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Although the non-selective β-blocker, propranolol, improves bone density with PTH treatment in mice, the mechanism of this effect is unclear. To address this, we used a combination of in vitro and in vivo approaches to address how propranolol influences bone remodeling in the context of PTH treatment. In female C57BL/6J mice, intermittent PTH and propranolol administration had complementary effects in the trabecular bone of the distal femur and L5 vertebra, with combination treatment achieving micro-architectural parameters beyond that of PTH alone. Combined treatment improved the serum bone formation marker, P1NP, but did not impact other histomorphometric parameters relating to osteoblast function at the L5. In vitro, propranolol amplified the acute, PTH-induced, intracellular calcium signal in osteoblast-like cells. The most striking finding, however, was suppression of PTH-induced bone resorption. Despite this, PTH-induced receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA and protein levels were unaltered by propranolol, which led us to hypothesize that propranolol could act directly on osteoclasts. Using in situ methods, we found Adrb2 expression in osteoclasts in vivo, suggesting β-blockers may directly impact osteoclasts. Consistent with this, we found propranolol directly suppresses osteoclast differentiation in vitro. Taken together, this work suggests a strong anti-osteoclastic effect of non-selective β-blockers in vivo, indicating that combining propranolol with PTH could be beneficial to patients with extremely low bone density.

Lymphomatoid papulosis (LyP) is one of primary cutaneous CD30-positive large T-cell lymphoproliferative disorders (CD30 + PCLPD) that seldom arises in the oral region. We report here an extremely rare case of LyP arising on the lip. A 25-year-old female developed a painless mass on her right upper lip, which gradually grew and became painful. She visited her dermatologist who diagnosed it as herpes labialis and administered valacyclovir, but the lesion was unchanged. Then, she was referred to our department. At first visit, the mass was elastic, slightly hard, scarlet in color, and 15 mm in diameter. As T-cell lymphoma was initially diagnosed following incisional biopsy, we referred her to the Departments of Hematology and also Dermatology at our hospital. Bone marrow examination showed no neoplastic changes, and positron emission tomography-computed tomography revealed no other lesion except on the lip. Although new lesions appeared on her upper lip during the examinations, the lesions spontaneously disappeared without further treatment after the biopsy. The final diagnosis was CD30 + PCLPD pathologically and LyP clinically. No recurrence was found during the 10-month follow-up period.

Background Cancer arises from cumulative genetic or epigenetic aberrations, or the destabilization of central signaling pathways that regulate cell proliferation, differentiation, cell cycle, gene transcription, migration, angiogenesis and apoptosis. Investigating the cancer-specific genetic background is important to get deeper apprehension of cancer biology. In this review, we aimed to identify head and neck squamous cell carcinoma (HNSCC)-specific genes and identified DKK3 gene as a candidate. Highlight DKK3 belongs to the DKK family (DKK1, DKK2, DKK3 and DKK4), which codes for an evolutionally conserved secreted glycoprotein that is characterized by two distinct cysteine rich domains and functions as an antagonist of the oncogenic Wnt signaling pathway. It has been reported that DKK3 expression is decreased in many kinds of cancers, and it is thus thought to be a tumor suppressor gene. However, our investigations have demonstrated unique expression and function of DKK3 in HNSCC. DKK3 protein expression is predominantly positive in HNSCC, and DKK3-positive patients show significantly shorter disease-free survival rates, whereas DKK3-negative cases do not show metastasis. Molecular biological analyses demonstrated that DKK3 over expression significantly increased HNSCC cell proliferation, migration, and invasion via increased phosphorylation of AKT. Moreover, DKK3 knockdown in HNSCC cells significantly decreased these malignant potentials through decreased AKT phosphorylation. Conclusion Our previously published data, alongside those from other reports, indicate that DKK3 may have an additional oncogenic function other than tumor suppression.

Activation of Wnt signaling leads to high bone density. The R-spondin family of four secreted glycoproteins (Rspo1-4) amplifies Wnt signaling. In humans, RSPO3 variants are strongly associated with bone density, but how RSPO3 affects skeletal homeostasis is not fully understood. Here we show that in mice Rspo3 haplo-insufficiency or its targeted deletion in osteoprogenitors lead to an increase in bone formation and bone mass. Contrary to expectations, Rspo3 haplo-insufficiency results in canonical Wnt signaling activation. Using mouse embryonic fibroblasts we show that Rspo3 deficiency leads to activation of Erk signaling, stabilizing β-catenin. Furthermore, Rspo3 haplo-insufficiency impairs Dkk1 efficacy in blocking canonical Wnt signaling and prevents the in vivo inhibition of bone formation and bone mass induced by osteoblast-targeted expression of Dkk1. We conclude that Rspo3 haplo-insufficiency/deficiency boosts canonical Wnt signaling by activating Erk signaling and impairing Dkk1’s inhibitory activity, which in turn lead to increased bone formation and bone mass.

Human genetic evidence demonstrates that WNT1 mutations cause osteogenesis imperfecta (OI) and early‐onset osteoporosis, implicating WNT1 as a major regulator of bone metabolism. However, its main cellular source and mechanisms of action in bone remain elusive. We generated global and limb bud mesenchymal cell–targeted deletion of Wnt1 in mice. Heterozygous deletion of Wnt1 resulted in mild trabecular osteopenia due to decreased osteoblast function. Targeted deletion of Wnt1 in mesenchymal progenitors led to spontaneous fractures due to impaired osteoblast function and increased bone resorption, mimicking the severe OI phenotype in humans with homozygous WNT1 mutations. Importantly, we showed for the first time that Wnt1 signals strictly in a juxtacrine manner to induce osteoblast differentiation and to suppress osteoclastogenesis, in part via canonical Wnt signaling. In conclusion, mesenchymal cell‐derived Wnt1, acting in short range, is an essential regulator of bone homeostasis and an intriguing target for therapeutic interventions for bone diseases. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

