BAP1 promotes osteoclast function by metabolic reprogramming.

Treatment of osteoporosis commonly diminishes osteoclast number which suppresses bone formation thus compromising fracture prevention. Bone formation is not suppressed, however, when bone degradation is reduced by retarding osteoclast functional resorptive capacity, rather than differentiation. We find deletion of deubiquitinase, BRCA1-associated protein 1 (Bap1), in myeloid cells (Bap1∆LysM), arrests osteoclast function but not formation. Bap1∆LysM osteoclasts fail to organize their cytoskeleton which is essential for bone degradation consequently increasing bone mass in both male and female mice. The deubiquitinase activity of BAP1 modifies osteoclast function by metabolic reprogramming. Bap1 deficient osteoclast upregulate the cystine transporter, Slc7a11, by enhanced H2Aub occupancy of its promoter. SLC7A11 controls cellular reactive oxygen species levels and redirects the mitochondrial metabolites away from the tricarboxylic acid cycle, both being necessary for osteoclast function. Thus, in osteoclasts BAP1 appears to regulate the epigenetic-metabolic axis and is a potential target to reduce bone degradation while maintaining osteogenesis in osteoporotic patients.

Insights into pulmonary phosphate homeostasis and osteoclastogenesis emerge from the study of pulmonary alveolar microlithiasis.

Pulmonary alveolar microlithiasis is an autosomal recessive lung disease caused by a deficiency in the pulmonary epithelial Npt2b sodium-phosphate co-transporter that results in accumulation of phosphate and formation of hydroxyapatite microliths in the alveolar space. The single cell transcriptomic analysis of a pulmonary alveolar microlithiasis lung explant showing a robust osteoclast gene signature in alveolar monocytes and the finding that calcium phosphate microliths contain a rich protein and lipid matrix that includes bone resorbing osteoclast enzymes and other proteins suggested a role for osteoclast-like cells in the host response to microliths. While investigating the mechanisms of microlith clearance, we found that Npt2b modulates pulmonary phosphate homeostasis through effects on alternative phosphate transporter activity and alveolar osteoprotegerin, and that microliths induce osteoclast formation and activation in a receptor activator of nuclear factor-κB ligand and dietary phosphate dependent manner. This work reveals that Npt2b and pulmonary osteoclast-like cells play key roles in pulmonary homeostasis and suggest potential new therapeutic targets for the treatment of lung disease.

Pulmonary osteoclast-like cells in silica induced pulmonary fibrosis.

The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored the mechanisms of silica-induced pulmonary fibrosis in a mouse model using multiple modalities including whole-lung single-nucleus RNA sequencing. These analyses revealed that in addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor-κB ligand (RANKL) in pulmonary lymphocytes and alveolar type II cells. Furthermore, anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated silica-induced pulmonary fibrosis. We conclude that silica induces osteoclast-like differentiation of distinct recruited and tissue resident monocyte populations, leading to progressive lung injury, likely due to sustained elaboration of bone resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.

A bone to pick-cellular and molecular mechanisms of bone pain in sickle cell disease.

The bone is one of the most commonly affected organs in sickle cell disease (SCD). Repeated ischemia, oxidative stress and inflammation within the bone is largely responsible for promoting bone pain. As more individuals with SCD survive into adulthood, they are likely to experience a synergistic impact of both aging and SCD on their bone health. As bone health deteriorates, bone pain will likely exacerbate. Recent mechanistic and observational studies emphasize an intricate relationship between bone remodeling and the peripheral nervous system. Under pathological conditions, abnormal bone remodeling plays a key role in the propagation of bone pain. In this review, we first summarize mechanisms and burden of select bone complications in SCD. We then discuss processes that contribute to pathological bone pain that have been described in both SCD as well as non-sickle cell animal models. We emphasize the role of bone-nervous system interactions and pitfalls when designing new therapies especially for the sickle cell population. Lastly, we also discuss future basic and translational research in addressing questions about the complex role of stress erythropoiesis and inflammation in the development of SCD bone complications, which may lead to promising therapies and reduce morbidity in this vulnerable population.

