipogenic differentiation. Stem Cells 2017;35:1624-1635.

Physical Signals May Affect Mesenchymal Stem Cell Differentiation via Epigenetic Controls.

Marrow mesenchymal stem cells supply bone osteoblasts and adipocytes. Exercise effects to increase bone and decrease fat involve transfer of signals from the cytoplasm into the nucleus to regulate gene expression. We propose that exercise control of stem cell fate relies on structural connections that terminate in the nucleus and involve intranuclear actin structures that regulate epigenetic gene expression.

Intranuclear Actin Regulates Osteogenesis.

Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and global effects on gene transcription. We here show that in mesenchymal stem cells (MSCs), cytochalasin D treatment causes rapid cofilin-/importin-9-dependent transfer of G-actin into the nucleus. The continued presence of intranuclear actin, which forms rod-like structures that stain with phalloidin, is associated with induction of robust expression of the osteogenic genes osterix and osteocalcin in a Runx2-dependent manner, and leads to acquisition of osteogenic phenotype. Adipogenic differentiation also occurs, but to a lesser degree. Intranuclear actin leads to nuclear export of Yes-associated protein (YAP); maintenance of nuclear YAP inhibits Runx2 initiation of osteogenesis. Injection of cytochalasin into the tibial marrow space of live mice results in abundant bone formation within the space of 1 week. In sum, increased intranuclear actin forces MSC into osteogenic lineage through controlling Runx2 activity; this process may be useful for clinical objectives of forming bone.

Cell Mechanosensitivity to Extremely Low-Magnitude Signals Is Enabled by a LINCed Nucleus.

A cell's ability to recognize and adapt to the physical environment is central to its survival and function, but how mechanical cues are perceived and transduced into intracellular signals remains unclear. In mesenchymal stem cells (MSCs), high-magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of focal adhesion (FA) kinase (FAK)/mTORC2/Akt signaling generated at FAs. Physiologic systems also rely on a persistent barrage of low-level signals to regulate behavior. Exposing MSC to extremely low-magnitude mechanical signals (LMS) suppresses adipocyte formation despite the virtual absence of substrate strain (<0.001%), suggesting that LMS-induced dynamic accelerations can generate force within the cell. Here, we show that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating FAK and Akt signaling followed by FAK-dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the FAs, LMS-induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear-actin cytoskeleton mechanocoupling by disrupting linker of nucleoskeleton and cytoskeleton (LINC) complexes inhibited these LMS-induced signals as well as prevented LMS repression of adipogenic differentiation, highlighting that LINC connections are critical for sensing LMS. In contrast, FAK activation by HMS was unaffected by LINC decoupling, consistent with signal initiation at the FA mechanosome. These results indicate that the MSC responds to its dynamic physical environment not only with "outside-in" signaling initiated by substrate strain, but vibratory signals enacted through the LINC complex enable matrix independent "inside-inside" signaling.

Mechanically activated Fyn utilizes mTORC2 to regulate RhoA and adipogenesis in mesenchymal stem cells.

Mechanical strain provides an anti-adipogenic, pro-osteogenic stimulus to mesenchymal stem cells (MSC) through generating intracellular signals and via cytoskeletal restructuring. Recently, mTORC2 has been shown to be a novel mechanical target critical for the anti-adipogenic signal leading to preservation of ß-catenin. As mechanical activation of mTORC2 requires focal adhesions (FAs), we asked whether proximal signaling involved Src and FAK, which are early responders to integrin-FA engagement. Application of mechanical strain to marrow-derived MSCs was unable to activate mTORC2 when Src family kinases were inhibited. Fyn, but not Src, was specifically required for mechanical activation of mTORC2 and was recruited to FAs after strain. Activation of mTORC2 was further diminished following FAK inhibition, and as FAK phosphorylation (Tyr-397) required Fyn activity, provided evidence of Fyn/FAK cooperativity. Inhibition of Fyn also prevented mechanical activation of RhoA as well as mechanically induced actin stress fiber formation. We thus asked whether RhoA activation by strain was dependent on mTORC2 downstream of Fyn. Inhibition of mTORC2 or its downstream substrate, Akt, both prevented mechanical RhoA activation, indicating that Fyn/FAK affects cytoskeletal structure via mTORC2. We then sought to ascertain whether this Fyn-initiated signal pathway modulated MSC lineage decisions. siRNA knockdown of Fyn, but not Src, led to rapid attainment of adipogenic phenotype with significant increases in adipocyte protein 2, peroxisome proliferator-activated receptor gamma, adiponectin, and perilipin. As such, Fyn expression in mdMSCs contributes to basal cytoskeletal architecture and, when associated with FAs, functions as a proximal mechanical effector for environmental signals that influence MSC lineage allocation.

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.

Mechanical regulation of glycogen synthase kinase 3ß (GSK3ß) in mesenchymal stem cells is dependent on Akt protein serine 473 phosphorylation via mTORC2 protein.

