Osteoclast Receptor Research Articles

Pdk3's role in RANKL-induced osteoclast differentiation: insights from a bone marrow macrophage model.

Osteoporosis (OP) is a chronic disease characterized by decreased bone mass, loss of skeletal structural integrity and increased susceptibility to fracture. Available studies have shown that the pyruvate dehydrogenase kinase (PDK) family is associated with osteoclastogenesis and bone loss, but the specific role of Pdk3 in bone pathology has not been systematically investigated. A cell OP model was established in receptor activator for nuclear factor-κB Ligand (RANKL)-induced bone marrow macrophages (BMMs). Hereafter, the expression levels of Pdk3 and osteoclastogenesis feature genes including nuclear factor of activated T cells 1 (Nfatc1), Cathepsin K (Ctsk), osteoclast associated Ig-like receptor (Oscar) in BMMs-derived osteoclasts were examined based on real-time quantitative PCR and western blotting methods. Further, the phosphorylation of ERK, P65 and JAK/STAT and their correlation was Pdk3 was gauged. In particular, changes in the activity of these signaling pathways were observed by silencing experiments of the Pdk3 gene (using small interfering RNA). Finally, the effects of Pdk3 gene silencing on signaling pathway activity, osteoclastogenesis, and related inflammatory and apoptotic indicators were observed by transfection with PDK3-specific siRNA. Following RANKL exposure, the levels of Pdk3 and osteoclastogenesis feature genes were all elevated, and a positive correlation between Pdk3 and osteoclastogenesis feature genes was seen. Meanwhile, ERK, P65 and JAK/STAT phosphorylation was increased by RANKL, and Pdk3 was confirmed to be positively correlated with the phosphorylation of ERK, P65 and JAK/STAT. Additionally, in RANKL-exposed osteoclasts, Pdk3 knockdown diminished the phosphorylation of ERK, P65 and JAK/STAT, reduced the expressions of osteoclastogenesis feature genes. Importantly, knockdown of Pdk3 also reduced the expression of inflammatory cytokines and resulted in elevated levels of Bax and Casp3 expression, as well as downregulation of Bcl2 expression. This study reveals for the first time the role of Pdk3 in RANKL-induced osteoclastogenesis and OP. These findings provide a foundation for future studies on the role of Pdk3 in other bone diseases and provide new ideas for the development of OP therapeutics targeting Pdk3.

Antipsychotic-induced bone loss: the role of dopamine, serotonin and adrenergic receptor signalling.

Antipsychotics are commonly used in treating psychiatric disorders. These medications primarily target dopamine the serotonin receptors, they have some affinity to adrenergic, histamine, glutamate and muscarinic receptors. There is clinical evidence that antipsychotic use decreases BMD and increases fracture risk, with dopamine, serotonin and adrenergic receptor-signalling becoming an increasing area of focus where the presence of these receptors in osteoclasts and osteoblasts have been demonstrated. Osteoclasts and osteoblasts are the most important cells in the bone remodelling and the bone regeneration process where the activity of these cells determine the bone resorption and formation process in order to maintain healthy bone. However, an imbalance in osteoclast and osteoblast activity can lead to decreased BMD and increased fracture risk, which is also believed to be exacerbated by antipsychotics use. Therefore, the aim of this review is to provide an overview of the mechanisms of action of first, second and third generation antipsychotics and the expression profiles of dopamine, serotonin and adrenergic receptors during osteoclastogenesis and osteoblastogenesis.

HSP90 drives the Rab11a-mediated vesicular transport of the cell surface receptors in osteoclasts.

