Novel insights into the coupling of osteoclasts and resorption to bone formation.

Bone remodeling consists of resorption by osteoclasts (OCs) and formation by osteoblasts (OBs). Precise coordination of these activities is required for the resorbed bone to be replaced with an equal amount of new bone in order to maintain skeletal mass throughout the lifespan. This coordination of remodeling processes is referred to as the "coupling" of resorption to bone formation. In this review, we discuss the essential role for OCs in coupling resorption to bone formation, mechanisms for this coupling, and how coupling becomes less efficient or disrupted in conditions of bone loss. Lastly, we provide perspectives on targeting coupling to treat human bone disease.

Efferocytosis and Bone Dynamics.

PURPOSE OF REVIEW: This review summarizes the recently published scientific evidence regarding the role of efferocytosis in bone dynamics and skeletal health. RECENT FINDINGS: Several types of efferocytes have been identified within the skeleton, with macrophages being the most extensively studied. Efferocytosis is not merely a 'clean-up' process vital for maintaining skeletal homeostasis; it also plays a crucial role in promoting resolution pathways and orchestrating bone dynamics, such as osteoblast-osteoclast coupling during bone remodeling. Impaired efferocytosis has been associated with aging-related bone loss and various skeletal pathologies, including osteoporosis, osteoarthritis, rheumatoid arthritis, and metastatic bone diseases. Accordingly, emerging evidence suggests that targeting efferocytic mechanisms has the potential to alleviate these conditions. While efferocytosis remains underexplored in the skeleton, recent discoveries have shed light on its pivotal role in bone dynamics, with important implications for skeletal health and pathology. However, there are several knowledge gaps and persisting technical limitations that must be addressed to fully unveil the contributions of efferocytosis in bone.

Functional Heterogeneity Within Osteoclast Populations-a Critical Review of Four Key Publications that May Change the Paradigm of Osteoclasts.

PURPOSE OF REVIEW: In this review, we critically evaluate the literature for osteoclast heterogeneity, including heterogeneity in osteoclast behavior, which has hitherto been unstudied and has only recently come to attention. We give a critical review centered on four recent high-impact papers on this topic and aim to shed light on the elusive biology of osteoclasts and focus on the variant features of osteoclasts that diverge from the classical viewpoint. RECENT FINDINGS: Osteoclasts originate from the myeloid lineage and are best known for their unique ability to resorb bone. For decades, osteoclasts have been defined simply as multinucleated cells positive for tartrate-resistant acid phosphatase activity and quantified relative to the bone perimeter or surface in histomorphometric analyses. However, several recent, high-profile studies have demonstrated the existence of heterogeneous osteoclast populations, with variable origins and functions depending on the microenvironment. This includes long-term persisting osteoclasts, inflammatory osteoclasts, recycling osteoclasts (osteomorphs), and bone resorption modes. Most of these findings have been revealed through murine studies and have helped identify new targets for human studies. These studies have also uncovered distinct sets of behavioral patterns in heterogeneous osteoclast cultures. The underlying osteoclast heterogeneity likely drives differences in bone remodeling, altering patient risk for osteoporosis and fracture. Thus, identifying the underlying key features of osteoclast heterogeneity may help in better targeting bone diseases.

Developments in sclerostin biology: regulation of gene expression, mechanisms of action, and physiological functions.

The SOST gene, which encodes the protein sclerostin, was identified through genetic linkage analysis of sclerosteosis and van Buchem's disease patients. Sclerostin is a secreted glycoprotein that binds to the low-density lipoprotein receptor-related proteins 4, 5, and 6 to inhibit Wnt signaling. Since the initial discovery of sclerostin, much understanding has been gained into the role of this protein in the regulation of skeletal biology. In this article, we discuss the latest findings in the regulation of SOST expression, sclerostin mechanisms of action, and the potential utility of targeting sclerostin in conditions of low bone mass.

Macrophages and Bone Remodeling.

Bone remodeling in the adult skeleton facilitates the removal and replacement of damaged and old bone to maintain bone quality. Tight coordination of bone resorption and bone formation during remodeling crucially maintains skeletal mass. Increasing evidence suggests that many cell types beyond osteoclasts and osteoblasts support bone remodeling, including macrophages and other myeloid lineage cells. Herein, we discuss the origin and functions for macrophages in the bone microenvironment, tissue resident macrophages, osteomacs, as well as newly identified osteomorphs that result from osteoclast fission. We also touch on the role of macrophages during inflammatory bone resorption. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Modest Effects of Osteoclast-Specific ERα Deletion after Skeletal Maturity.