Background: The incidence of mixed hepatitis C virus (HCV) genotype infection is variable, and a few reports exist regarding the efficacy of direct-acting antivirals (DAA) therapy for mixed genotype. We aimed to investigate the prevalence of mixed genotype and its impact on the virologic response to DAA therapy. Methods: A total of 365 patients with chronic HCV infection who completed antiviral therapy were recruited. Nested polymerase chain reaction with universal and specific primers of genotypes 1b and 2 and direct sequencing were used for HCV genotyping. Results: Direct sequencing with universal primers defined genotypes 1b (n = 230), 2a (n = 95), and 2b (n = 40). Direct sequencing of genotype 2 was performed in patients with genotype 1b, and direct sequencing of genotype 1b in patients with genotype 2. Four patients with genotype 1b underwent amplification for genotype 2, and direct sequencing identified genotypes 1b (n = 1), 2a (n = 1), and 2b (n = 2). None with genotype 2 underwent amplification for genotype 1b. Three cases were confirmed to have mixed genotype. Conclusions: Mixed genotype was rare, and hence the impact of mixed genotype on treatment outcome with DAA therapy is expected to be minimal.

The outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified in 2019; thereafter, the COVID-19 outbreak became a health emergency of international concern. The impact of COVID-19 on liver-transplant recipients is unclear. Thus, it is currently unknown whether liver-transplant recipients are at a higher risk of developing complications related to COVID-19. Here, we report the case of liver-transplant recipients who were infected with SARS-CoV-2. A 20-year-old man who had undergone living-donor liver transplantation from his father at 5 years of age because of congenital biliary atresia was referred to our hospital for SARS-CoV-2 infection. Chest computed tomography did not show any abnormalities; however, laboratory results revealed liver dysfunction. He received tacrolimus as maintenance therapy that was continued at the same dose. He has not developed severe pulmonary disease and was discharged after 10 days of hospitalization. Limited data are available on post-transplant patients with COVID-19, and this case of a young patient without metabolic comorbidities did not show any association of severe COVID-19 under tacrolimus treatment. The progression of COVID-19 in liver-transplant recipients is complex, and COVID-19 risk should be evaluated in each patient until the establishment of optimal guidelines.

Although the surface of hands in humans is known to harbor high levels of antimicrobial activity, reports investigating the relationship between antimicrobial activity and morbidity in infectious diseases are lacking. Additionally, the precise components involved in this activity are not known. Therefore, in this study, a method was developed to quantitatively measure the antimicrobial activity of the components found on the surface of hands, which was then compared with the medical history of the participants for infectious diseases. As a result, the antimicrobial activity of the surface of the hands was found to be positively associated with the history of infection in individuals. Furthermore, a comprehensive analysis of the components on the surface of hands indicated that organic acids and antimicrobial peptides are highly correlated with antimicrobial activity. The high amounts of lactic acid found on the surface of hands suggested it is an important factor in the hand surface infection barrier. Here we showed that the application of lactic acid within the range of the amounts present on the hand surface was found to significantly improve the antimicrobial activity of the hands. Taken together, these results demonstrate that this new method can be used to quantify antimicrobial activity, which opens new avenues for the development of hand hygiene practices by enhancing the antimicrobial activity on the surface of hands using natural ingredients against pathogens.

Irisin, a skeletal-muscle secreted myokine, facilitates muscle-bone crosstalk and skeletal remodeling in part by its action on osteoblasts and osteocytes. In this study, we investigated whether irisin directly regulates osteoclasts. In vitro, irisin (2–10 ng/mL) increased osteoclast differentiation in C57BL/6J mouse bone marrow progenitors; however, this increase was blocked by a neutralizing antibody to integrin αVβ5. Irisin also increased bone resorption on several substrates in situ. RNAseq revealed differential gene expression induced by irisin including upregulation of markers for osteoclast differentiation and resorption, as well as osteoblast-stimulating ‘clastokines’. Forced expression of the irisin precursor Fndc5 in transgenic C57BL/6J mice resulted in lower bone mass at three ages and greater in vitro osteoclastogenesis from Fndc5-transgenic bone marrow progenitors. This study demonstrates that irisin acts directly on osteoclast progenitors to increase differentiation and promote bone resorption, supporting the tenet that irisin not only stimulates bone remodeling but may also be an important counter-regulatory hormone.

Early B cell factor 1 (Ebf1) is a transcription factor that regulates B cell, neuronal cell and adipocyte differentiation. We and others have shown that Ebf1 is expressed in osteoblasts and that global deletion of Ebf1 results in increased bone formation in vivo. However, as Ebf1 is expressed in multiple tissues and cell types, it has remained unclear, which of the phenotypic changes in bone are derived from bone cells. The aim of this study was to determine the cell-autonomous and differentiation stage-specific roles of Ebf1 in osteoblasts. In vitro, haploinsufficient Ebf1+/− calvarial cells showed impaired osteoblastic differentiation indicated by lower alkaline phosphatase (ALP) activity and reduced mRNA expression of osteoblastic genes, while overexpression of Ebf1 in wild type mouse calvarial cells led to enhanced osteoblast differentiation with increased expression of Osterix (Osx). We identified a putative Ebf1 binding site in the Osterix promoter by ChIP assay in MC3T3-E1 osteoblasts and showed that Ebf1 was able to activate Osx-luc reporter construct that included this Ebf1 binding site, suggesting that Ebf1 indeed regulates osteoblast differentiation by inducing Osterix expression. To reconcile our previous data and that of others with our novel findings, we hypothesized that Ebf1 could have a dual role in osteoblast differentiation promoting early but inhibiting late stages of differentiation and osteoblast function. To test this hypothesis in vivo, we generated conditional Ebf1 knockout mice, in which Ebf1 deletion was targeted to early or late osteoblasts by crossing Ebf1fl/fl mice with Osx- or Osteocalcin (hOC)-Cre mouse lines, respectively. Deletion of Ebf1 in early Ebf1Osx−/− osteoblasts resulted in significantly increased bone volume and trabecular number at 12 weeks by μCT analysis, while Ebf1hOC−/− mice did not have a bone phenotype. To conclude, our data demonstrate that Ebf1 promotes early osteoblast differentiation by regulating Osterix expression. However, Ebf1 inhibits bone accrual in the Osterix expressing osteoblasts in vivo but it is redundant in the maintenance of mature osteoblast function.