Rab11a, which ubiquitously localizes to early and recycling endosomes, is required for regulating the vesicular transport of cellular cargos. Interestingly, our previous study revealed that Rab11a served as a negative regulator of osteoclastogenesis by facilitating the lysosomal proteolysis of (1) colony-stimulating factor-1 (c-fms) receptor and (2) receptor activator of nuclear factor-κB (RANK) receptor, thereby resulting in inhibition of osteoclast (OC) differentiation, maturation, and bone-resorbing activity. However, the molecular mechanisms of how Rab11a negatively affected osteoclastogenesis were largely unknown. Heat shock protein (HSP90), including two isoforms HSP90αand HSP90β, necessitates the stability, maturation, and activity of a broad range of its clients, and is essentially required for a vast array of signal transduction pathways in nonstressful conditions. Furthermore, cumulative evidence suggests that HSP90 is a vital element of the vesicular transport network. Indeed, our recent study revealed thatHSP90, a novel effector protein of Rab11b, modulatedRab11b-mediated osteoclastogenesis. In this study, we also found that Rab11a interacted with both HSP90α and HSP90β in OCs. Upon blockade of HSP90 ATPase activity by a specific inhibitor(17-allylamino-demethoxygeldanamycin), we showed that (1) the ATPase domain of HSP90 was a prerequisite for the interaction between HSP90 and Rab11a, and (2) the interaction of HSP90 to Rab11a sufficiently maintained the inhibitory effects of Rab11a on osteoclastogenesis. Altogether, our findings undoubtedly indicate a novel role of HSP90 in regulating Rab11a-mediated osteoclastogenesis.

PGE2 activates EP4 in subchondral bone osteoclasts to regulate osteoarthritis

Prostaglandin E2 (PGE2), a major cyclooxygenase-2 (COX-2) product, is highly secreted by the osteoblast lineage in the subchondral bone tissue of osteoarthritis (OA) patients. However, NSAIDs, including COX-2 inhibitors, have severe side effects during OA treatment. Therefore, the identification of novel drug targets of PGE2 signaling in OA progression is urgently needed. Osteoclasts play a critical role in subchondral bone homeostasis and OA-related pain. However, the mechanisms by which PGE2 regulates osteoclast function and subsequently subchondral bone homeostasis are largely unknown. Here, we show that PGE2 acts via EP4 receptors on osteoclasts during the progression of OA and OA-related pain. Our data show that while PGE2 mediates migration and osteoclastogenesis via its EP2 and EP4 receptors, tissue-specific knockout of only the EP4 receptor in osteoclasts (EP4LysM) reduced disease progression and osteophyte formation in a murine model of OA. Furthermore, OA-related pain was alleviated in the EP4LysM mice, with reduced Netrin-1 secretion and CGRP-positive sensory innervation of the subchondral bone. The expression of platelet-derived growth factor-BB (PDGF-BB) was also lower in the EP4LysM mice, which resulted in reduced type H blood vessel formation in subchondral bone. Importantly, we identified a novel potent EP4 antagonist, HL-43, which showed in vitro and in vivo effects consistent with those observed in the EP4LysM mice. Finally, we showed that the Gαs/PI3K/AKT/MAPK signaling pathway is downstream of EP4 activation via PGE2 in osteoclasts. Together, our data demonstrate that PGE2/EP4 signaling in osteoclasts mediates angiogenesis and sensory neuron innervation in subchondral bone, promoting OA progression and pain, and that inhibition of EP4 with HL-43 has therapeutic potential in OA.

The Neuropeptide VIP Limits Human Osteoclastogenesis: Clinical Associations with Bone Metabolism Markers in Patients with Early Arthritis.

We aimed to evaluate the direct action of VIP on crucial molecules involved in human osteoclast differentiation and function. We also investigated the relationship between VIP serum levels and bone remodeling mediators in early arthritis patients. The expression of VIP receptors and osteoclast gene markers in monocytes and in vitro differentiated osteoclasts was studied by real-time PCR. NFATc1 activity was measured using a TransAM® kit. Osteoclastogenesis was confirmed by quantification of tartrate-resistant acid phosphatase positive multinucleated cells. OsteoAssay® Surface Multiple Well Plate was used to evaluate bone-resorbing activity. The ring-shaped actin cytoskeleton and the VPAC1 and VPAC2 expression were analyzed by immunofluorescence. We described the presence of VIP receptors in monocytes and mature osteoclasts. Osteoclasts that formed in the presence of VIP showed a decreased expression of osteoclast differentiation gene markers and proteolytic enzymes involved in bone resorption. VIP reduced the resorption activity and decreased both β3 integrin expression and actin ring formation. Elevated serum VIP levels in early arthritis patients were associated with lower BMD loss and higher serum OPG concentration. These results demonstrate that VIP exerts an anti-osteoclastogenic action impairing both differentiation and resorption activity mainly through the negative regulation of NFATc1, evidencing its bone-protective effects in humans.

