Meghan E. McGee-Lawrence

Epigenetic control of skeletal development by the histone methyltransferase Ezh2

Background: Osteogenic differentiation is initiated by transcriptional and post-transcriptional epigenetic mechanisms. Results: Inhibition of H3K27 methyltransferase EZH2 enhances osteogenic commitment of human mesenchymal progenitors, and its depletion in mouse mesenchymal cells causes multiple skeletal abnormalities. Conclusion: EZH2 is required for skeletal patterning and bone formation. Significance: EZH2-dependent epigenetic mechanisms control osteogenesis both in vitro and in vivo. Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.

Fatty Infiltration of Skeletal Muscle: Mechanisms and Comparisons with Bone Marrow Adiposity

Skeletal muscle and bone share common embryological origins from mesodermal cell populations and also display common growth trajectories early in life. Moreover, muscle and bone are both mechanoresponsive tissues, and the mass and strength of both tissues decline with age. The decline in muscle and bone strength that occurs with aging is accompanied in both cases by an accumulation of adipose tissue. In bone, adipocyte (AC) accumulation occurs in the marrow cavities of long bones and is known to increase with estrogen deficiency, mechanical unloading, and exposure to glucocorticoids. The factors leading to accumulation of intra- and intermuscular fat (myosteatosis) are less well understood, but recent evidence indicates that increases in intramuscular fat are associated with disuse, altered leptin signaling, sex steroid deficiency, and glucocorticoid treatment, factors that are also implicated in bone marrow adipogenesis. Importantly, accumulation of ACs in skeletal muscle and accumulation of intramyocellular lipid are linked to loss of muscle strength, reduced insulin sensitivity, and increased mortality among the elderly. Resistance exercise and whole body vibration can prevent fatty infiltration in skeletal muscle and also improve muscle strength. Therapeutic strategies to prevent myosteatosis may improve muscle function and reduce fall risk in the elderly, potentially impacting the incidence of bone fracture.

Suppressed bone remodeling in black bears conserves energy and bone mass during hibernation.

ABSTRACT Decreased physical activity in mammals increases bone turnover and uncouples bone formation from bone resorption, leading to hypercalcemia, hypercalcuria, bone loss and increased fracture risk. Black bears, however, are physically inactive for up to 6 months annually during hibernation without losing cortical or trabecular bone mass. Bears have been shown to preserve trabecular bone volume and architectural parameters and cortical bone strength, porosity and geometrical properties during hibernation. The mechanisms that prevent disuse osteoporosis in bears are unclear as previous studies using histological and serum markers of bone remodeling show conflicting results. However, previous studies used serum markers of bone remodeling that are known to accumulate with decreased renal function, which bears have during hibernation. Therefore, we measured serum bone remodeling markers (BSALP and TRACP) that do not accumulate with decreased renal function, in addition to the concentrations of serum calcium and hormones involved in regulating bone remodeling in hibernating and active bears. Bone resorption and formation markers were decreased during hibernation compared with when bears were physically active, and these findings were supported by histomorphometric analyses of bone biopsies. The serum concentration of cocaine and amphetamine regulated transcript (CART), a hormone known to reduce bone resorption, was 15-fold higher during hibernation. Serum calcium concentration was unchanged between hibernation and non-hibernation seasons. Suppressed and balanced bone resorption and formation in hibernating bears contributes to energy conservation, eucalcemia and the preservation of bone mass and strength, allowing bears to survive prolonged periods of extreme environmental conditions, nutritional deprivation and anuria. Highlighted Article: Hibernating bears have suppressed bone remodeling, contributing to energy conservation, eucalcemia and the preservation of bone mass and strength, promoting survival during prolonged periods of extreme environmental conditions.