Objective Most malignant tumors require remodeling extracellular matrices (ECMs) for invasive growth and metastasis. Cancer cells and stromal cells remodel ECM. We investigated the relationship between regional lymph node (LN) metastasis and expression of ECM-remodeling factors in oral squamous cell carcinoma (OSCC). Methods Using primary OSCC and cervical LNs obtained surgically, we performed immunohistochemical evaluation of the ECM-remodeling factors, lysyl oxidase (LOX), MT1-MMP, S100A8, and TIMP-1 in primary tumor and marginal sinus histiocytosis (MSH) in LNs, and determined the statistical significance of the positive rates between metastatic and metastasis-free groups. Results Marginal sinus histiocytosis was more frequently formed in the metastatic group compared to the metastasis-free group. Lymphatic metastasis correlated with the immunopositivity rates of tumor cells expressing LOX, MT1-MMP, and TIMP-1, and of stromal cells expressing TIMP-1. The case rates of MSH containing macrophages positive for LOX and MT1-MMP in the metastasis group were significantly higher than in the metastasis-free group. ECM-remodeling-associated macrophages accumulate in marginal sinus in conjunction with lymphatic metastasis. Conclusion Expression of LOX, MT1-MMP, and TIMP-1 in the parenchyma, and stromal expression of TIMP-1 in primary tumor may predict lymphatic metastasis. LOX and MT1-MMP have a possibility to participate in formation of pre-metastatic niche in LNs.

Although the non-selective β-blocker, propranolol, improves bone density with PTH treatment in mice, the mechanism of this effect is unclear. To address this, we used a combination of in vitro and in vivo approaches to address how propranolol influences bone remodeling in the context of PTH treatment. In female C57BL/6J mice, intermittent PTH and propranolol had complementary effects in the trabecular bone of the distal femur and L5 vertebra, with combination treatment achieving micro-architectural parameters beyond that of PTH alone. Combined treatment improved the serum bone formation marker, P1NP, but did not impact other histomorphometric parameters relating to osteoblast function at the L5. In vitro, propranolol amplified the acute, PTH-induced, intracellular calcium signal in osteoblast-like cells. The most striking finding, however, was suppression of PTH-induced bone resorption. Despite this, PTH-induced receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA and protein levels were unaltered by propranolol, which led us to hypothesize that propranolol could act directly on osteoclasts. Using in situ methods, we found Adrb2 expression in osteoclasts in vivo, suggesting β-blockers may directly impact osteoclasts. Taken together, this work suggests a strong anti-osteoclastic effect of non-selective β-blockers in vivo, indicating that combining propranolol with PTH could be beneficial to patients with extremely low bone density.

Myeloma bone disease is a devastating complication of multiple myeloma (MM) and is caused by dysregulation of bone remodeling processes in the bone marrow microenvironment. Previous studies showed that microRNA-138 (miR-138) is a negative regulator of osteogenic differentiation of mesenchymal stromal cells (MSCs) and that inhibiting its function enhances bone formation in vitro. In this study, we explored the role of miR-138 in myeloma bone disease and evaluated the potential of systemically delivered locked nucleic acid (LNA)-modified anti-miR-138 oligonucleotides in suppressing myeloma bone disease. We showed that expression of miR-138 was significantly increased in MSCs from MM patients (MM-MSCs) and myeloma cells compared to those from healthy subjects. Furthermore, inhibition of miR-138 resulted in enhanced osteogenic differentiation of MM-MSCs in vitro and increased the number of endosteal osteoblastic lineage cells (OBCs) and bone formation rate in mouse models of myeloma bone disease. RNA sequencing of the OBCs identified TRPS1 and SULF2 as potential miR-138 targets that were de-repressed in anti-miR-138-treated mice. In summary, these data indicate that inhibition of miR-138 enhances bone formation in MM and that pharmacological inhibition of miR-138 could represent a new therapeutic strategy for treatment of myeloma bone disease.

In the brain, the ventral hypothalamus (VHT) regulates energy and bone metabolism. Whether this regulation uses the same or different neuronal circuits is unknown. Alteration of AP1 signaling in the VHT increases energy expenditure, glucose utilization, and bone density, yet the specific neurons responsible for each or all of these phenotypes are not identified. Using neuron-specific, genetically targeted AP1 alterations as a tool in adult mice, we found that agouti-related peptide-expressing (AgRP-expressing) or proopiomelanocortin-expressing (POMC-expressing) neurons, predominantly present in the arcuate nucleus (ARC) within the VHT, stimulate whole-body energy expenditure, glucose utilization, and bone formation and density, although their effects on bone resorption differed. In contrast, AP1 alterations in steroidogenic factor 1-expressing (SF1-expressing) neurons, present in the ventromedial hypothalamus (VMH), increase energy but decrease bone density, suggesting that these effects are independent. Altered AP1 signaling also increased the level of the neuromediator galanin in the hypothalamus. Global galanin deletion (VHT galanin silencing using shRNA) or pharmacological galanin receptor blockade counteracted the observed effects on energy and bone. Thus, AP1 antagonism reveals that AgRP- and POMC-expressing neurons can stimulate body metabolism and increase bone density, with galanin acting as a central downstream effector. The results obtained with SF1-expressing neurons, however, indicate that bone homeostasis is not always dictated by the global energy status, and vice versa.

Atypical antipsychotic (AA) drugs, including risperidone (RIS), are used to treat schizophrenia, bipolar disorder, and autism, and are prescribed off-label for other mental health issues. AA drugs are associated with severe metabolic side effects of obesity and type 2 diabetes. Cross-sectional and longitudinal data also show that risperidone causes bone loss and increases fracture risk in both men and women. There are several potential mechanisms of bone loss from RIS. One is hypogonadism due to hyperprolactinemia from dopamine receptor antagonism. However, many patients have normal prolactin levels; moreover we demonstrated that bone loss from RIS in mice can be blocked by inhibition of β-adrenergic receptor activation with propranolol, suggesting the sympathetic nervous system (SNS) plays a pathological role. Further, when, we treated ovariectomized (OVX) and sham operated mice daily for 8 weeks with RIS or vehicle we demonstrated that RIS causes significant trabecular bone loss in both sham operated and OVX mice. RIS directly suppressed osteoblast number in both sham and OVX mice, but increased osteoclast number and surface in OVX mice alone, potentially accounting for the augmented bone loss. Thus, hypogonadism alone cannot explain RIS induced bone loss. In the current study, we show that dopamine and RIS are present in the bone marrow compartment and that RIS can exert its effects directly on bone cells via dopamine receptors. Our findings of both direct and indirect effects of AA drugs on bone are relevant for current and future clinical and translational studies investigating the mechanism of skeletal changes from AA drugs.