The Novel Role of Checkpoint Inhibitor PD-1H/VISTA in Osteoclast Activation and Multiple Myeloma Bone Disease

IntroductionMultiple myeloma (MM)-induced bone disease remains one of its most devastating complications, caused by increased bone resorption by overactivated osteoclasts coupled with impaired bone formation. MM cells produce osteoclast-activating factors that induce osteoclast activation and extensive bone resorption. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells (Fu J etc. JCI. 2016). We also identified that the checkpoint inhibitor, programmed death-1 homolog (PD-1H/VISTA), serves as the MMP-13 receptor in osteoclasts and mediates MMP-13-dependent osteoclastogenic function which is largely blocked in Pd-1h -/-osteoclasts (Fu J etc. ASH 2019, 2020). While the inhibitory role of PD-1H/VISTA in T-cells has recently been described (ElTanbouly MA etc. Science 2020), its cellular binding proteins remain unclear, and its role in osteoclast activation andMM bone disease have not been addressed.Methods and ResultsTo identify its interacting proteins, PD-1H-His 6 recombinant protein was expressed in mouse bone marrow mononuclear cells, the associated proteins pulled down by Ni-NTA agarose beads from cell lysates and identified by mass spectrometry. Functional annotation charting of the 75 proteins enriched in PD-1H pull-down samples (with signal ratio of PD-1H-His 6 pull-down vs control >2) indicated that almost 30% of the interacting targets were either cytoskeletal or cytoskeleton-associated proteins.Given that the F-actin cytoskeleton undergoes dynamic reorganization during osteoclast differentiation and plays critical roles in bone resorption, we further addressed the role of PD-1H in F-actin cytoskeleton regulation. Initially, osteoclasts form F-actin-rich adhesive structures, termed podosomes. At later stages, podosomes collectively rearrange into clusters and rings and finally into sealing belts to mediate osteoclast spreading, migration and bone resorption (Teitelbaum SL. Ann N Y Acad Sci. 2011). By confocal immunofluorescence microscopy, we found that PD-1H co-localized with F-actin podosome clusters, rings and sealing belts during osteoclast differentiation (Figure 1A). The functional role of PD-1H in F-actin cytoskeleton reorganization was addressed using Pd-1h -/- osteoclast wherein Pd-1h knockout lead to the disruption of podosome clusters at early stages relative to WT controls, while at later stages, Pd-1h -/-osteoclasts exhibited significantly fewer F-actin rings and belts (Figure 1B). Further, binding of MMP-13 to PD-1H increased the number of osteoclasts forming F-actin rings and belts, as well as the size of F-actin belts, which was blocked in Pd-1h -/- osteoclasts.To determine the role of PD-1H in the development of myeloma-induced lytic bone lesions, 5TGM1 myeloma cells were bilaterally intratibially injected into Pd-1h wtRag2 -/- or Pd-1h -/-Rag2 -/- mice (n=10) to induce lytic bone lesions. Three weeks following intratibial 5TGM1 injection, tibiae were harvested for micro-computed tomography. Subsequent quantitative histomorphology analyses of the trabecular and cortical bones confirmed that the knockout of Pd-1h reduced MM-induced bone destruction with significantly less decrease in trabecular bone volume (Tb. [BV/TV]), trabecular bone number (Tb. N.), trabecular bone thickness (Tb. Th.), as well as less increase in trabecular bone spacing (Tb. Sp.) and bone specific surface (Tb. [BS/BV]) compared to Pd-1h wtRag2 -/- mice. Similar effects were observed in cortical bone with less decrease in cortical bone thickness (CT. Th.), cortical bone area fraction (CT. [BA/TA]), and cortical tissue mineral density (CT. TMD) in 5TGM1 bearing Pd-1h -/-Rag2 -/- mice vs Pd-1h wtRag2 -/- mice (Table 1).ConclusionsTaken together, our study, for the first time, reveals the novel role of checkpoint inhibitor, PD-1H/VISTA, in osteoclasts and myeloma bone disease. PD-1H associates with cytoskeleton proteins and regulates the F-actin cytoskeleton reorganization which is critical for osteoclast bone resorption activity. Further, PD-1H mediates MMP-13-induced osteoclast fusion, F-actin belts formation, and osteoclast activation. Pd-1h -/-in recipient mice significantly impairs MM-induced bone loss, demonstrating that PD-1H/VISTA plays a critical role in MM bone disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A novel role of HSP90 in regulating osteoclastogenesis by abrogating Rab11b-driven transport