ThPOK Inhibits Osteoclast Formation Via NFATc1 Transcription and Function.

Both LRF (Zbtb7a) and ThPOK (Zbtb7b) belong to the POK (BTB/POZ and Kruppel) family of transcription repressors that participate in development, differentiation, and oncogenesis. Although LRF mediates osteoclast differentiation by regulating NFATc1 expression, the principal established function of ThPOK is transcriptional control of T-cell lineage commitment. Whether ThPOK affects osteoclast formation or function is not known. We find that marrow macrophage ThPOK expression diminishes with exposure to receptor activator of NF-kB ligand (RANKL), but ThPOK deficiency does not affect osteoclast differentiation. On the other hand, enhanced ThPOK, in macrophages, substantially impairs osteoclastogenesis. Excess ThPOK binds the NFATc1 promoter and suppresses its transcription, suggesting a mechanism for its osteoclast inhibitory effect. Despite suppression of osteoclastogenesis by excess ThPOK being associated with diminished NFATc1, osteoclast formation is not rescued by NFATc1 overexpression. Thus, ThPOK appears to inhibit NFATc1 transcription and its osteoclastogenic capacity, while its depletion has no effect on the bone-resorptive cell. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Adipose tissue is a critical regulator of osteoarthritis.

Osteoarthritis (OA), the leading cause of pain and disability worldwide, disproportionally affects individuals with obesity. The mechanisms by which obesity leads to the onset and progression of OA are unclear due to the complex interactions among the metabolic, biomechanical, and inflammatory factors that accompany increased adiposity. We used a murine preclinical model of lipodystrophy (LD) to examine the direct contribution of adipose tissue to OA. Knee joints of LD mice were protected from spontaneous or posttraumatic OA, on either a chow or high-fat diet, despite similar body weight and the presence of systemic inflammation. These findings indicate that adipose tissue itself plays a critical role in the pathophysiology of OA. Susceptibility to posttraumatic OA was reintroduced into LD mice using implantation of a small adipose tissue depot derived from wild-type animals or mouse embryonic fibroblasts that undergo spontaneous adipogenesis, implicating paracrine signaling from fat, rather than body weight, as a mediator of joint degeneration.

Intercellular Mitochondria Transfer to Macrophages Regulates White Adipose Tissue Homeostasis and Is Impaired in Obesity.

Recent studies suggest that mitochondria can be transferred between cells to support the survival of metabolically compromised cells. However, whether intercellular mitochondria transfer occurs in white adipose tissue (WAT) or regulates metabolic homeostasis in vivo remains unknown. We found that macrophages acquire mitochondria from neighboring adipocytes in vivo and that this process defines a transcriptionally distinct macrophage subpopulation. A genome-wide CRISPR-Cas9 knockout screen revealed that mitochondria uptake depends on heparan sulfates (HS). High-fat diet (HFD)-induced obese mice exhibit lower HS levels on WAT macrophages and decreased intercellular mitochondria transfer from adipocytes to macrophages. Deletion of the HS biosynthetic gene Ext1 in myeloid cells decreases mitochondria uptake by WAT macrophages, increases WAT mass, lowers energy expenditure, and exacerbates HFD-induced obesity in vivo. Collectively, this study suggests that adipocytes and macrophages employ intercellular mitochondria transfer as a mechanism of immunometabolic crosstalk that regulates metabolic homeostasis and is impaired in obesity.

Ablation of Fat Cells in Adult Mice Induces Massive Bone Gain.