Osteocytes within the mineralized bone matrix control bone remodeling by regulating osteoblast and osteoclast activity. Osteocytes express the aging suppressor Klotho, but the functional role of this protein in skeletal homeostasis is unknown. Here we identify Klotho expression in osteocytes as a potent regulator of bone formation and bone mass. Targeted deletion of Klotho from osteocytes led to a striking increase in bone formation and bone volume coupled with enhanced osteoblast activity, in sharp contrast to what is observed in Klotho hypomorphic (kl/kl) mice. Conversely, overexpression of Klotho in cultured osteoblastic cells inhibited mineralization and osteogenic activity during osteocyte differentiation. Further, the induction of chronic kidney disease with high-turnover renal osteodystrophy led to downregulation of Klotho in bone cells. This appeared to offset the skeletal impact of osteocyte-targeted Klotho deletion. Thus, our findings establish a key role of osteocyte-expressed Klotho in regulating bone metabolism and indicate a new mechanism by which osteocytes control bone formation.

Collagen triple helix repeat-containing1 (Cthrc1) has previously been implicated in osteogenic differentiation and positive regulation of bone mass, however, the underlying mechanisms remain unclear. Here we characterized the bone phenotype of a novel Cthrc1 null mouse strain using bone histomorphometry, μCT analysis and functional readouts for bone strength. In male Cthrc1 null mice both trabecular bone as well as cortical bone formation was impaired, whereas in female Cthrc1 null mice only trabecular bone parameters were altered. Novel and highly specific monoclonal antibodies revealed that CTHRC1 is expressed by osteocytes and osteoblasts, but not osteoclasts. Furthermore, Cthrc1 null mice exhibited increased bone resorption with increased number of osteoclast and increased osteoclast activity together with enhanced expression of osteoclastogenic genes such as c-Fos, Rankl, Trap, and Nfatc1. Differentiation of bone marrow-derived monocytes isolated from Cthrc1 null mice differentiated into osteoclasts as effectively as those from wildtype mice. In the presence of CTHRC1 osteoclastogenic differentiation of bone marrow-derived monocytes was dramatically inhibited as was functional bone resorption by osteoclasts. This process was accompanied by downregulation of osteoclastogenic marker genes, indicating that extrinsically derived CTHRC1 is required for such activity. In vitro, CTHRC1 had no effect on osteogenic differentiation of bone marrow stromal cells, however, calvarial osteoblasts from Cthrc1 null mice exhibited reduced osteogenic differentiation compared to osteoblasts from wildtypes. In a collagen antibody-induced arthritis model Cthrc1 null mice suffered significantly more severe inflammation and joint destruction than wildtypes, suggesting that CTHRC1 expressed by the activated synoviocytes has anti-inflammatory effects. Mechanistically, we found that CTHRC1 inhibited NFκB activation by preventing IκBα degradation while also inhibiting ERK1/2 activation. Collectively our studies demonstrate that CTHRC1 secreted from osteocytes and osteoblasts functions as an inhibitor of osteoclast differentiation via inhibition of NFκB-dependent signaling. Furthermore, our data suggest that CTHRC1 has potent anti-inflammatory properties that limit arthritic joint destruction.

The ventral hypothalamus (VHT) integrates several physiological cues to maintain glucose homeostasis and energy balance. Aging is associated with increased glucose intolerance but the underlying mechanisms responsible for age-related metabolic decline, including neuronal signaling in the VHT, remain elusive. We have shown that mice with VHT-targeted overexpression of ∆FosB, a splice variant of the AP1 transcription factor FosB, exhibit increased energy expenditure, leading to decreased adiposity. Here, we show that VHT-targeted overexpression of ∆FosB also improves glucose tolerance, increases insulin sensitivity in target organs and thereby suppresses insulin secretion. These effects are also observed by the overexpression of dominant negative JunD, demonstrating that they occur via AP1 antagonism within the VHT. Furthermore, the improved glucose tolerance and insulin sensitivity persisted in aged animals overexpressing ∆FosB in the VHT. These beneficial effects on glucose metabolism were abolished by peripheral sympathectomy and α-adrenergic, but not β-adrenergic, blockade. Taken together, our results show that antagonizing AP1 transcription activity in the VHT leads to a marked improvement in whole body glucose homeostasis via activation of the SNS, conferring protection against age-related impairment in glucose metabolism. These findings may open novel avenues for therapeutic intervention in diabetes and age-related glucose intolerance.

Lactation induces bone loss to provide sufficient calcium in the milk, a process that involves osteoclastic bone resorption but also osteocytes and perilacunar resorption. The exact mechanisms by which osteocytes contribute to bone loss remain elusive. Osteocytes express genes required in osteoclasts for bone resorption, including cathepsin K (Ctsk), and lactation elevates their expression. We show that Ctsk deletion in osteocytes prevented the increase in osteocyte lacunar area seen during lactation, as well as the effects of lactation to increase osteoclast numbers and decrease trabecular bone volume, cortical thickness and mechanical properties. In addition, Ctsk deletion in osteocytes increased bone Parathyroid Hormone related Peptide (PTHrP), prevented the decrease in serum Parathyroid Hormone (PTH) induced by lactation, but amplified the increase in serum 1,25(OH)2D. The net result of these changes is to maintain serum and milk calcium levels in the normal range, ensuring normal offspring skeletal development. Our studies confirm the fundamental role of osteocytic perilacunar remodeling in physiological states of lactation and provides genetic evidence that osteocyte-derived Ctsk contributes not only to osteocyte perilacunar remodeling, but also to the regulation of PTH, PTHrP, 1,25-Dyhydroxyvitamin D (1,25(OH)2D), osteoclastogenesis and bone loss in response to the high calcium demand associated with lactation.