Histone deacetylase 3 supports endochondral bone formation by controlling cytokine signaling and matrix remodeling

Histone deacetylase 3 prevents chondrocytes from producing inflammatory mediators that prevent bone development. Growing bones need histone deacetylase Histone deacetylase (HDAC) inhibitors may be therapeutic in various diseases, but their use causes birth defects and is detrimental to growing bones or the repair of injured bones. Cartilage provides the bone-promoting matrix and bone-forming progenitor cells required for the formation of long bones. Carpio et al. found that HDAC3 promotes pre- and postnatal bone growth by restricting the secretion of inflammatory factors from cartilage cells called chondrocytes. Mice lacking chondrocyte-specific Hdac3 died in utero, and inducible transgenic mice lacking postnatal HDAC3 in chondrocytes had impaired long bone development. Chondrocytes from these mice had increased acetylation of a proinflammatory transcription factor as well as of histones in and near loci encoding secreted proinflammatory factors that promote matrix degradation and the proliferation and activity of bone-resorbing osteoclasts. The findings explain why HDAC inhibitors cause skeletal defects and are ill-advised for children and pregnant women as well as for patients with bone fractures. Histone deacetylase (HDAC) inhibitors are efficacious epigenetic-based therapies for some cancers and neurological disorders; however, each of these drugs inhibits multiple HDACs and has detrimental effects on the skeleton. To better understand how HDAC inhibitors affect endochondral bone formation, we conditionally deleted one of their targets, Hdac3, pre- and postnatally in type II collagen α1 (Col2α1)–expressing chondrocytes. Embryonic deletion was lethal, but postnatal deletion of Hdac3 delayed secondary ossification center formation, altered maturation of growth plate chondrocytes, and increased osteoclast activity in the primary spongiosa. HDAC3-deficient chondrocytes exhibited increased expression of cytokine and matrix-degrading genes (Il-6, Mmp3, Mmp13, and Saa3) and a reduced abundance of genes related to extracellular matrix production, bone development, and ossification (Acan, Col2a1, Ihh, and Col10a1). Histone acetylation increased at and near genes that had increased expression. The acetylation and activation of nuclear factor κB (NF-κB) were also increased in HDAC3-deficient chondrocytes. Increased cytokine signaling promoted autocrine activation of Janus kinase (JAK)–signal transducer and activator of transcription (STAT) and NF-κB pathways to suppress chondrocyte maturation, as well as paracrine activation of osteoclasts and bone resorption. Blockade of interleukin-6 (IL-6)–JAK–STAT signaling, NF-κB signaling, and bromodomain extraterminal proteins, which recognize acetylated lysines and promote transcriptional elongation, significantly reduced Il-6 and Mmp13 expression in HDAC3-deficient chondrocytes and secondary activation in osteoclasts. The JAK inhibitor ruxolitinib also reduced osteoclast activity in Hdac3 conditional knockout mice. Thus, HDAC3 controls the temporal and spatial expression of tissue-remodeling genes and inflammatory responses in chondrocytes to ensure proper endochondral ossification during development.

Enhancer of Zeste Homolog 2 Inhibition Stimulates Bone Formation and Mitigates Bone Loss Caused by Ovariectomy in Skeletally Mature Mice.

Perturbations in skeletal development and bone degeneration may result in reduced bone mass and quality, leading to greater fracture risk. Bone loss is mitigated by bone protective therapies, but there is a clinical need for new bone-anabolic agents. Previous work has demonstrated that Ezh2 (enhancer of zeste homolog 2), a histone 3 lysine 27 (H3K27) methyltransferase, suppressed differentiation of osteogenic progenitors. Here, we investigated whether inhibition of Ezh2 can be leveraged for bone stimulatory applications. Pharmacologic inhibition and siRNA knockdown of Ezh2 enhanced osteogenic commitment of MC3T3 preosteoblasts. Next generation RNA sequencing of mRNAs and real time quantitative PCR profiling established that Ezh2 inactivation promotes expression of bone-related gene regulators and extracellular matrix proteins. Mechanistically, enhanced gene expression was linked to decreased H3K27 trimethylation (H3K27me3) near transcriptional start sites in genome-wide sequencing of chromatin immunoprecipitations assays. Administration of an Ezh2 inhibitor modestly increases bone density parameters of adult mice. Furthermore, Ezh2 inhibition also alleviated bone loss in an estrogen-deficient mammalian model for osteoporosis. Ezh2 inhibition enhanced expression of Wnt10b and Pth1r and increased the BMP-dependent phosphorylation of Smad1/5. Thus, these data suggest that inhibition of Ezh2 promotes paracrine signaling in osteoblasts and has bone-anabolic and osteoprotective potential in adults.