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays a pivotal role in folding, activating and assembling a variety of client proteins. In addition, HSP90 has recently emerged as a crucial regulator of vesicular transport of cellular proteins. In our previous study, we revealed Rab11b negatively regulated osteoclastogenesis by promoting the lysosomal proteolysis of c-fms and RANK surface receptors via the axis of early endosome-late endosome-lysosomes. In this study, using an in vitro model of osteoclasts differentiated from murine macrophage-like RAW-D cells, we revealed that Rab11b interacted with both HSP90 isoforms, HSP90 alpha (HSP90α) and HSP90 beta (HSP90β), suggesting that Rab11b is an HSP90 client. Using at specific blocker for HSP90 ATPase activity, 17-allylamino-demethoxygeldanamycin (17-AAG), we found that the HSP90 ATPase domain is indispensable for maintaining the interaction between HSP90 and Rab11b in osteoclasts. Nonetheless, its ATPase activity is not required for regulating the turnover of endogenous Rab11b. Interestingly, blocking the interaction between HSP90 and Rab11b by either HSP90-targeting small interfering RNA (siHSP90) or 17-AAG abrogated the inhibitory effects of Rab11b on osteoclastogenesis by suppressing the Rab11b-mediated transport of c-fms and RANK surface receptors to lysosomes via the axis of early endosome-late endosome-lysosomes, alleviating the Rab11b-mediated proteolysis of these surface receptors in osteoclasts. Based on our observations, we propose a HSP90/Rab11b-mediated regulatory mechanism for osteoclastogenesis by directly modulating the c-fms and RANK surface receptors in osteoclasts, thereby contributing to the maintenance of bone homeostasis.

The Effect of Inflammation on Bone.

Bone remodeling is the continual process to renew the adult skeleton through the sequential action of osteoblasts and osteoclasts. Nuclear factor RANK, an osteoclast receptor, and its ligand RANKL, expressed on the surface of osteoblasts, result in coordinated control of bone remodeling. Inflammation, a feature of illness and injury, plays a distinct role in skewing this process toward resorption. It does so via the interaction of inflammatory mediators and their related peptides with osteoblasts and osteoclasts, as well as other immune cells, to alter the expression of RANK and RANKL. Such chemical mediators include TNFα, glucocorticoids, histamine, bradykinin, PGE2, systemic RANKL from immune cells, and interleukins 1 and 6. Conditions, such as periodontal disease and alveolar bone erosion, aseptic prosthetic loosening, rheumatoid arthritis, and some sports related injuries are characterized by the result of this process. A thorough understanding of bone response to injury and disease, and ability to detect such biomarkers, as well as imaging to identify early structural and mechanical property changes in bone architecture, is important in improving management and outcomes of bone related pathology. While gut health and vitamin and mineral availability appear vitally important, nutraceuticals also have an impact on bone health. To date most pharmaceutical intervention targets inflammatory cytokines, although strategies to favorably alter inflammation induced bone pathology are currently limited. Further research is required in this field to advance early detection and treatments.

The Antiresorptive Effect of GIP, But Not GLP-2, Is Preserved in Patients With Hypoparathyroidism-A Randomized Crossover Study.

ABSTRACTGlucose‐dependent insulinotropic polypeptide (GIP) and glucagon‐like peptide‐2 (GLP‐2) are gut hormones secreted postprandially. In healthy humans, both hormones decrease bone resorption accompanied by a rapid reduction in parathyroid hormone (PTH). The aim of this study was to investigate whether the changes in bone turnover after meal intake and after GIP‐ and GLP‐2 injections, respectively, are mediated via a reduction in PTH secretion. This was tested in female patients with hypoparathyroidism given a standardized liquid mixed‐meal test (n = 7) followed by a peptide injection test (n = 4) using a randomized crossover design. We observed that the meal‐ and GIP‐ but not the GLP‐2‐induced changes in bone turnover markers were preserved in the patients with hypoparathyroidism. To understand the underlying mechanisms, we examined the expression of the GIP receptor (GIPR) and the GLP‐2 receptor (GLP‐2R) in human osteoblasts and osteoclasts as well as in parathyroid tissue. The GIPR was expressed in both human osteoclasts and osteoblasts, whereas the GLP‐2R was absent or only weakly expressed in osteoclasts. Furthermore, both GIPR and GLP‐2R were expressed in parathyroid tissue. Our findings suggest that the GIP‐induced effect on bone turnover may be mediated directly via GIPR expressed in osteoblasts and osteoclasts and that this may occur independent of PTH. In contrast, the effect of GLP‐2 on bone turnover seems to depend on changes in PTH and may be mediated through GLP‐2R in the parathyroid gland. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Pros and Cons of Denosumab Treatment for Osteoporosis and Implication for RANKL Aptamer Therapy.