Energy metabolism and bone homeostasis share several neuronal regulatory pathways. Within the ventral hypothalamus (VHT), the orexigenic neurons co-express Agouti-related peptide (AgRP) and neuropeptide Y (NPY) and the anorexigenic neurons co-express, α-melanocyte stimulating hormone derived from proopiomelanocortin (POMC), and cocaine and amphetamine-regulated transcript (CART). These neurons regulate both processes, yet their relative contribution is unknown. Previously, using genetically targeted activator protein (AP1) alterations as a tool, we showed in adult mice that AgRP or POMC neurons are capable of inducing whole-body energy catabolism and bone accrual, with different effects on bone resorption. Here, we investigated whether co-residing neurons exert similar regulatory effects. We show that AP1 antagonists targeted to NPY-producing or CART-producing neurons in adult mice stimulate energy expenditure, reduce body weight gain and adiposity and promote trabecular bone formation and mass, yet again via different effects on bone resorption, as measured by serum level of carboxy-terminal collagen type I crosslinks (CTX). In addition, AP1 antagonists promote neurite expansion, increasing neurite number, length, and surface area in primary hypothalamic neuronal cultures. Overall, our data demonstrate that the orexigenic NPY and anorexigenic CART neurons both have the capacity to stimulate energy burning state and increase bone mass. © 2020 American Society for Bone and Mineral Research.

Androgen deficiency is a leading cause of male osteoporosis, with bone loss driven by an inadequate level of bone formation relative to the extent of bone resorption. Abaloparatide, an osteoanabolic PTH receptor agonist used to treat women with postmenopausal osteoporosis at high risk for fracture, increases bone formation and bone strength in estrogen-deficient animals without increasing bone resorption. This study examined the effects of abaloparatide on bone formation, bone mass, and bone strength in androgen-deficient orchiectomized (ORX) rats, a male osteoporosis model. Four-month-old Sprague-Dawley rats underwent ORX or sham surgery. Eight weeks later, sham-operated rats received vehicle (saline; n = 10) while ORX rats (n = 10/group) received vehicle (Veh) or abaloparatide at 5 or 25 μg/kg (ABL5 or ABL25) by daily s.c. injection for 8 weeks, followed by sacrifice. Dynamic bone histomorphometry indicated that the tibial diaphysis of one or both abaloparatide groups had higher periosteal mineralizing surface, intracortical bone formation rate (BFR), endocortical BFR, and cortical thickness vs Veh controls. Vertebral trabecular BFR was also higher in both abaloparatide groups vs Veh, and the ABL25 group had higher trabecular osteoblast surface without increased osteoclast surface. By micro-CT, the vertebra and distal femur of both abaloparatide-groups had improved trabecular bone volume and micro-architecture, and the femur diaphysis of the ABL25 group had greater cortical thickness with no increase in porosity vs Veh. Biomechanical testing indicated that both abaloparatide-groups had stronger vertebrae and femoral necks vs Veh controls. These findings provide preclinical support for evaluating abaloparatide as an investigational treatment for male osteoporosis.

Chondroitin sulfate (CS) has been accepted as an ingredient in health foods for the treatment of symptoms related to arthritis and cartilage repair. However, CS is poorly absorbed through the gastrointestinal tract because of its high negative electric charges and molecular weight (MW). In this study, poly-ion complex (PIC) formation was found in aqueous solutions through electrostatic interaction between CS and polyamines-organic molecules having two or more primary amino groups ubiquitously distributed in natural products at high concentrations. Characteristic properties of various PICs generated by mixing CS and natural polyamines, including unusual polyamines, were studied based on the turbidity for PIC formation, the dynamic light scattering for the size of PIC particles, and ζ-potential measurements for the surface charges of PIC particles. The efficiency of PIC formation between CS and spermine increased in a CS MW-dependent manner, with 15 kDa CS being critical for the formation of PIC (particle size: 3.41 µm) having nearly neutral surface charge (ζ-potential: -0.80 mV). Comparatively, mixing tetrakis(3-aminopropyl)ammonium and 15 kDa of CS afforded significant levels of PIC (particle size: 0.42±0.16 µm) despite a strongly negative surface charge (-34.67±1.15 mV). Interestingly, the oral absorption efficiency of CS was greatly improved only when PIC possessing neutral surface charges was administered to mice. High formation efficiency and electrically neutral surface charge of PIC particles are important factors for oral CS bioavailability.

Chondrocytes proliferate and mature into hypertrophic chondrocytes. Vascular invasion into the cartilage occurs in the terminal hypertrophic chondrocyte layer, and terminal hypertrophic chondrocytes die by apoptosis or transdifferentiate into osteoblasts. Runx2 is essential for osteoblast differentiation and chondrocyte maturation. Runx2-deficient mice are composed of cartilaginous skeletons and lack the vascular invasion into the cartilage. However, the requirement of Runx2 in the vascular invasion into the cartilage, mechanism of chondrocyte transdifferentiation to osteoblasts, and its significance in bone development remain to be elucidated. To investigate these points, we generated Runx2fl/flCre mice, in which Runx2 was deleted in hypertrophic chondrocytes using Col10a1 Cre. Vascular invasion into the cartilage was similarly observed in Runx2fl/fl and Runx2fl/flCre mice. Vegfa expression was reduced in the terminal hypertrophic chondrocytes in Runx2fl/flCre mice, but Vegfa was strongly expressed in osteoblasts in the bone collar, suggesting that Vegfa expression in bone collar osteoblasts is sufficient for vascular invasion into the cartilage. The apoptosis of terminal hypertrophic chondrocytes was increased and their transdifferentiation was interrupted in Runx2fl/flCre mice, leading to lack of primary spongiosa and osteoblasts in the region at E16.5. The osteoblasts appeared in this region at E17.5 in the absence of transdifferentiation, and the number of osteoblasts and the formation of primary spongiosa, but not secondary spongiosa, reached to levels similar those in Runx2fl/fl mice at birth. The bone structure and volume and all bone histomophometric parameters were similar between Runx2fl/fl and Runx2fl/flCre mice after 6 weeks of age. These findings indicate that Runx2 expression in terminal hypertrophic chondrocytes is not required for vascular invasion into the cartilage, but is for their survival and transdifferentiation into osteoblasts, and that the transdifferentiation is necessary for trabecular bone formation in embryonic and neonatal stages, but not for acquiring normal bone structure and volume in young and adult mice., PLoS Genetics, 16(11), art.no.1009169; 2020