Adipocytes control bone mass, but the mechanism is unclear. To explore the effect of postnatal adipocyte elimination on bone cells, we mated mice expressing an inducible primate diphtheria toxin receptor (DTR) to those bearing adiponectin (ADQ)-Cre. DTR activation eliminates peripheral and marrow adipocytes in these DTRADQ mice. Within 4 days of DTR activation, the systemic bone mass of DTRADQ mice began to increase due to stimulated osteogenesis, with a 1,000% expansion by 10-14 days post-DTR treatment. This adipocyte ablation-mediated enhancement of skeletal mass reflected bone morphogenetic protein (BMP) receptor activation following the elimination of its inhibitors, associated with simultaneous epidermal growth factor (EGF) receptor signaling. DTRADQ-induced osteosclerosis is not due to ablation of peripheral adipocytes but likely reflects the elimination of marrow ADQ-expressing cells. Thus, anabolic drugs targeting BMP receptor inhibitors with short-term EGF receptor activation may be a means of profoundly increasing skeletal mass to prevent or reverse pathological bone loss.

Hepatic lipids promote liver metastasis.

Obesity predisposes to cancer and a virtual universality of nonalcoholic fatty liver disease (NAFLD). However, the impact of hepatic steatosis on liver metastasis is enigmatic. We find that while control mice were relatively resistant to hepatic metastasis, those which were lipodystrophic or obese, with NAFLD, had a dramatic increase in breast cancer and melanoma liver metastases. NAFLD promotes liver metastasis by reciprocal activation initiated by tumor-induced triglyceride lipolysis in juxtaposed hepatocytes. The lipolytic products are transferred to cancer cells via fatty acid transporter protein 1, where they are metabolized by mitochondrial oxidation to promote tumor growth. The histology of human liver metastasis indicated the same occurs in humans. Furthermore, comparison of isolates of normal and fatty liver established that steatotic lipids had enhanced tumor-stimulating capacity. Normalization of glucose metabolism by metformin did not reduce steatosis-induced metastasis, establishing the process is not mediated by the metabolic syndrome. Alternatively, eradication of NAFLD in lipodystrophic mice by adipose tissue transplantation reduced breast cancer metastasis to that of control mice, indicating the steatosis-induced predisposition is reversible.

Myeloid-specific Asxl2 deletion limits diet-induced obesity by regulating energy expenditure.

We previously established that global deletion of the enhancer of trithorax and polycomb (ETP) gene, Asxl2, prevents weight gain. Because proinflammatory macrophages recruited to adipose tissue are central to the metabolic complications of obesity, we explored the role of ASXL2 in myeloid lineage cells. Unexpectedly, mice without Asxl2 only in myeloid cells (Asxl2ΔLysM) were completely resistant to diet-induced weight gain and metabolically normal despite increased food intake, comparable activity, and equivalent fecal fat. Asxl2ΔLysM mice resisted HFD-induced adipose tissue macrophage infiltration and inflammatory cytokine gene expression. Energy expenditure and brown adipose tissue metabolism in Asxl2ΔLysM mice were protected from the suppressive effects of HFD, a phenomenon associated with relatively increased catecholamines likely due to their suppressed degradation by macrophages. White adipose tissue of HFD-fed Asxl2ΔLysM mice also exhibited none of the pathological remodeling extant in their control counterparts. Suppression of macrophage Asxl2 expression, via nanoparticle-based siRNA delivery, prevented HFD-induced obesity. Thus, ASXL2 controlled the response of macrophages to dietary factors to regulate metabolic homeostasis, suggesting modulation of the cells' inflammatory phenotype may impact obesity and its complications.

Congenital lipodystrophy induces severe osteosclerosis.

Berardinelli-Seip congenital generalized lipodystrophy is associated with increased bone mass suggesting that fat tissue regulates the skeleton. Because there is little mechanistic information regarding this issue, we generated "fat-free" (FF) mice completely lacking visible visceral, subcutaneous and brown fat. Due to robust osteoblastic activity, trabecular and cortical bone volume is markedly enhanced in these animals. FF mice, like Berardinelli-Seip patients, are diabetic but normalization of glucose tolerance and significant reduction in circulating insulin fails to alter their skeletal phenotype. Importantly, the skeletal phenotype of FF mice is completely rescued by transplantation of adipocyte precursors or white or brown fat depots, indicating that adipocyte derived products regulate bone mass. Confirming such is the case, transplantation of fat derived from adiponectin and leptin double knockout mice, unlike that obtained from their WT counterparts, fails to normalize FF bone. These observations suggest a paucity of leptin and adiponectin may contribute to the increased bone mass of Berardinelli-Seip patients.