Osteoporosis is age-related deterioration in bone mass and micro-architecture. Denosumab is a novel human monoclonal antibody for osteoporosis. It is a receptor activator of nuclear factor-κB ligand (RANKL) inhibitor, which binds to and inhibits osteoblast-produced RANKL, in turn reduces the binding between RANKL and osteoclast receptor RANK, therefore decreases osteoclast-mediated bone resorption and turnover. However, adverse events have also been reported after denosumab treatment, including skin eczema, flatulence, cellulitis and osteonecrosis of the jaw (ONJ). Extensive researches on the mechanism of adverse reactions caused by denosumab have been conducted and may provide new insights into developing new RANKL inhibitors that achieve better specificity and safety. Aptamers are single-stranded oligonucleotides that can bind to target molecules with high specificity and affinity. They are screened from large single-stranded synthetic oligonucleotides and enriched by a technology named SELEX (systematic evolution of ligands by exponential enrichment). With extra advantages such as high stability, low immunogenicity and easy production over antibodies, aptamers are hypothesized to be promising candidates for therapeutic drugs targeting RANKL to counteract osteoporosis. In this review, we focus on the pros and cons of denosumab treatment in osteoporosis and the implication for novel aptamer treatment.

Sphingosine-1-phosphate (S1P) receptors: Promising drug targets for treating bone-related diseases.

Sphingosine‐1‐phosphate (S1P) is a natural bioactive lipid molecule and a common first or second messenger in the cardiovascular and immune systems. By binding with its receptors, S1P can serve as mediator of signalling during cell migration, differentiation, proliferation and apoptosis. Although the predominant role of S1P in bone regeneration has been noted in many studies, this role is not as well‐known as its roles in the cardiovascular and immune systems. In this review, we summarize previous research on the role of S1P receptors (S1PRs) in osteoblasts and osteoclasts. In addition, S1P is regarded as a bridge between bone resorption and formation, which brings hope to patients with bone‐related diseases. Finally, we discuss S1P and its receptors as therapeutic targets for treating osteoporosis, inflammatory osteolysis and bone metastasis based on the biological effects of S1P in osteoclastic/osteoblastic cells, immune cells and tumour cells.

Discovery of Branebrutinib (BMS-986195): A Strategy for Identifying a Highly Potent and Selective Covalent Inhibitor Providing Rapid in Vivo Inactivation of Bruton's Tyrosine Kinase (BTK).

Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase, is a member of the Tec family of kinases and is essential for B cell receptor (BCR) mediated signaling. BTK also plays a critical role in the downstream signaling pathways for the Fcγ receptor in monocytes, the Fcε receptor in granulocytes, and the RANK receptor in osteoclasts. As a result, pharmacological inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as rheumatoid arthritis and lupus. This article will outline the evolution of our strategy to identify a covalent, irreversible inhibitor of BTK that has the intrinsic potency, selectivity, and pharmacokinetic properties necessary to provide a rapid rate of inactivation systemically following a very low dose. With excellent in vivo efficacy and a very desirable tolerability profile, 5a (branebrutinib, BMS-986195) has advanced into clinical studies.