Mechanical signals can inactivate glycogen synthase kinase 3ß (GSK3ß), resulting in stabilization of ß-catenin. This signaling cascade is necessary for the inhibition of adipogenesis in mesenchymal stem cells (MSC) that is produced by a daily strain regimen. We investigated whether Akt is the mechanically activated kinase responsible for phosphorylation and inactivation of GSK3ß in MSC. Mechanical strain (2% magnitude, 0.17 Hz) induced phosphorylation of Akt at Ser-473 and Thr-308 in parallel with phosphorylation of GSK3ß at Ser-9. Inhibiting Akt (Akt1/2 kinase inhibitor treatment or Akt knockdown) prevented strain-induced phosphorylation of GSK3ß at Ser-9. Inhibition of PI3K prevented Thr-308 phosphorylation, but strain-induced Ser-473 phosphorylation was measurable and induced phosphorylation of GSK3ß, suggesting that Ser-473 phosphorylation is sufficient for the downstream mechanoresponse. As Rictor/mTORC2 (mammalian target of rapamycin complex 2) is known to transduce phosphorylation of Akt at Ser-473 by insulin, we investigated whether it contributes to strain-induced Ser-473 phosphorylation. Phosphorylation of Ser-473 by both mechanical and insulin treatment in MSC was prevented by the mTOR inhibitor KU0063794. When mTORC2 was blocked, mechanical GSK3ß inactivation was prevented, whereas insulin inhibition of GSK3ß was still measured in the absence of Ser-473 phosphorylation, presumably through phosphorylation of Akt at Thr-308. In sum, mechanical input initiates a signaling cascade that is uniquely dependent on mTORC2 activation and phosphorylation of Akt at Ser-473, an effect sufficient to cause inactivation of GSK3ß. Thus, mechanical regulation of GSK3ß downstream of Akt is dependent on phosphorylation of Akt at Ser-473 in a manner distinct from that of growth factors. As such, Akt reveals itself to be a pleiotropic signaling molecule whose downstream targets are differentially regulated depending upon the nature of the activating input.

Mechanically induced focal adhesion assembly amplifies anti-adipogenic pathways in mesenchymal stem cells.

The fate of pluripotent mesenchymal stem cells (MSC) is determined through integration of chemical, spatial, and physical signals. The suppression of MSC adipogenesis by mechanical stimuli, which requires Akt-induced inhibition of glycogen synthase kinase 3ß (GSK3ß) with ß-catenin activation, can be enhanced by repetitive dosing within a single day. Here, we demonstrate that reapplication of cyclic strain within a 24-hour period leads to amplification of both Akt activation and its subsequent inhibition of GSK3ß, such that total cycle number can be reduced while still inhibiting adipogenesis. Amplification of Akt signaling is facilitated by a dynamic restructuring of the cell in response to mechanical signals, as evidenced by a transient increase in focal adhesion (FA) number and increased RhoA activity. Preventing FA assembly or development of tension blocks activation of Akt by mechanical signals, but not by insulin. This indicates that the FA infrastructure is essential to the physical, but not necessarily the chemical, sensitivity, and responsiveness of the cell. Exploiting the transient nature of cytoskeletal remodeling may represent a process to enhance cell responsiveness to mechanical input and ultimately define the fate of MSCs with a minimal input.

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.

A Study of Paraganglioma Cases With Non-European Ancestry.

Capable of generating excess catecholamines, untreated extra-adrenal paragangliomas (PGLs) result in severe cardiovascular morbidity and mortality. Increasingly, a hereditary basis can be identified to underlie PGLs, though such data are largely absent in populations of non-European descent. We present two patients with PGL, both exhibiting similar age, sex, and geographic ancestry. Our patients are unrelated, Kinyarwanda-speaking females from the Democratic Republic of the Congo. The first patient presented with lower extremity edema and poorly controlled hypertension and was found to have multifocal PGL in the abdomen and bladder, proven by biopsy and treated with surgical excision. Our second patient presented with palpitations, shortness of breath, headache, and hypertension, was found to have mediastinal PGL, and underwent surgical excision. Genetic testing was negative in both cases. The first patient has not shown recurrence based on active surveillance with imaging and biochemical testing. There is a concern for recurrence in the second patient, eight years after diagnosis, which is currently being investigated. Our second patient lived at a high altitude for most of her life, pointing toward a possible role of hypoxia in the pathogenesis of her tumor development. Our cases raise questions that require active inquiry regarding additional environmental and/or genetic factors that might predispose to PGLs in uncommon anatomic sites and in understudied, vulnerable populations.

A Normal FGF23 Does Not Preclude Tumor-Induced Osteomalacia.

Tumor-induced osteomalacia (TIO) is a rare cause of impaired bone mineralization mediated by the osteocyte-derived, phosphaturic hormone: fibroblast growth factor 23 (FGF23). The case is presented of a previously healthy 45-year-old man who developed fragility fractures at multiple sites (initially metatarsals, eventually ribs, hips, spine, scapula, and sacrum) resulting in rapid functional deterioration, weakness, and the inability to bear weight and ambulate without a walker. Workup for secondary causes of bone loss was negative except for mild hypogonadotropic hypogonadism with normal pituitary MRI and hypophosphatemia that persisted despite aggressive supplementation. Testosterone was initiated but discontinued 6 months later because of deep vein thrombosis and pulmonary embolism, likely provoked by his new sedentary state, in addition to smoking history and possibly testosterone usage. Serum FGF23 was nonelevated at 138 mRU/mL (44-215). A genetic panel for OI variants was negative for a causal mutation. At the age of 48, 3 years after his initial fracture, he was referred to our academic endocrine clinic. We ruled out additional mutations that lead to hypophosphatemic rickets, including phosphate-regulating endopeptidase homolog, X-linked. PET/CT looking for a potential TIO locus revealed uptake in the left suprapatellar recess. Biopsy was consistent with a phosphaturic mesenchymal tumor. FGF23 was repeated for a preoperative baseline and now found to be elevated at 289 mRU/mL. In retrospect, it is likely that the initial level was inappropriately elevated for the degree of hypophosphatemia. After resection, he experienced marked improvement in physical function, decreased pain, and resolution of renal phosphate wasting. The principals of establishing a robust clinical diagnosis of TIO should be emphasized, excluding other entities and avoiding pitfalls in the interpretation of laboratory testing. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

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.