Kynurenine, a Tryptophan Metabolite that Accumulates with Age, Induces Bone Loss†

Age‐dependent bone loss occurs in humans and in several animal species, including rodents. The underlying causal mechanisms are probably multifactorial, although an age‐associated increase in the generation of reactive oxygen species has been frequently implicated. We previously reported that aromatic amino acids function as antioxidants, are anabolic for bone, and that they may potentially play a protective role in an aging environment. We hypothesized that upon oxidation the aromatic amino acids would not only lose their anabolic effects but also potentially become a catabolic byproduct. When measured in vivo in C57BL/6 mice, the tryptophan oxidation product and kynurenine precursor, N‐formylkynurenine (NFK), was found to increase with age. We tested the direct effects of feeding kynurenine (kyn) on bone mass and also tested the short‐term effects of intraperitoneal kyn injection on bone turnover in CD‐1 mice. μCT analyses showed kyn‐induced bone loss. Levels of serum markers of osteoclastic activity (pyridinoline [PYD] and RANKL) increased significantly with kyn treatment. In addition, histological and histomorphometric studies showed an increase in osteoclastic activity in the kyn‐treated groups in both dietary and injection‐based studies. Further, kyn treatment significantly increased bone marrow adiposity, and BMSCs isolated from the kyn‐injected mice exhibited decreased mRNA expression of Hdac3 and its cofactor NCoR1 and increased expression of lipid storage genes Cidec and Plin1. A similar pattern of gene expression is observed with aging. In summary, our data show that increasing kyn levels results in accelerated skeletal aging by impairing osteoblastic differentiation and increasing osteoclastic resorption. These data would suggest that kyn could play a role in age‐induced bone loss.

Whole-Body Vibration Mimics the Metabolic Effects of Exercise in Male Leptin Receptor–Deficient Mice

Whole-body vibration (WBV) has gained attention as a potential exercise mimetic, but direct comparisons with the metabolic effects of exercise are scarce. To determine whether WBV recapitulates the metabolic and osteogenic effects of physical activity, we exposed male wild-type (WT) and leptin receptor-deficient (db/db) mice to daily treadmill exercise (TE) or WBV for 3 months. Body weights were analyzed and compared with WT and db/db mice that remained sedentary. Glucose and insulin tolerance testing revealed comparable attenuation of hyperglycemia and insulin resistance in db/db mice following TE or WBV. Both interventions reduced body weight in db/db mice and normalized muscle fiber diameter. TE or WBV also attenuated adipocyte hypertrophy in visceral adipose tissue and reduced hepatic lipid content in db/db mice. Although the effects of leptin receptor deficiency on cortical bone structure were not eliminated by either intervention, exercise and WBV increased circulating levels of osteocalcin in db/db mice. In the context of increased serum osteocalcin, the modest effects of TE and WBV on bone geometry, mineralization, and biomechanics may reflect subtle increases in osteoblast activity in multiple areas of the skeleton. Taken together, these observations indicate that WBV recapitulates the effects of exercise on metabolism in type 2 diabetes.

Scapula fracture incidence in reverse total shoulder arthroplasty using screws above or below metaglene central cage: clinical and biomechanical outcomes