Estrogen regulates bone mass in women and men, but the underlying cellular mechanisms of estrogen action on bone remain unclear. Although both estrogen receptor (ER)α and ERß are expressed in bone cells, ERα is the dominant receptor for skeletal estrogen action. Previous studies using either global or cell-specific ERα deletion provided important insights, but each of these approaches had limitations. Specifically, either high circulating sex steroid levels in global ERα knockout mice or the effects of deletion of ERα during growth and development in constitutive cell-specific knockout mice have made it difficult to clearly define the role of ERα in specific cell types in the adult skeleton. We recently generated and characterized mice with tamoxifen-inducible ERα deletion in osteocytes driven by the 8-kb Dmp1 promoter (ERαΔOcy mice), revealing detrimental effects of osteocyte-specific ERα deletion on trabecular bone volume (-20.1%) and bone formation rate (-18.9%) in female, but not male, mice. Here, we developed and characterized analogous mice with inducible ERα deletion in osteoclasts using the Cathepsin K promoter (ERαΔOcl mice). In a study design identical to that with the previously described ERαΔOcy mice, adult female, but not male, ERαΔOcl mice showed a borderline (-10.2%, p = 0.084) reduction in trabecular bone volume, no change in osteoclast numbers, but a significant increase in serum CTx levels, consistent with increased osteoclast activity. These findings in ERαΔOcl mice differ from previous studies of constitutive osteoclast-specific ERα deletion, which led to clear deficits in trabecular bone and increased osteoclast numbers. Collectively, these data indicate that in adult mice, estrogen action in the osteocyte is likely more important than via the osteoclast and that ERα deletion in osteoclasts from conception onward has more dramatic skeletal effects than inducible osteoclastic ERα deletion in adult mice. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Estrogen-regulated miRs in bone enhance osteoblast differentiation and matrix mineralization.

Estrogen signaling is critical for the development and maintenance of healthy bone, and age-related decline in estrogen levels contributes to the development of post-menopausal osteoporosis. Most bones consist of a dense cortical shell and an internal mesh-like network of trabecular bone that respond differently to internal and external cues such as hormonal signaling. To date, no study has assessed the transcriptomic differences that occur specifically in cortical and trabecular bone compartments in response to hormonal changes. To investigate this, we employed a mouse model of post-menopausal osteoporosis (ovariectomy, OVX) and estrogen replacement therapy (ERT). mRNA and miR sequencing revealed distinct transcriptomic profiles between cortical and trabecular bone in the setting of OVX and ERT. Seven miRs were identified as likely contributors to the observed estrogen-mediated mRNA expression changes. Of these, four miRs were prioritized for further study and decreased predicted target gene expression in bone cells, enhanced the expression of osteoblast differentiation markers, and altered the mineralization capacity of primary osteoblasts. As such, candidate miRs and miR mimics may have therapeutic relevance for bone loss resulting from estrogen depletion without the unwanted side effects of hormone replacement therapy and therefore represent novel therapeutic approaches to combat diseases of bone loss.

The effects of direct inhibition of geranylgeranyl pyrophosphate synthase on osteoblast differentiation.

Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. These effects have been attributed to the depletion of geranylgeranyl pyrophosphate (GGPP). In this study, we tested whether specific inhibition of GGPP synthase (GGPPS) with digeranyl bisphosphonate (DGBP) would similarly lead to increased osteoblast differentiation. DGBP concentration dependently decreased intracellular GGPP levels in MC3T3-E1 pre-osteoblasts and primary rat calvarial osteoblasts, leading to impaired Rap1a geranylgeranylation. In contrast to our hypothesis, 1 µM DGBP inhibited matrix mineralization in the MC3T3-E1 pre-osteoblasts. Consistent with this, DGBP inhibited the expression of alkaline phosphatase and osteocalcin in primary osteoblasts. By inhibiting GGPPS, DGBP caused an accumulation of the GGPPS substrate farnesyl pyrophosphate (FPP). This effect was observed throughout the time course of MC3T3-E1 pre-osteoblast differentiation. Interestingly, DGBP treatment led to activation of the glucocorticoid receptor in MC3T3-E1 pre-osteoblast cells, consistent with recent findings that FPP activates nuclear hormone receptors. These findings demonstrate that direct inhibition of GGPPS, and the resulting specific depletion of GGPP, does not stimulate osteoblast differentiation. This suggests that in addition to depletion of GGPP, statin-stimulated osteoblast differentiation may depend on the depletion of upstream isoprenoids, including FPP.

miRNAs in osteoclast biology.

