Tmem263 deletion disrupts the GH/IGF-1 axis and causes dwarfism and impairs skeletal acquisition.

Genome-wide association studies (GWAS) have identified a large number of candidate genes believed to affect longitudinal bone growth and bone mass. One of these candidate genes, TMEM263, encodes a poorly characterized plasma membrane protein. Single nucleotide polymorphisms in TMEM263 are associated with bone mineral density in humans and mutations are associated with dwarfism in chicken and severe skeletal dysplasia in at least one human fetus. Whether this genotype-phenotype relationship is causal, however, remains unclear. Here, we determine whether and how TMEM263 is required for postnatal growth. Deletion of the Tmem263 gene in mice causes severe postnatal growth failure, proportional dwarfism, and impaired skeletal acquisition. Mice lacking Tmem263 show no differences in body weight within the first 2 weeks of postnatal life. However, by P21 there is a dramatic growth deficit due to a disrupted growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis, which is critical for longitudinal bone growth. Tmem263-null mice have low circulating IGF-1 levels and pronounced reductions in bone mass and growth plate length. The low serum IGF-1 in Tmem263-null mice is associated with reduced hepatic GH receptor (GHR) expression and GH-induced JAK2/STAT5 signaling. A deficit in GH signaling dramatically alters GH-regulated genes and feminizes the liver transcriptome of Tmem263-null male mice, with their expression profile resembling wild-type female, hypophysectomized male, and Stat5b-null male mice. Collectively, our data validates the causal role for Tmem263 in regulating postnatal growth and raises the possibility that rare mutations or variants of TMEM263 may potentially cause GH insensitivity and impair linear growth.

A mouse model of Weaver syndrome displays overgrowth and excess osteogenesis reversible with KDM6A/6B inhibition.

Weaver syndrome is a Mendelian disorder of the epigenetic machinery (MDEM) caused by germline pathogenic variants in EZH2, which encodes the predominant H3K27 methyltransferase and key enzymatic component of Polycomb repressive complex 2 (PRC2). Weaver syndrome is characterized by striking overgrowth and advanced bone age, intellectual disability, and distinctive facies. We generated a mouse model for the most common Weaver syndrome missense variant, EZH2 p.R684C. Ezh2R684C/R684C mouse embryonic fibroblasts (MEFs) showed global depletion of H3K27me3. Ezh2R684C/+ mice had abnormal bone parameters, indicative of skeletal overgrowth, and Ezh2R684C/+ osteoblasts showed increased osteogenic activity. RNA-Seq comparing osteoblasts differentiated from Ezh2R684C/+, and Ezh2+/+ BM-mesenchymal stem cells (BM-MSCs) indicated collective dysregulation of the BMP pathway and osteoblast differentiation. Inhibition of the opposing H3K27 demethylases KDM6A and KDM6B substantially reversed the excessive osteogenesis in Ezh2R684C/+ cells both at the transcriptional and phenotypic levels. This supports both the ideas that writers and erasers of histone marks exist in a fine balance to maintain epigenome state and that epigenetic modulating agents have therapeutic potential for the treatment of MDEMs.

Tmem263 deletion disrupts the GH/IGF-1 axis and causes dwarfism and impairs skeletal acquisition.

Genome-wide association studies (GWAS) have identified a large number of candidate genes believed to affect longitudinal bone growth and bone mass. One of these candidate genes, TMEM263, encodes a poorly characterized plasma membrane protein. Single nucleotide polymorphisms in TMEM263 are associated with bone mineral density in humans and mutations are associated with dwarfism in chicken and severe skeletal dysplasia in at least one human fetus. Whether this genotype-phenotype relationship is causal, however, remains unclear. Here, we determine whether and how TMEM263 is required for postnatal growth. Deletion of the Tmem263 gene in mice causes severe postnatal growth failure, proportional dwarfism, and impaired skeletal acquisition. Mice lacking Tmem263 show no differences in body weight within the first two weeks of postnatal life. However, by P21 there is a dramatic growth deficit due to a disrupted GH/IGF-1 axis, which is critical for longitudinal bone growth. Tmem263-null mice have low circulating IGF-1 levels and pronounced reductions in bone mass and growth plate length. The low serum IGF-1 in Tmem263-null mice is associated with reduced hepatic GH receptor (GHR) expression and GH-induced JAK2/STAT5 signaling. A deficit in GH signaling dramatically alters GH-regulated genes and feminizes the liver transcriptome of Tmem263-null male mice, with their expression profile resembling a wild-type female, hypophysectomized male, and Stat5b-null male mice. Collectively, our data validates the causal role for Tmem263 in regulating postnatal growth and raises the possibility that rare mutations or variants of TMEM263 may potentially cause GH insensitivity and impair linear growth.