Absence of vitamin D receptor in mature osteoclasts results in altered osteoclastic activity and bone loss

Mature osteoclasts express the vitamin D receptor (VDR) and are able to synthesise and respond to 1,25(OH)2D3 via CYP27B1 enzyme activity. Whether vitamin D signalling within osteoclasts is necessary for the regulation of osteoclastic bone resorption in an in vivo setting is unclear. To determine the requirement for the VDR- and CYP27B1-mediated activity in mature osteoclasts, conditional deletion mouse models were created whereby either Vdr or Cyp27b1 gene was inactivated by breeding either Vdrfl/fl or Cyp27b1fl/fl mice with Cathepsin K-Cre transgenic mice (CstkCre) to generate CtskCre/Vdr−/− and CtskCre/Cyp27b1−/− mice respectively. To account for potential CtskCre-meaited off-target deletion of Vdr, Dmp1Cre were also used determine the effect of Vdr deletion in osteocytes. Furthermore, CtskCre/Vdr−/− mice were ovariectomised (OVX) to assess the role of VDR in osteoclasts under bone-loss conditions and bone marrow precursor cells were cultured under osteoclastogenic conditions to assess osteoclast formation. Six-week-old CtskCre/Vdr−/− female mice demonstrated a 15% decrease in femoral BV/TV (p<0.05). In contrast, BV/TV remained unchanged in CtskCre/Cyp27b1−/− mice as well as in Dmp1Cre/VDR−/− mice. When CtskCre/Vdr−/− mice were subjected to OVX, the bone loss that occurred in CtskCre/Vdr−/− was predominantly due to a diminished volume of thinner trabeculae when compared to control levels. These changes in bone volume in CtskCre/Vdr−/− mice occurred without an observable histological change in osteoclast numbers or size. However, while cultured bone marrow-derived osteoclasts from CtskCre/Vdr−/− mice were marginally increased when compared to VDRfl/fl mice, elevated expression of genes such as Cathepsin K, Nfatc1 and VATPase was observed. Collectively, these data indicate that the absence of VDR in mature osteoclasts causes exacerbated bone loss in young mice and during OVX which is associated with enhanced osteoclastic activity and without increased osteoclastogenesis.

Activation of the P2Y2 receptor regulates bone cell function by enhancing ATP release.

Bone cells constitutively release ATP into the extracellular environment where it acts locally via P2 receptors to regulate bone cell function. Whilst P2Y2 receptor stimulation regulates bone mineralisation, the functional effects of this receptor in osteoclasts remain unknown. This investigation used the P2Y2 receptor knockout (P2Y2R-/- ) mouse model to investigate the role of this receptor in bone. MicroCT analysis of P2Y2R-/- mice demonstrated age-related increases in trabecular bone volume (≤48%), number (≤30%) and thickness (≤17%). In vitro P2Y2R-/- osteoblasts displayed a 3-fold increase in bone formation and alkaline phosphatase activity, whilst P2Y2R-/- osteoclasts exhibited a 65% reduction in resorptive activity. Serum cross-linked C-telopeptide levels (CTX, resorption marker) were also decreased (≤35%). The resorption defect in P2Y2R-/- osteoclasts was rescued by the addition of exogenous ATP, suggesting that an ATP deficit could be a key factor in the reduced function of these cells. In agreement, we found that basal ATP release was reduced up to 53% in P2Y2R-/- osteoclasts. The P2Y2 receptor agonists, UTP and 2-thioUTP, increased osteoclast activity and ATP release in wild-type but not in P2Y2R-/- cells. This indicates that the P2Y2 receptor may regulate osteoclast function indirectly by promoting ATP release. UTP and 2-thioUTP also stimulate ATP release from osteoblasts suggesting that the P2Y2 receptor exerts a similar function in these cells. Taken together, our findings are consistent with the notion that the primary action of P2Y2 receptor signalling in bone is to regulate extracellular ATP levels.

Does systemic inflammation and immune activation contribute to fracture risk in HIV?

There is increasing evidence pointing toward an important role of heightened immune activation and inflammation in people living with HIV contributing to the development of non-AIDS comorbidities. This review aims to explore low bone mineral density (BMD) in HIV with a focus on the underlying mechanisms and relationships between the immune and skeletal systems. Baseline immune activation and inflammation negatively impact BMD at antiretroviral therapy (ART) initiation. B- and T-cell alterations in HIV lead to an imbalance in the osteoblastic osteoprotegerin (OPG) and osteoclastic receptor activator of NF-κB ligand (RANKL) cytokines which favours osteoclastogenesis and bone resorption. These findings suggest an important role for immune-mediated mechanisms in the pathogenesis of low BMD in HIV. Bone homeostasis is in part regulated by cells of the immune system through complex interactions with the RANK/RANKL/OPG axis. Disturbances in the normal functioning of T, B cells, and monocytes in HIV and the resulting proinflammatory state may contribute to dysregulation of this finely controlled balance leading to increased bone loss. Pre-ART levels of immune activation and inflammation have a consistently negative effect on BMD and further suggest the immunocentric basis of bone loss in HIV alongside supporting the benefits of earlier ART initiation. Further longitudinal studies will help determine the effect this will have on fracture risk in people living with HIV.