MicroRNAs (miRNAs) are a class of short RNA molecules that mediate the regulation of gene activity through interactions with target mRNAs and subsequent silencing of gene expression. It has become increasingly clear the miRNAs regulate many diverse aspects of bone biology, including bone formation and bone resorption processes. The role of miRNAs specifically in osteoclasts has been of recent investigation, due to clinical interest in discovering new paradigms to control excessive bone resorption, as is observed in multiple conditions including aging, estrogen deprivation, cancer metastases or glucocorticoid use. Therefore understanding the role that miRNAs play during osteoclastic differentiation is of critical importance. In this review, we highlight and discuss general aspects of miRNA function in osteoclasts, including exciting data demonstrating that miRNAs encapsulated in extracellular vesicles (EVs) either originating from osteoclasts, or signaling to osteoclast from divergent sites, have important roles in bone homeostasis.

Identification of osteoclast-osteoblast coupling factors in humans reveals links between bone and energy metabolism.

Bone remodeling consists of resorption by osteoclasts followed by formation by osteoblasts, and osteoclasts are a source of bone formation-stimulating factors. Here we utilize osteoclast ablation by denosumab (DMAb) and RNA-sequencing of bone biopsies from postmenopausal women to identify osteoclast-secreted factors suppressed by DMAb. Based on these analyses, LIF, CREG2, CST3, CCBE1, and DPP4 are likely osteoclast-derived coupling factors in humans. Given the role of Dipeptidyl Peptidase-4 (DPP4) in glucose homeostasis, we further demonstrate that DMAb-treated participants have a significant reduction in circulating DPP4 and increase in Glucagon-like peptide (GLP)-1 levels as compared to the placebo-treated group, and also that type 2 diabetic patients treated with DMAb show significant reductions in HbA1c as compared to patients treated either with bisphosphonates or calcium and vitamin D. Thus, our results identify several coupling factors in humans and uncover osteoclast-derived DPP4 as a potential link between bone remodeling and energy metabolism.

Multiple myeloma increases nerve growth factor and other pain-related markers through interactions with the bone microenvironment.

Interactions between multiple myeloma (MM) and bone marrow (BM) are well documented to support tumour growth, yet the cellular mechanisms underlying pain in MM are poorly understood. We have used in vivo murine models of MM to show significant induction of nerve growth factor (NGF) by the tumour-bearing bone microenvironment, alongside other known pain-related characteristics such as spinal glial cell activation and reduced locomotion. NGF was not expressed by MM cells, yet bone stromal cells such as osteoblasts expressed and upregulated NGF when cultured with MM cells, or MM-related factors such as TNF-α. Adiponectin is a known MM-suppressive BM-derived factor, and we show that TNF-α-mediated NGF induction is suppressed by adiponectin-directed therapeutics such as AdipoRON and L-4F, as well as NF-κB signalling inhibitor BMS-345541. Our study reveals a further mechanism by which cellular interactions within the tumour-bone microenvironment contribute to disease, by promoting pain-related properties, and suggests a novel direction for analgesic development.

miR-219a-5p Regulates Rorß During Osteoblast Differentiation and in Age-related Bone Loss.