Peroxisome proliferator activated receptor-γ in osteoblasts controls bone formation and fat mass by regulating sclerostin expression.

The nuclear receptor peroxisome proliferator activated receptor-γ (PPARγ) is a key contributor to metabolic function via its adipogenic and insulin-sensitizing functions, but it has negative effects on skeletal homeostasis. Here, we questioned whether the skeletal and metabolic actions of PPARγ are linked. Ablating Pparg expression in osteoblasts and osteocytes produced a high bone mass phenotype, secondary to increased osteoblast activity, and a reduction in subcutaneous fat mass because of reduced fatty acid synthesis and increased fat oxidation. The skeletal and metabolic phenotypes in Pparg mutants proceed from the regulation of sclerostin production by PPARγ. Mutants exhibited reductions in skeletal Sost expression and serum sclerostin levels while increasing production normalized both phenotypes. Importantly, disrupting the production of sclerostin synergized with the insulin-sensitizing actions of a PPARγ agonist while preventing bone loss. These data suggest that modulating sclerostin action may prevent bone loss associated with anti-diabetic therapies and augment their metabolic actions.

Novel mouse model of Weaver syndrome displays overgrowth and excess osteogenesis reversible with KDM6A/6B inhibition.

Weaver syndrome is a Mendelian disorder of the epigenetic machinery (MDEM) caused by germline pathogenic variants in EZH2, which encodes the predominant H3K27 methyltransferase and key enzymatic component of Polycomb repressive complex 2 (PRC2). Weaver syndrome is characterized by striking overgrowth and advanced bone age, intellectual disability, and distinctive facies. We generated a mouse model for the most common Weaver syndrome missense variant, EZH2 p.R684C. Ezh2R684C/R684C mouse embryonic fibroblasts (MEFs) showed global depletion of H3K27me3. Ezh2R684C/+ mice had abnormal bone parameters indicative of skeletal overgrowth, and Ezh2R684C/+ osteoblasts showed increased osteogenic activity. RNA-seq comparing osteoblasts differentiated from Ezh2R684C/+ and Ezh2+/+ bone marrow mesenchymal stem cells (BM-MSCs) indicated collective dysregulation of the BMP pathway and osteoblast differentiation. Inhibition of the opposing H3K27 demethylases Kdm6a/6b substantially reversed the excessive osteogenesis in Ezh2R684C/+ cells both at the transcriptional and phenotypic levels. This supports both the ideas that writers and erasers of histone marks exist in a fine balance to maintain epigenome state, and that epigenetic modulating agents have therapeutic potential for the treatment of MDEMs.

Mitochondrial ß-oxidation of adipose-derived fatty acids by osteoblasts fuels parathyroid hormone-induced bone formation.

The energetic costs of bone formation require osteoblasts to coordinate their activities with tissues, like adipose, that can supply energy-dense macronutrients. In the case of intermittent parathyroid hormone (PTH) treatment, a strategy used to reduce fracture risk, bone formation is preceded by a change in systemic lipid homeostasis. To investigate the requirement for fatty acid oxidation by osteoblasts during PTH-induced bone formation, we subjected mice with osteoblast-specific deficiency of mitochondrial long-chain ß-oxidation as well as mice with adipocyte-specific deficiency for the PTH receptor or adipose triglyceride lipase to an anabolic treatment regimen. PTH increased the release of fatty acids from adipocytes and ß-oxidation by osteoblasts, while the genetic mouse models were resistant to the hormone's anabolic effect. Collectively, these data suggest that PTH's anabolic actions require coordinated signaling between bone and adipose, wherein a lipolytic response liberates fatty acids that are oxidized by osteoblasts to fuel bone formation.