BACKGROUND Reverse total shoulder arthroplasty (RTSA) is a viable treatment option for rotator cuff tear arthropathy but carries a complication risk of scapular fracture. We hypothesized that using screws above the central glenoid axis for metaglene fixation creates a stress riser contributing to increased scapula fracture incidence. Clinical type III scapular fracture incidence was determined with screw placement correlation: superior screw vs. screws placed exclusively below the glenoid midpoint. Cadaveric RTSA biomechanical modeling was employed to analyze scapular fractures. METHODS We reviewed 318 single-surgeon single-implant RTSAs with screw correlation to identify type III scapular fractures. Seventeen cadaveric scapula specimens were matched for bone mineral density, metaglenes implanted, and fixation with 2 screw configurations: inferior screws alone (group 1INF) vs. inferior screws with one additional superior screw (group 2SUP). Biomechanical load to failure was analyzed. RESULTS Of 206 patients, 9 (4.4%) from the superior screw group experienced scapula fractures (type III); 0 fractures (0/112; 0%) were identified in the inferior screw group. Biomechanically, superior screw constructs (group 2SUP) demonstrated significantly (P < .05) lower load to failure (1077 N vs. 1970 N) compared with constructs with no superior screws (group 1INF). There was no significant age or bone mineral density discrepancy. CONCLUSION Clinical scapular fracture incidence significantly decreased (P < .05) for patients with no screws placed above the central cage compared with patients with superior metaglene screws. Biomechanical modeling demonstrates significant construct compromise when screws are used above the central cage, fracturing at nearly half the ultimate load of the inferior screw constructs. We recommend use of inferior screws, all positioned below the central glenoid axis, unless necessary to stabilize the metaglene construct.

Regulation of the Glycerol Transporter, Aquaporin-3, by Histone Deacetylase-3 and p53 in Keratinocytes

Aquaporin- (AQP) 3, a water and glycerol channel, plays an important role in epidermal function, with studies showing its involvement in keratinocyte proliferation, differentiation, and migration and in epidermal wound healing and barrier repair. Increasing speculation about the use of histone deacetylase (HDAC) inhibitors to treat skin diseases led us to investigate HDACs role in the regulation of AQP3. The broad-spectrum HDAC inhibitor suberoylanilide hydroxamic acid induced AQP3 mRNA and protein expression in a dose- and time-dependent manner in normal keratinocytes. The SAHA-induced increase in AQP3 levels resulted in enhanced [3H]glycerol uptake in normal but not in AQP3-knockout keratinocytes, confirming that the expressed AQP3 was functional. Use of HDAC inhibitors with different specificities limited our exploration of the responsible HDAC member to HDAC1, HDAC2, or HDAC3. Cre-recombinase-mediated knockdown and overexpression of HDAC3 suggested a role for HDAC3 in suppressing AQP3 expression basally. Further investigation implicated p53 as a transcription factor involved in regulating HDAC inhibitor-induced AQP3 expression. Thus, our study supports the regulation of AQP3 expression by HDAC3 and p53. Because suberoylanilide hydroxamic acid is already approved to treat cutaneous T-cell lymphoma, it could potentially be used as a therapy for skin diseases like psoriasis, where AQP3 is abnormally expressed.

Arctic Ground Squirrels Limit Bone Loss during the Prolonged Physical Inactivity Associated with Hibernation

Prolonged disuse (e.g., physical inactivity) typically results in increased bone porosity, decreased mineral density, and decreased bone strength, leading to increased fracture risk in many mammals. However, bears, marmots, and two species of ground squirrels have been shown to preserve macrostructural bone properties and bone strength during long seasons of hibernation while they remain mostly inactive. Some small hibernators (e.g., 13-lined ground squirrels) show microstructural bone loss (i.e., osteocytic osteolysis) during hibernation, which is not seen in larger hibernators (e.g., bears and marmots). Arctic ground squirrels (Urocitellus parryii) are intermediate in size between 13-lined ground squirrels and marmots and are perhaps the most extreme rodent hibernator, hibernating for up to 8 mo annually with body temperatures below freezing. The goal of this study was to quantify the effects of hibernation and inactivity on cortical and trabecular bone properties in arctic ground squirrels. Cortical bone geometrical properties (i.e., thickness, cross-sectional area, and moment of inertia) at the midshaft of the femur were not different in animals sampled over the hibernation and active seasons. Femoral ultimate stress tended to be lower in hibernators than in summer animals, but toughness was not affected by hibernation. The area of osteocyte lacunae was not different between active and hibernating animals. There was an increase in osteocytic lacunar porosity in the hibernation group due to increased lacunar density. Trabecular bone volume fraction in the proximal tibia was unexpectedly greater in the hibernation group than in the active group. This study shows that, similar to other hibernators, arctic ground squirrels are able to preserve many bone properties during hibernation despite being physically inactive for up to 8 mo.