Developing novel approaches to treat skeletal disorders requires an understanding of how critical molecular factors regulate osteoblast differentiation and bone remodeling. We have reported that (1) retinoic acid receptor-related orphan receptor beta (Rorß) is upregulated in bone samples isolated from aged mice and humans in vivo; (2) Rorß expression is inhibited during osteoblastic differentiation in vitro; and (3) genetic deletion of Rorß in mice results in preservation of bone mass during aging. These data establish that Rorß inhibits osteogenesis and that strict control of Rorß expression is essential for bone homeostasis. Because microRNAs (miRNAs) are known to play important roles in the regulation of gene expression in bone, we explored whether a predicted subset of nine miRNAs regulates Rorß expression during both osteoblast differentiation and aging. Mouse osteoblastic cells were differentiated in vitro and assayed for Rorß and miRNA expression. As Rorß levels declined with differentiation, the expression of many of these miRNAs, including miR-219a-5p, was increased. We further demonstrated that miR-219a-5p was decreased in bone samples from old (24-month) mice, as compared with young (6-month) mice, concomitant with increased Rorß expression. Importantly, we also found that miR-219a-5p expression was decreased in aged human bone biopsies compared with young controls, demonstrating that this phenomenon also occurs in aging bone in humans. Inhibition of miR-219a-5p in mouse calvarial osteoblasts led to increased Rorß expression and decreased alkaline phosphatase expression and activity, whereas a miR-219a-5p mimic decreased Rorß expression and increased osteogenic activity. Finally, we demonstrated that miR-219a-5p physically interacts with Rorß mRNA in osteoblasts, defining Rorß as a true molecular target of miR-219a-5p. Overall, our findings demonstrate that miR-219a-5p is involved in the regulation of Rorß in both mouse and human bone. © 2018 American Society for Bone and Mineral Research.

Targeting senescent cells alleviates obesity-induced metabolic dysfunction.

Adipose tissue inflammation and dysfunction are associated with obesity-related insulin resistance and diabetes, but mechanisms underlying this relationship are unclear. Although senescent cells accumulate in adipose tissue of obese humans and rodents, a direct pathogenic role for these cells in the development of diabetes remains to be demonstrated. Here, we show that reducing senescent cell burden in obese mice, either by activating drug-inducible "suicide" genes driven by the p16Ink4a promoter or by treatment with senolytic agents, alleviates metabolic and adipose tissue dysfunction. These senolytic interventions improved glucose tolerance, enhanced insulin sensitivity, lowered circulating inflammatory mediators, and promoted adipogenesis in obese mice. Elimination of senescent cells also prevented the migration of transplanted monocytes into intra-abdominal adipose tissue and reduced the number of macrophages in this tissue. In addition, microalbuminuria, renal podocyte function, and cardiac diastolic function improved with senolytic therapy. Our results implicate cellular senescence as a causal factor in obesity-related inflammation and metabolic derangements and show that emerging senolytic agents hold promise for treating obesity-related metabolic dysfunction and its complications.

Osteoprotection Through the Deletion of the Transcription Factor Rorß in Mice.

There is a clinical need to identify new molecular targets for the treatment of osteoporosis, particularly those that simultaneously inhibit bone resorption while stimulating bone formation. We have previously shown in overexpression studies that retinoic acid receptor-related orphan receptor ß (Rorß) suppresses in vitro osteoblast differentiation. In addition, the expression of Rorß is markedly increased in bone marrow-derived mesenchymal stromal cells with aging in both mice and humans. Here we establish a critical role for Rorß in regulating bone metabolism using a combination of in vitro and in vivo studies. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing to demonstrate that loss of Rorß in osteoblasts enhances Wnt signaling, specifically through increased recruitment of ß-catenin to T-cell factor/lymphoid enhancer factor (Tcf/Lef) DNA binding sites in the promoters of the Wnt target genes Tcf7 and Opg. This resulted in increased osteogenic gene expression and suppressed osteoclast formation through increased osteoprotegerin (OPG) secretion in Rorß-deficient cells. Consistent with our in vitro data, genetic deletion of Rorß in both female and male mice resulted in preserved bone mass and microarchitecture with advancing age due to increased bone formation with a concomitant decrease in resorption. The improved skeletal phenotype in the Rorß-/- mice was also associated with increased bone protein levels of TCF7 and OPG. These data demonstrate that loss of Rorß has beneficial skeletal effects by increasing bone formation and decreasing bone resorption, at least in part through ß-catenin-dependent activation of the Wnt pathway. Thus, inhibition of Rorß represents a novel approach to potentially prevent or reverse osteoporosis. © 2017 American Society for Bone and Mineral Research.

Senolytics improve physical function and increase lifespan in old age.