Endocrine Functions of Sclerostin.

Sclerostin, the product of the SOST gene has primarily been studied for its profound impact on bone mass. By interacting with LRP5 and LRP6, the glycoprotein suppresses the propagation of Wnt signals to ß-catenin and thereby suppresses new bone formation. In this review, we discuss emerging data which suggest that sclerostin also acts outside the skeleton to influence metabolism. In humans, serum sclerostin levels are associated with body mass index and indices of metabolic function. Likewise, genetic mouse models of Sost gene deficiency indicate sclerostin influences adipocyte development and insulin signaling. These data raise the possibility that sclerostin neutralization may be effective at treating two epidemic conditions: osteoporosis and obesity.

A specialized metabolic pathway partitions citrate in hydroxyapatite to impact mineralization of bones and teeth.

Citrate is a critical metabolic substrate and key regulator of energy metabolism in mammalian cells. It has been known for decades that the skeleton contains most (>85%) of the body's citrate, but the question of why and how this metabolite should be partitioned in bone has received singularly little attention. Here, we show that osteoblasts use a specialized metabolic pathway to regulate uptake, endogenous production, and the deposition of citrate into bone. Osteoblasts express high levels of the membranous Na+-dependent citrate transporter solute carrier family 13 member 5 (Slc13a5) gene. Inhibition or genetic disruption of Slc13a5 reduced osteogenic citrate uptake and disrupted mineral nodule formation. Bones from mice lacking Slc13a5 globally, or selectively in osteoblasts, showed equivalent reductions in cortical thickness, with similarly compromised mechanical strength. Surprisingly, citrate content in mineral from Slc13a5-/- osteoblasts was increased fourfold relative to controls, suggesting the engagement of compensatory mechanisms to augment endogenous citrate production. Indeed, through the coordinated functioning of the apical membrane citrate transporter SLC13A5 and a mitochondrial zinc transporter protein (ZIP1; encoded by Slc39a1), a mediator of citrate efflux from the tricarboxylic acid cycle, SLC13A5 mediates citrate entry from blood and its activity exerts homeostatic control of cytoplasmic citrate. Intriguingly, Slc13a5-deficient mice also exhibited defective tooth enamel and dentin formation, a clinical feature, which we show is recapitulated in primary teeth from children with SLC13A5 mutations. Together, our results reveal the components of an osteoblast metabolic pathway, which affects bone strength by regulating citrate deposition into mineral hydroxyapatite.

Mitochondrial fatty acid ß-oxidation is important for normal osteoclast formation in growing female mice.

Skeletal remodeling is an energy demanding process that is linked to nutrient availability and the levels of metabolic hormones. While recent studies have examined the metabolic requirements of bone formation by osteoblasts, much less is known about the energetic requirements of bone resorption by osteoclasts. The abundance of mitochondria in mature osteoclasts suggests that the production of an acidified micro-environment conducive to the ionization of hydroxyapatite, secretion of matrix-degrading enzymes, and motility during resorption requires significant energetic capacity. To investigate the contribution of mitochondrial long chain fatty acid ß-oxidation to osteoclast development, we disrupted the expression of carnitine palmitoyltransferase-2 (Cpt2) in myeloid-lineage cells. Fatty acid oxidation increases dramatically in bone marrow cultures stimulated with RANKL and M-CSF and microCT analysis revealed that the genetic inhibition of long chain fatty acid oxidation in osteoclasts significantly increases trabecular bone volume in female mice secondary to reduced osteoclast numbers. In line with these data, osteoclast precursors isolated from Cpt2 mutants exhibit reduced capacity to form large-multinucleated osteoclasts, which was not rescued by exogenous glucose or pyruvate, and signs of an energetic stress response. Together, our data demonstrate that mitochondrial long chain fatty acid oxidation by the osteoclast is required for normal bone resorption as its inhibition produces an intrinsic defect in osteoclast formation.

Pharmacological TRPC6 inhibition improves survival and muscle function in mice with Duchenne muscular dystrophy.