Bovine parathyroid hormone enhances osteoclast bone resorption by modulating V-ATPase through PTH1R.

The vacuolar-type H+ adenosine triphosphatase (V-ATPase) plays an important role in cellular acidification and bone resorption by osteoclasts. However, the direct effect of bovine parathyroid hormone (bPTH) on V-ATPase has not yet been elucidated. The aim of the present study was to assess the effects of bPTH on V-ATPase and osteoclasts. Osteoclasts from bone marrow (BM)-derived monocytes of C57BL/6 mice were cultured with or without bPTH. The mRNA and protein expression levels of the V-ATPase a3-subunit and d2-subunit (by RT-qPCR and western blot analysis), V-ATPase activity (using the V type ATPase Activity Assay kit) and the bone resorption function of osteoclasts (by bone resorption assay) were examined following treatment with various concentrations of bPTH (0.1, 1.0, 10 and 100 ng/ml) alone or with bPTH and its inhibitor, bafilomycin A1. Furthermore, the expression of parathyroid hormone (PTH) receptors in osteoclasts was also detected. The results revealed that the mRNA and protein expression levels of V-ATPase a3-subunit and d2-subunit increased in a dose-dependent manner, paralleling the level of bPTH present. In addition, an increase in the concentration of bPTH was accompanied by the increased resorption capability of osteoclasts, whereas bone resorption was inhibited in the presence of bafilomycin A1. In addition, we confirmed the existence of parathyroid hormone 1 receptor (PTH1R) in osteoclasts using three different methods (RT-qPCR, western blot analysis and immunofluorescence staining). We found that bPTH enhanced the bone resorption capability of osteoclasts by modulating the expression of V-ATPase subunits, intracellular acidification and V-ATPase activity. Thus, we propose that PTH has a direct effect on osteoblasts and osteoclasts, and that this effect is mediated through PTH1R, thus contributing to bone remodeling.

Letter to the Editor: Possible Decrease in GIP Levels May Explain Increased Plasma Dipeptidyl Peptidase-4 Activity-Associated Osteoporosis

We read with great interest the Early Release article in the Journal of Clinical Endocrinology & Metabolism (August 2015) with the title of “Plasma DPP4 activities are associated with osteoporosis in postmenopausal women with normal glucose tolerance” by Zheng et al (1). The authors prepared an outstanding study in the field of bone metabolism on the topic of dipeptidyl peptidase-4 (DPP4) activities and osteoporosis. However, the article raises some important points about this effect. The authors mentioned that increased DPP4 activities are associated with osteoporosis, regardless of decrease in the active fasting glucagon-like peptide-1 level. Gastric inhibitory polypeptide (GIP) is released from the K cells of the upper intestine, and secreted GIP is rapidly inactivated by DPP-4 (2). Bollag et al (3) reported that GIP receptors are present in bone and osteoblast-like cells, and collagen type 1 expression and alkaline phosphatase activity in osteoblastic-like cells are increased by GIP. Intermittent injection of GIP significantly increased bone mineral density in ovariectomized rats (4). Zhong et al (5) demonstrated the presence of GIP receptors in osteoclasts, in which GIP seems to suppress bone resorption and the resorptive activity of mature osteoclasts. Using GIP receptor knockout mice, Xie et al (6) demonstrated a reduction in bone formation, deteriorated bone microstructure, and biomechanical properties and an increase in bone resorption with a low bone mass. On the other hand, GIP-overexpressing transgenic mice caused elevated bone density (7). We would like to emphasize that increased DPP4 activity-induced GIP reduction may have a role in increased DPP4-associated osteoporosis. In this study, the authors did not report any information about the GIP levels. We think that itwouldbemore reasonable if theauthors add thedataof theabove-mentioned suggestions,whichwould strengthen their study. Finally, we would like to congratulate Zheng et al (1) on their outstanding study. The authors chose not to comment on this letter.