Fat-Produced Adipsin Regulates Inflammatory Arthritis.

We explored the relationship of obesity and inflammatory arthritis (IA) by selectively expressing diphtheria toxin in adipose tissue yielding "fat-free" (FF) mice completely lacking white and brown fat. FF mice exhibit systemic neutrophilia and elevated serum acute phase proteins suggesting a predisposition to severe IA. Surprisingly, FF mice are resistant to K/BxN serum-induced IA and attendant bone destruction. Despite robust systemic basal neutrophilia, neutrophil infiltration into joints of FF mice does not occur when challenged with K/BxN serum. Absence of adiponectin, leptin, or both has no effect on joint disease, but deletion of the adipokine adipsin (complement factor D) completely prevents serum-induced IA. Confirming that fat-expressed adipsin modulates the disorder, transplantation of wild-type (WT) adipose tissue into FF mice restores susceptibility to IA, whereas recipients of adipsin-deficient fat remain resistant. Thus, adipose tissue regulates development of IA through a pathway in which adipocytes modify neutrophil responses in distant tissues by producing adipsin.

Congenital disorders of bone and blood.

Bone and marrow are the two facets of the same organ, in which bone and hematopoietic cells coexist and interact. Marrow and skeletal tissue influence each-other and a variety of genetic disorders directly targets both of them, which may result in combined hematopoietic failure and skeletal malformations. Other conditions primarily affect one organ with secondary influences on the other. For instance, various forms of congenital anemias reduce bone mass and induce osteoporosis, while osteoclast failure in osteopetrosis prevents marrow development reducing medullary cavities and causing anemia and pancytopenia. Understanding the pathophysiology of these conditions may facilitate diagnosis and management, although many disorders are presently incurable. This article describes several congenital bone diseases and their relationship to hematopoietic tissue.

Novel Pure αVß3 Integrin Antagonists That Do Not Induce Receptor Extension, Prime the Receptor, or Enhance Angiogenesis at Low Concentrations.

The integrin αVß3 receptor has been implicated in several important diseases, but no antagonists are approved for human therapy. One possible limitation of current small-molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small-molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVß3-mediated cell adhesion to αVß3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVß3 antagonist cilengitide of paradoxically enhancing aortic sprout angiogenesis at concentrations below its IC50, which correlates with cilengitide's enhancement of tumor growth in vivo.

ASXL1 impairs osteoclast formation by epigenetic regulation of NFATc1.

Additional sex comb-like 1 (ASXL1) mutations are commonly associated with myeloid malignancies and are markers of aggressive disease. The fact that ASXL1 is necessary for myeloid differentiation raises the possibility it also regulates osteoclasts. We find deletion of ASXL1 in myeloid cells results in bone loss with increased abundance of osteoclasts. Because ASXL1 is an enhancer of trithorax and polycomb (ETP) protein, we asked if it modulates osteoclast differentiation by maintaining balance between positive and negative epigenetic regulators. In fact, loss of ASXL1 induces concordant loss of inhibitory H3K27me3 with gain of H3K4me3 at key osteoclast differentiation genes, including nuclear factor for activated T cells 1 (NFATc1) and itgb3 In the setting of ASXL1 deficiency, increased NFATc1 binds to the Blimp1 (Prdm1) promoter thereby enhancing expression of this pro-osteoclastogenic gene. The global reduction of K27 trimethylation in ASXL1-deficient osteoclasts is also attended by a 40-fold increase in expression of the histone demethylase Jumonji domain-containing 3 (Jmjd3). Jmjd3 knockdown in ASXL1-deficient osteoclast precursors increases H3K27me3 on the NFATc1 promoter and impairs osteoclast formation. Thus, in addition to promoting myeloid malignancies, ASXL1 controls epigenetic reprogramming of osteoclasts to regulate bone resorption and mass.