Physical function declines in old age, portending disability, increased health expenditures, and mortality. Cellular senescence, leading to tissue dysfunction, may contribute to these consequences of aging, but whether senescence can directly drive age-related pathology and be therapeutically targeted is still unclear. Here we demonstrate that transplanting relatively small numbers of senescent cells into young mice is sufficient to cause persistent physical dysfunction, as well as to spread cellular senescence to host tissues. Transplanting even fewer senescent cells had the same effect in older recipients and was accompanied by reduced survival, indicating the potency of senescent cells in shortening health- and lifespan. The senolytic cocktail, dasatinib plus quercetin, which causes selective elimination of senescent cells, decreased the number of naturally occurring senescent cells and their secretion of frailty-related proinflammatory cytokines in explants of human adipose tissue. Moreover, intermittent oral administration of senolytics to both senescent cell-transplanted young mice and naturally aged mice alleviated physical dysfunction and increased post-treatment survival by 36% while reducing mortality hazard to 65%. Our study provides proof-of-concept evidence that senescent cells can cause physical dysfunction and decreased survival even in young mice, while senolytics can enhance remaining health- and lifespan in old mice.

Sclerostin expression and functions beyond the osteocyte.

Sclerostin, the product of the SOST gene, is a secreted inhibitor of Wnt signaling that is produced by osteocytes to regulate bone formation. While it is often considered an osteocyte-specific protein, SOST expression has been reported in numerous other cell types, including hypertrophic chondrocytes and cementocytes. Of interest, SOST/sclerostin expression is altered in certain pathogenic conditions, including osteoarthritis and rheumatic joint disease, and it is unclear whether sclerostin plays a protective role or whether sclerostin may mediate disease pathogenesis. Therefore, as anti-sclerostin antibodies are being developed for the treatment of osteoporosis, it is important to understand the functions of sclerostin beyond the regulation of bone formation.

Transplanted Senescent Cells Induce an Osteoarthritis-Like Condition in Mice.

Osteoarthritis (OA) is the leading form of arthritis in the elderly, causing pain, disability, and immobility. OA has been associated with accumulation of senescent cells in or near joints. However, evidence for a causal link between OA and cellular senescence is lacking. Here, we present a novel senescent cell transplantation model involving injection of small numbers of senescent or nonsenescent cells from the ear cartilage of luciferase-expressing mice into the knee joint area of wild-type mice. By using bioluminescence and 18FDG PET imaging, we could track the injected cells in vivo for more than 10 days. Transplanting senescent cells into the knee region caused leg pain, impaired mobility, and radiographic and histological changes suggestive of OA. Transplanting nonsenescent cells had less of these effects. Thus, senescent cells can induce an OA-like state and targeting senescent cells could be a promising strategy for treating OA.

Targeting cellular senescence prevents age-related bone loss in mice.

Aging is associated with increased cellular senescence, which is hypothesized to drive the eventual development of multiple comorbidities. Here we investigate a role for senescent cells in age-related bone loss through multiple approaches. In particular, we used either genetic (i.e., the INK-ATTAC 'suicide' transgene encoding an inducible caspase 8 expressed specifically in senescent cells) or pharmacological (i.e., 'senolytic' compounds) means to eliminate senescent cells. We also inhibited the production of the proinflammatory secretome of senescent cells using a JAK inhibitor (JAKi). In aged (20- to 22-month-old) mice with established bone loss, activation of the INK-ATTAC caspase 8 in senescent cells or treatment with senolytics or the JAKi for 2-4 months resulted in higher bone mass and strength and better bone microarchitecture than in vehicle-treated mice. The beneficial effects of targeting senescent cells were due to lower bone resorption with either maintained (trabecular) or higher (cortical) bone formation as compared to vehicle-treated mice. In vitro studies demonstrated that senescent-cell conditioned medium impaired osteoblast mineralization and enhanced osteoclast-progenitor survival, leading to increased osteoclastogenesis. Collectively, these data establish a causal role for senescent cells in bone loss with aging, and demonstrate that targeting these cells has both anti-resorptive and anabolic effects on bone. Given that eliminating senescent cells and/or inhibiting their proinflammatory secretome also improves cardiovascular function, enhances insulin sensitivity, and reduces frailty, targeting this fundamental mechanism to prevent age-related bone loss suggests a novel treatment strategy not only for osteoporosis, but also for multiple age-related comorbidities.

Corrigendum: Targeting cellular senescence prevents age-related bone loss in mice.

This corrects the article DOI: 10.1038/nm.4385.