Energy metabolism and bone homeostasis share several regulatory pathways. The AP1 transcription factor ΔFosB and leptin both regulate energy metabolism and bone, yet whether their pathways intersect is not known. Transgenic mice overexpressing ΔFosB under the control of the Enolase 2 (ENO2) promoter exhibit high bone mass, high energy expenditure, low fat mass, and low circulating leptin levels. Because leptin is a regulator of bone and ΔFosB acts on leptin-responsive ventral hypothalamic (VHT) neurons to induce bone anabolism, we hypothesized that regulation of leptin may contribute to the central actions of ΔFosB in the VHT. To address this question, we used adeno-associated virus (AAV) expression of ΔFosB in the VHT of leptin-deficient ob/ob mice and genetic crossing of ENO2-ΔFosB with ob/ob mice. In both models, leptin deficiency prevented ΔFosB-triggered reduction in body weight, increase in energy expenditure, increase in glucose utilization, and reduction in pancreatic islet size. In contrast, leptin deficiency failed to prevent ΔFosB-triggered increase in bone mass. Unlike leptin deficiency, galanin deficiency blocked both the metabolic and the bone ΔFosB-induced effects. Overall, our data demonstrate that, while the catabolic energy metabolism effects of ΔFosB require intact leptin and galanin signaling, the bone mass-accruing effects of ΔFosB require galanin but are independent of leptin. © 2019 American Society for Bone and Mineral Research.

© 2018 Irisin is secreted by muscle, increases with exercise, and mediates certain favorable effects of physical activity. In particular, irisin has been shown to have beneficial effects in adipose tissues, brain, and bone. However, the skeletal response to exercise is less clear, and the receptor for irisin has not been identified. Here we show that irisin binds to proteins of the αV class of integrins, and biophysical studies identify interacting surfaces between irisin and αV/β5 integrin. Chemical inhibition of the αV integrins blocks signaling and function by irisin in osteocytes and fat cells. Irisin increases both osteocytic survival and production of sclerostin, a local modulator of bone remodeling. Genetic ablation of FNDC5 (or irisin) completely blocks osteocytic osteolysis induced by ovariectomy, preventing bone loss and supporting an important role of irisin in skeletal remodeling. Identification of the irisin receptor should greatly facilitate our understanding of irisin's function in exercise and human health. Irisin, through its integrin receptor, promotes skeletal remodeling with implications for stemming bone loss.

Previously, we reported sexually dimorphic bone mass and body composition phenotypes in Igfbp2−/− mice (−/−), where male mice exhibited decreased bone and increased fat mass, whereas female mice displayed increased bone but no changes in fat mass. To investigate the interaction between IGF-binding protein (IGFBP)-2 and estrogen, we subjected Igfbp2 −/− and +/+ female mice to ovariectomy (OVX) or sham surgery at 8 weeks of age. At 20 weeks of age, mice underwent metabolic cage analysis and insulin tolerance tests before killing. At harvest, femurs were collected for microcomputed tomography, serum for protein levels, brown adipose tissue (BAT) and inguinal white adipose tissue (IWAT) adipose depots for histology, gene expression, and mitochondrial respiration analysis of whole tissue. In +/+ mice, serum IGFBP-2 dropped 30% with OVX. In the absence of IGFBP-2, OVX had no effect on preformed BAT; however, there was significant “browning” of the IWAT depot coinciding with less weight gain, increased insulin sensitivity, lower intraabdominal fat, and increased bone loss due to higher resorption and lower formation. Likewise, after OVX, energy expenditure, physical activity and BAT mitochondrial respiration were decreased less in the OVX−/− compared with OVX+/+. Mitochondrial respiration of IWAT was reduced in OVX+/+ yet remained unchanged in OVX−/− mice. These changes were associated with significant increases in Fgf21 and Foxc2 expression, 2 proteins known for their insulin sensitizing and browning of WAT effects. We conclude that estrogen deficiency has a profound effect on body and bone composition in the absence of IGFBP-2 and may be related to changes in fibroblast growth factor 21.

Atypical antipsychotic (AA) drugs cause significant metabolic side effects, and clinical data are emerging that demonstrate increased fracture risk and bone loss after treatment with the AA, risperidone (RIS). The pharmacology underlying the adverse effects on bone is unknown. However, RIS action in the central nervous system could be responsible because the sympathetic nervous system (SNS) is known to uncouple bone remodeling. RIS treatment in mice significantly lowered trabecular bone volume fraction (bone volume/total volume), owing to increased osteoclast-mediated erosion and reduced osteoblast-mediated bone formation. Daytime energy expenditure was also increased and was temporally associated with the plasma concentration of RIS. Even a single dose of RIS transiently elevated expression of brown adipose tissue markers of SNS activity and thermogenesis, Pgc1a and Ucp1. Rankl, an osteoclast recruitment factor regulated by the SNS, was also increased 1 hour after a single dose of RIS. Thus, we inferred that bone loss from RIS was regulated, at least in part, by the SNS. To test this, we administered RIS or vehicle to mice that were also receiving the nonselective β-blocker propranolol. Strikingly, RIS did not cause any changes in trabecular bone volume/total volume, erosion, or formation while propranolol was present. Furthermore, β2-adrenergic receptor null (Adrb2(-/-)) mice were also protected from RIS-induced bone loss. This is the first report to demonstrate SNS-mediated bone loss from any AA. Because AA medications are widely prescribed, especially to young adults, clinical studies are needed to assess whether β-blockers will prevent bone loss in this vulnerable population.