Gene mutations causing loss of dystrophin result in the severe muscle disease known as Duchenne muscular dystrophy (DMD). Despite efforts at genetic repair, DMD therapy remains largely palliative. Loss of dystrophin destabilizes the sarcolemmal membrane, inducing mechanosensitive cation channels to increase calcium entry and promote cell damage and, eventually, muscle dysfunction. One putative channel is transient receptor potential canonical 6 (TRPC6); we have shown that TRPC6 contributed to abnormal force and calcium stress-responses in cardiomyocytes from mice lacking dystrophin that were haplodeficient for utrophin (mdx/utrn+/- [HET] mice). Here, we show in both the HET mouse and the far more severe homozygous mdx/utrn-/- mouse that TRPC6 gene deletion or its selective pharmacologic inhibition (by BI 749327) prolonged survival 2- to 3-fold, improving skeletal and cardiac muscle and bone defects. Gene pathways reduced by BI 749327 treatment most prominently regulated fat metabolism and TGF-ß1 signaling. These results support the testing of TRPC6 inhibitors in human trials for other diseases as a novel DMD therapy.

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells.

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large extracellular matrix proteins that compose bone tissue and then concentrate the ions necessary for its mineralization. Data on the metabolic requirements of bone formation are emerging rapidly. While much remains to be learned, it is expected that derangements in the intermediary metabolism contribute to skeletal disease. Here, a protocol is outlined to assess the capacity of osteoblastic cells to oxidize 14C-labeled fatty acids to 14CO2 and acid-soluble metabolites. Fatty acids represent a rich-energy reserve that can be taken up from the circulation after feeding or after their liberation from adipose tissue stores. The assay, performed in T-25 tissue culture flasks, is helpful for the study of gene gain or loss-of-function on fatty acid utilization and the effect of anabolic signals in the form of growth factors or morphogens necessary for the maintenance of bone mass. Details on the ability to adapt the protocol to assess the oxidation of glucose or amino acids like glutamine are also provided.

VascuViz: a multimodality and multiscale imaging and visualization pipeline for vascular systems biology.

Despite advances in imaging, image-based vascular systems biology has remained challenging because blood vessel data are often available only from a single modality or at a given spatial scale, and cross-modality data are difficult to integrate. Therefore, there is an exigent need for a multimodality pipeline that enables ex vivo vascular imaging with magnetic resonance imaging, computed tomography and optical microscopy of the same sample, while permitting imaging with complementary contrast mechanisms from the whole-organ to endothelial cell spatial scales. To achieve this, we developed 'VascuViz'-an easy-to-use method for simultaneous three-dimensional imaging and visualization of the vascular microenvironment using magnetic resonance imaging, computed tomography and optical microscopy in the same intact, unsectioned tissue. The VascuViz workflow permits multimodal imaging with a single labeling step using commercial reagents and is compatible with diverse tissue types and protocols. VascuViz's interdisciplinary utility in conjunction with new data visualization approaches opens up new vistas in image-based vascular systems biology.

3D-printed oxygen-releasing scaffolds improve bone regeneration in mice.

Low oxygen (O2) diffusion into large tissue engineered scaffolds hinders the therapeutic efficacy of transplanted cells. To overcome this, we previously studied hollow, hyperbarically-loaded microtanks (µtanks) to serve as O2 reservoirs. To adapt these for bone regeneration, we fabricated biodegradable µtanks from polyvinyl alcohol and poly (lactic-co-glycolic acid) and embedded them to form 3D-printed, porous poly-ε-caprolactone (PCL)-µtank scaffolds. PCL-µtank scaffolds were loaded with pure O2 at 300-500 psi. When placed at atmospheric pressures, the scaffolds released O2 over a period of up to 8 h. We confirmed the inhibitory effects of hypoxia on the osteogenic differentiation of human adipose-derived stem cells (hASCs and we validated that µtank-mediated transient hyperoxia had no toxic impacts on hASCs, possibly due to upregulation of endogenous antioxidant regulator genes. We assessed bone regeneration in vivo by implanting O2-loaded, hASC-seeded, PCL-µtank scaffolds into murine calvarial defects (4 mm diameters × 0.6 mm height) and subcutaneously (4 mm diameter × 8 mm height). In both cases we observed increased deposition of extracellular matrix in the O2 delivery group along with greater osteopontin coverages and higher mineral deposition. This study provides evidence that even short-term O2 delivery from PCL-µtank scaffolds may enhance hASC-mediated bone tissue regeneration.