The Urocortin System: A Novel Bone Regulatory Triad, Working In Opposition to RANKL/RANK/OPG

Bone is in a continual state of flux, with old bone being continually replaced by new. This bone turnover, or remodelling, needs to be highly regulated to prevent disorders associated with aberrant bone mass. This process is controlled principally by two cell types, osteoclasts and osteoblasts, which are responsible for bone resorption and deposition respectively. A crucial, well established regulatory mechanism involved in the control of these cells is the RANKL/RANK/ OPG pathway. With this, an osteoblast derived ligand, RANKL, binds to an osteoclastic receptor RANK, producing increased osteoclastogenesis and resorption. In contrast the Ucn system has only recently been found in bone cells, where an osteoblast and osteoclast derived ligand Ucn1, binds to an osteoclast derived receptor CRF-R2β, resulting in inhibition of osteoclastogenesis and resorption. Both systems possess a representative osteoblast derived binding factor with the potential to terminate the ligand signal. In this review we will briefly describe the discovery of the two systems and then go on to compare and contrast the respective components of these two bone regulatory mechanisms. We will review the pathways employed to produce their bone metabolising effects, and finally, we will speculate upon new areas of research which could be exploited to alleviate conditions associated with abnormal bone mass.

An insulin-sensitizing thiazolidinedione, which minimally activates PPARγ, does not cause bone loss.

Rosiglitazone is an insulin-sensitizing thiazolidinedione (TZD) that activates the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ). Although rosiglitazone effectively treats type II diabetes mellitus (T2DM), it carries substantial complications, including increased fracture risk. This predisposition to fracture is consistent with the fact that PPARγ preferentially promotes formation of adipocytes at the cost of osteoblasts. Rosiglitazone-activated PPARγ, however, also stimulates osteoclast formation. A new TZD analog with low affinity for binding and activation of PPARγ but whose insulin-sensitizing properties mirror those of rosiglitazone has been recently developed. Because of its therapeutic implications, we investigated the effects of this new TZD analog (MSDC-0602) on skeletal homeostasis, in vitro and in vivo. Confirming it activates the nuclear receptor in osteoclasts, rosiglitazone enhances expression of the PPARγ target gene, CD36. MSDC-0602, in contrast, minimally activates PPARγ and does not alter CD36 expression in the bone-resorptive cells. Consistent with this finding, rosiglitazone increases receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation and number, whereas MSDC-0602 fails to do so. To determine if this new TZD analog is bone sparing, in vivo, we fed adult male C57BL/6 mice MSDC-0602 or rosiglitazone. Six months of a rosiglitazone diet results in a 35% decrease in bone mass with increased number of osteoclasts, whereas that of MSDC-0602-fed mice is indistinguishable from control. Thus, PPARγ sparing eliminates the skeletal side effects of TZDs while maintaining their insulin-sensitizing properties.

Androgens and estrogens in skeletal sexual dimorphism

Bone is an endocrine tissue expressing androgen and estrogen receptors as well as steroid metabolizing enzymes. The bioactivity of circulating sex steroids is modulated by sex hormone-binding globulin and local conversion in bone tissue, for example, from testosterone (T) to estradiol (E2) by aromatase, or to dihydrotestosterone by 5α-reductase enzymes. Our understanding of the structural basis for gender differences in bone strength has advanced considerably over recent years due to increasing use of (high resolution) peripheral computed tomography. These microarchitectural insights form the basis to understand sex steroid influences on male peak bone mass and turnover in cortical vs trabecular bone. Recent studies using Cre/LoxP technology have further refined our mechanistic insights from global knockout mice into the direct contributions of sex steroids and their respective nuclear receptors in osteoblasts, osteoclasts, osteocytes, and other cells to male osteoporosis. At the same time, these studies have reinforced the notion that androgen and estrogen deficiency have both direct and pleiotropic effects via interaction with, for example, insulin-like growth factor 1, inflammation, oxidative stress, central nervous system control of bone metabolism, adaptation to mechanical loading, etc., This review will summarize recent advances on these issues in the field of sex steroid actions in male bone homeostasis.