Osteocytes secrete paracrine factors that regulate the balance between bone formation and destruction. Among these molecules, sclerostin (encoded by the gene SOST) inhibits osteoblastic bone formation and is an osteoporosis drug target. The molecular mechanisms underlying SOST expression remain largely unexplored. Here, we report that histone deacetylase 5 (HDAC5) negatively regulates sclerostin levels in osteocytes in vitro and in vivo. HDAC5 shRNA increases, whereas HDAC5 overexpression decreases SOST expression in the novel murine Ocy454 osteocytic cell line. HDAC5 knockout mice show increased levels of SOST mRNA, more sclerostin-positive osteocytes, decreased Wnt activity, low trabecular bone density, and reduced bone formation by osteoblasts. In osteocytes, HDAC5 binds and inhibits the function of MEF2C, a crucial transcription factor for SOST expression. Using chromatin immunoprecipitation, we have mapped endogenous MEF2C binding in the SOST gene to a distal intergenic enhancer 45 kB downstream from the transcription start site. HDAC5 deficiency increases SOST enhancer MEF2C chromatin association and H3K27 acetylation and decreases recruitment of corepressors NCoR and HDAC3. HDAC5 associates with and regulates the transcriptional activity of this enhancer, suggesting direct regulation of SOST gene expression by HDAC5 in osteocytes. Finally, increased sclerostin production achieved by HDAC5 shRNA is abrogated by simultaneous knockdown of MEF2C, indicating that MEF2C is a major target of HDAC5 in osteocytes.

Misty mice (m/m) have a loss of function mutation in Dock7 gene, a guanine nucleotide exchange factor, resulting in low bone mineral density, uncoupled bone remodeling and reduced bone formation. Dock7 has been identified as a modulator of osteoblast number and in vitro osteogenic differentiation in calvarial osteoblast culture. In addition, m/m exhibit reduced preformed brown adipose tissue innervation and temperature as well as compensatory increase in beige adipocyte markers. While the low bone mineral density phenotype is in part due to higher sympathetic nervous system (SNS) drive in young mice, it is unclear what effect aging would have in mice homozygous for the mutation in the Dock7 gene. We hypothesized that age-related trabecular bone loss and periosteal envelope expansion would be altered in m/m. To test this hypothesis, we comprehensively characterized the skeletal phenotype of m/m at 16, 32, 52, and 78 wks of age. When compared to age-matched wild-type control mice (+/+), m/m had lower areal bone mineral density (aBMD) and areal bone mineral content (aBMC). Similarly, both femoral and vertebral BV/TV, Tb.N., and ConnD were decreased in m/m while there was also an increase in Tb.Sp. As low bone mineral density and decreased trabecular bone were already present at 16 wks of age in m/m and persisted throughout life, changes in age-related trabecular bone loss were not observed highlighting the role of Dock7 in controlling trabecular bone acquisition or bone loss prior to 16 wks of age. Cortical thickness was also lower in the m/m across all ages. Periosteal and endosteal circumferences were higher in m/m compared to +/+ at 16 wks. However, endosteal and periosteal expansion were attenuated in m/m, resulting in m/m having lower periosteal and endosteal circumferences by 78 wks of age compared to +/+, highlighting the critical role of Dock7 in appositional bone expansion. Histomorphometry revealed that osteoblasts were nearly undetectable in m/m and marrow adipocytes were elevated 3.5 fold over +/+ (p=0.014). Consistent with reduced bone formation, osteoblast gene expression of Alp, Col1a1, Runx-2, Sp7, and Bglap was significantly decreased in m/m whole bone. Furthermore, markers of osteoclasts were either unchanged or suppressed. Bone marrow stromal cell migration and motility were inhibited in culture and changes in senescence markers suggest that osteoblast function may also be inhibited with loss of Dock7 expression in m/m. Finally, increased Oil Red O staining in m/m ear mesenchymal stem cells during adipogenesis highlights a potential shift of cells from the osteogenic to adipogenic lineages. In summary, loss of Dock7 in the aging m/m resulted in an impairment of periosteal and endocortical envelope expansion, but did not alter age-related trabecular bone loss. These studies establish Dock7 as a critical regulator of both cortical and trabecular bone mass, and demonstrate for the first time a novel role of Dock7 in modulating compensatory changes in the periosteum with aging.

There is a growing and alarming prevalence of obesity and the metabolic syndrome in type I diabetic patients (T1DM), particularly in adolescence. In general, low bone mass, higher fracture risk, and increased marrow adipose tissue (MAT) are features of diabetic osteopathy in insulin-deficient subjects. On the other hand, type 2 diabetes (T2DM) is associated with normal or high bone mass, a greater risk of peripheral fractures, and no change in MAT. Therefore, we sought to determine the effect of weight gain on bone turnover in insulin-deficient mice. We evaluated the impact of a 6-week high-fat (HFD) rich in medium chain fatty acids or low-fat diet (LFD) on bone mass and MAT in a streptozotocin (STZ)-induced model using male C57BL/6J mice at 8 weeks of age. Dietary intervention was initiated after diabetes confirmation. At the endpoint, lower non-fasting glucose levels were observed in diabetic mice fed with high fat diet compared to diabetic mice fed the low fat diet (STZ-LFD). Compared to euglycemic controls, the STZ-LFD had marked polydipsia and polyphagia, as well as reduced lean mass, fat mass, and bone parameters. Interestingly, STZ-HFD mice had higher bone mass, namely less cortical bone loss and more trabecular bone than STZ-LFD. Thus, we found that a HFD, rich in medium chain fatty acids, protects against bone loss in a T1DM mouse model. Whether this may also translate to T1DM patients who are overweight or obese in respect to maintenance of bone mass remains to be determined through longitudinal studies.