PGC1ß Organizes the Osteoclast Cytoskeleton by Mitochondrial Biogenesis and Activation.

Osteoclasts are mitochondria-rich cells, but the role of these energy-producing organelles in bone resorption is poorly defined. To this end, we conditionally deleted the mitochondria-inducing co-activator, PGC1ß, in myeloid lineage cells to generate PGC1ßLysM mice. In contrast to previous reports, PGC1ß-deficient macrophages differentiate normally into osteoclasts albeit with impaired resorptive function due to cytoskeletal disorganization. Consequently, bone mass of PGC1ßLysM mice is double that of wild type. Mitochondrial biogenesis and function are diminished in PGC1ßLysM osteoclasts. All abnormalities are normalized by PGC1ß transduction. Furthermore, OXPHOS inhibitors reproduce the phenotype of PGC1ß deletion. PGC1ß's organization of the osteoclast cytoskeleton is mediated by expression of GIT1, which also promotes mitochondrial biogenesis. Thus, osteoclast mitochondria regulate the cell's resorptive activity by promoting cytoskeletal organization. © 2018 American Society for Bone and Mineral Research.

Inflammatory osteolysis: a conspiracy against bone.

There are many causes of inflammatory osteolysis, but regardless of etiology and cellular contexts, the osteoclast is the bone-degrading cell. Thus, the impact of inflammatory cytokines on osteoclast formation and function was among the most important discoveries advancing the treatment of focal osteolysis, leading to development of therapeutic agents that either directly block the bone-resorptive cell or do so indirectly via cytokine arrest. Despite these advances, a substantial number of patients with inflammatory arthritis remain resistant to current therapies, and even effective anti-inflammatory drugs frequently do not repair damaged bone. Thus, insights into events such as those impacted by inflammasomes, which signal through cytokine-dependent and -independent mechanisms, are needed to optimize treatment of inflammatory osteolysis.

Therapeutic implications of suppressing osteoclast formation versus function.

Anti-resorptive therapy is the principal means of treating osteoporotic disorders. The two families of presently available anti-resorptive drugs, namely bisphosphonates and denosumab, dampen activity of osteoclasts by reducing their number. In consequence, these agents also arrest bone remodelling eventuating suppressed formation as well as resorption. Evidence exists that osteoclasts recruit osteoblasts to sites of bone remodelling by mobilizing chemotactic proteins from matrix and direct secretion of such proteins that attract osteoblast precursors. Thus, anti-resorptive agents, such as the cathepsin K inhibitor odanacatib, that dampen osteoclast function but not number may also preserve osteoblast recruitment by preserving the bone resorptive cell.

Antagonizing Integrin ß3 Increases Immunosuppression in Cancer.

Integrin ß3 is critical for tumor invasion, neoangiogenesis, and inflammation, making it a promising cancer target. However, preclinical and clinical data of integrin ß3 antagonists have demonstrated no benefit or worse outcomes. We hypothesized that integrin ß3 could affect tumor immunity and evaluated tumors in mice with deletion of integrin ß3 in macrophage lineage cells (ß3KOM). ß3KOM mice had increased melanoma and breast cancer growth with increased tumor-promoting M2 macrophages and decreased CD8(+) T cells. Integrin ß3 antagonist, cilengitide, also enhanced tumor growth and increased M2 function. We uncovered a negative feedback loop in M2 myeloid cells, wherein integrin ß3 signaling favored STAT1 activation, an M1-polarizing signal, and suppressed M2-polarizing STAT6 activation. Finally, disruption of CD8(+) T cells, macrophages, or macrophage integrin ß3 signaling blocked the tumor-promoting effects of integrin ß3 antagonism. These results suggest that effects of integrin ß3 therapies on immune cells should be considered to improve outcomes. Cancer Res; 76(12); 3484-95. ©2016 AACR.