Bone-derived sclerostin and Wnt/ß-catenin signaling regulate PDGFRα+ adipoprogenitor cell differentiation.

The Wnt signaling antagonist, sclerostin, is a potent suppressor of bone acquisition that also mediates endocrine communication between bone and adipose. As a result, Sost-/- mice exhibit dramatic increases in bone formation but marked decreases in visceral and subcutaneous adipose that are secondary to alterations in lipid synthesis and utilization. While interrogating the mechanism by which sclerostin influences adipocyte metabolism, we observed paradoxical increases in the adipogenic potential and numbers of CD45- :Sca1+ :PDGFRα+ adipoprogenitors in the stromal vascular compartment of fat pads isolated from male Sost-/- mice. Lineage tracing studies indicated that sclerostin deficiency blocks the differentiation of PDGFRα+ adipoprogenitors to mature adipocytes in association with increased Wnt/ß-catenin signaling. Importantly, osteoblast/osteocyte-specific Sost gene deletion mirrors the accumulation of PDGFRα+ adipoprogenitors, reduction in fat mass, and improved glucose metabolism evident in Sost-/- mice. These data indicate that bone-derived sclerostin regulates multiple facets of adipocyte physiology ranging from progenitor cell commitment to anabolic metabolism.

Improved Radium-223 Therapy with Combination Epithelial Sodium Channel Blockade.

Background: Radium-223 dichloride ([223Ra]RaCl2) is the first approved alpha particle-emitting therapy and is indicated for treatment of bone metastatic castrate resistant prostate cancer. Approximately half of the dose is absorbed into the gastrointestinal (GI) tract within minutes of administration, limiting disease-site uptake and contributing to toxicity. Here, we investigate the role of enteric ion channels and their modulation for improved therapeutic efficacy and reduced side effects. Methods: Utilizing primary human duodenal organoids (enteroids) as in vitro models of the functional GI epithelium, we found that Amiloride (ENaC blocker) and NS-1619 (K+ channel activator) presented significant effects in 223Ra membranal transport. The radioactive drug distribution was evaluated for lead combinations in vivo, and in osteosarcoma and prostate cancer models. Results: Amiloride shifted 223Ra uptake in vivo from the gut, to nearly double the uptake at sites of bone remodeling. Bone tumor growth inhibition with the combination as measured by bioluminescent and X-ray imaging was significantly greater than single agents alone, and the combination resulted in no weight loss. Conclusion: This combination of approved agents may be readily implemented as a clinical approach to improve outcomes of bone metastatic cancer patients with the benefit of ameliorated tolerability.

Maternal High-Fat Diet Induces Long-Lasting Defects in Bone Structure in Rat Offspring Through Enhanced Osteoclastogenesis.

Maternal stressors during the prenatal and perinatal periods are associated with increased susceptibility for and severity of chronic disease phenotypes in adult offspring. In this study, we used a rat model of maternal high-fat diet (HFD) exposure during pregnancy and lactation to investigate the impact on skeletal homeostasis in offspring. In the distal femur, young male and female offspring (up to 3 weeks of age) from dams fed a HFD exhibited marked increases in trabecular bone volume relative to offspring from dams fed a chow diet, but this was followed by sustained bone loss. By 15 weeks of age, male offspring of HFD fed dams exhibited a 33% reduction in trabecular bone volume fraction that histomorphometric analyses revealed was due to a nearly threefold increase in the abundance of bone-resorbing osteoclasts, while there were no differences between female control and HFD offspring by 15 weeks of age. The osteoblastic differentiation of male offspring-derived bone marrow stromal cells was not affected by maternal diet. However, osteoclastic precursors isolated from the male offspring of HFD fed dams exhibited enhanced differentiation in vitro, forming larger osteoclasts with higher expression of the fusion marker DC-STAMP. This effect appears to be mediated by a cell autonomous increase in the sensitivity of precursors to RANKL. Taken together, these results suggest that maternal stressors like HFD exposure have persistent consequences for the skeletal health of offspring that may ultimately lead to a predisposition for osteopenia/osteoporosis.