Obesity during maturation can affect the growing skeleton directly and indirectly, although these effects and the mechanisms behind them are not fully understood. Our objective was to determine how a high-fat diet with or without metformin treatment affects skeletal development. We also sought to characterize changes that occur in white adipose tissue, circulating metabolites, lipids, and gut microbiota. A diet-induced obesity C57BL/6J mouse model was used to test the effects of obesity and metformin on bone using bone histomorphometry and microcomputed tomography. Bone marrow adipose tissue was quantified with osmium tetroxide microcomputed tomography and histology. Dual-energy x-ray absorptiometry was used to analyze body composition. Hematoxylin and eosin staining was used to assess changes in white adipose depots, mass spectrometry was used for circulating lipids and protein metabolite analysis, and ribosomal RNA sequencing was used for gut microbiome analysis. Mice fed a high fat-diet since wean displayed increased medullary areas and decreased osteoblast numbers in the long bones; this phenotype was partially normalized by metformin. Marrow and inguinal adipose expansion was also noted in obese mice, and this was partially normalized by metformin. A drug-by-diet interaction was noted for circulating lipid molecules, protein metabolites, and gut microbiome taxonomical units. Obesity was not detrimental to trabecular bone in growing mice, but bone marrow medullary expansion was observed, likely resulting from inhibition of osteoblastogenesis, and this was partially reversed by metformin treatment.

Nature Communications 7: Article number: 13176 (2017); Published 19 October 2016; Updated 22 February 2017 In this article, there are errors in the labelling of the y axis in Fig. 1b. The labels '50', '100' 'and ‘150’ should have been ‘500’, '1,000' and ‘1,500’, respectively. The correct version of this figure appears as Fig.

Insulin-like growth factor-1 (IGF-1) and its binding proteins are critical mediators of skeletal growth. Insulin-like growth factor-binding protein 4 (IGFBP-4) is highly expressed in osteoblasts and inhibits IGF-1 actions in vitro. Yet, in vivo studies suggest that it could potentiate IGF-1 and IGF-2 actions. In this study, we hypothesized that IGFBP-4 might potentiate the actions of IGF-1 on the skeleton. To test this, we comprehensively studied 8- and 16-week-old Igfbp4−/− mice. Both male and female adult Igfbp4−/− mice had marked growth retardation with reductions in body weight, body and femur lengths, fat proportion and lean mass at 8 and 16 weeks. Marked reductions in aBMD and aBMC were observed in 16-week-old Igfbp4−/− females, but not in males. Femoral trabecular BV/TV and thickness, cortical fraction and thickness in 16-week-old Igfbp4−/− females were significantly reduced. However, surprisingly, males had significantly more trabeculae with higher connectivity density than controls. Concordantly, histomorphometry revealed higher bone resorption and lower bone formation in Igfbp4−/− females. In contrast, Igfbp4−/− males had lower mineralized surface/bone surface. Femoral expression of Sost and circulating levels of sclerostin were reduced but only in Igfbp4−/− males. Bone marrow stromal cultures from mutants showed increased osteogenesis, whereas osteoclastogenesis was markedly increased in cells from Igfbp4−/− females but decreased in males. In sum, our results indicate that loss of Igfbp4 affects mesenchymal stromal cell differentiation, regulates osteoclastogenesis and influences both skeletal development and adult bone maintenance. Thus, IGFBP-4 modulates the skeleton in a gender-specific manner, acting as both a cell autonomous and cell non-autonomous factor.

Osteoblasts and adipocytes arise from a common mesenchymal precursor cell. The cell fate decision of a mesenchymal precursor cell is under the influence of molecular cues and signaling pathways that lead to the activation or repression of lineage-specific transcription factors. The molecular mechanisms determining osteoblast versus adipocyte lineage specificity in response to bone morphogenic protein (BMP) remain unclear. In this study, we describe the mechanism through which Zfp521 (ZNF521), a regulator of lineage progression in multiple immature cell populations, regulates lineage specification of mesenchymal progenitor cells during BMP-induced differentiation events. In vivo deletion or in vitro knockdown of Zfp521 in mesenchymal precursors resulted in increased expression of the adipocyte determinant factor Zfp423 (ZNF423). This was concurrent with the loss of histone H3K9 methylation and an increase in histone H3K9 acetylation at the Zfp423 promoter, which together are indicative of decreased gene repression. Indeed, we found that Zfp521 occupies and represses the promoter and intronic enhancer regions of Zfp423. Accordingly, conditional deletion of Zfp521 inhibited heterotopic bone formation in response to local injection of BMP2. In contrast, marrow adiposity within BMP2-induced bone was markedly enhanced in Zfp521-deficient mice, suggesting that precursor cells lacking Zfp521 differentiate preferentially into adipocytes instead of osteoblasts in response to BMP2. Consistent with a cell-autonomous role of Zfp521 in mesenchymal precursors, knockdown of Zfp521 in stromal cells prevented BMP2-induced osteoblast marker expression and simultaneously enhanced lipid accumulation and expression of adipocyte-related genes. Taken together, the data suggest that Zfp521 is a cell fate switch critical for BMP-induced osteoblast commitment and identify Zfp521 as the intrinsic repressor of Zfp423 and hence of adipocyte commitment during BMP-induced mesenchymal precursor differentiation.

Mechanical unloading, such as in a microgravity environment in space or during bed rest (for patients who require prolonged bed rest), leads to a decrease in bone mass because of the suppression of bone formation and the stimulation of bone resorption. To address the challenges presented by a prolonged stay in space and the forthcoming era of a super-aged society, it will be important to prevent the bone loss caused by prolonged mechanical unloading. Nuclear factor κB (NF-κB) transcription factors are activated by mechanical loading and inflammatory cytokines. Our objective was to elucidate the role of NF-κB pathways in bone loss that are caused by mechanical unloading. Eight-week-old wild-type (WT) and NF-κB1-deficient mice were randomly assigned to a control or mechanically unloaded with tail suspension group. After 2 weeks, a radiographic analysis indicated a decrease in bone mass in the tibias and femurs of the unloaded WT mice but not in the NF-κB1-deficient mice. An NF-κB1 deficiency suppressed the unloading-induced reduction in bone formation by maintaining the proportion and/or potential of osteoprogenitors or immature osteoblasts, and by suppression of bone resorption through the inhibition of intracellular signaling through the receptor activator of NF-κB ligand (RANKL) in osteoclast precursors. Thus, NF-κB1 is involved in two aspects of rapid reduction in bone mass that are induced by disuse osteoporosis in space or bed rest.