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.

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.

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).

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.

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.

Histone deacetylase 3 supports endochondral bone formation by controlling cytokine signaling and matrix remodeling.

Histone deacetylase (HDAC) inhibitors are efficacious epigenetic-based therapies for some cancers and neurological disorders; however, each of these drugs inhibits multiple HDACs and has detrimental effects on the skeleton. To better understand how HDAC inhibitors affect endochondral bone formation, we conditionally deleted one of their targets, Hdac3, pre- and postnatally in type II collagen α1 (Col2α1)-expressing chondrocytes. Embryonic deletion was lethal, but postnatal deletion of Hdac3 delayed secondary ossification center formation, altered maturation of growth plate chondrocytes, and increased osteoclast activity in the primary spongiosa. HDAC3-deficient chondrocytes exhibited increased expression of cytokine and matrix-degrading genes (Il-6, Mmp3, Mmp13, and Saa3) and a reduced abundance of genes related to extracellular matrix production, bone development, and ossification (Acan, Col2a1, Ihh, and Col10a1). Histone acetylation increased at and near genes that had increased expression. The acetylation and activation of nuclear factor κB (NF-κB) were also increased in HDAC3-deficient chondrocytes. Increased cytokine signaling promoted autocrine activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) and NF-κB pathways to suppress chondrocyte maturation, as well as paracrine activation of osteoclasts and bone resorption. Blockade of interleukin-6 (IL-6)-JAK-STAT signaling, NF-κB signaling, and bromodomain extraterminal proteins, which recognize acetylated lysines and promote transcriptional elongation, significantly reduced Il-6 and Mmp13 expression in HDAC3-deficient chondrocytes and secondary activation in osteoclasts. The JAK inhibitor ruxolitinib also reduced osteoclast activity in Hdac3 conditional knockout mice. Thus, HDAC3 controls the temporal and spatial expression of tissue-remodeling genes and inflammatory responses in chondrocytes to ensure proper endochondral ossification during development.

Identification of Senescent Cells in the Bone Microenvironment.

Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16Ink4a , profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence-associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age-related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16Ink4a expression by real-time quantitative polymerase chain reaction (rt-qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence-associated distension of satellites (SADS), ie, large-scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction-induced foci (TIFs) than osteocytes from young mice. Corresponding with the age-associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age-associated upregulation in myeloid cells. These data show that with aging, a subset of cells of various lineages within the bone microenvironment become senescent, although senescent myeloid cells and senescent osteocytes predominantly develop the SASP. Given the critical roles of osteocytes in orchestrating bone remodeling, our findings suggest that senescent osteocytes and their SASP may contribute to age-related bone loss. © 2016 American Society for Bone and Mineral Research.

Wnt Signaling Inhibits Osteoclast Differentiation by Activating Canonical and Noncanonical cAMP/PKA Pathways.

Although there has been extensive characterization of the Wnt signaling pathway in the osteoblast lineage, the effects of Wnt proteins on the osteoclast lineage are less well studied. We found that osteoclast lineage cells express canonical Wnt receptors. Wnt3a reduced osteoclast formation when applied to early bone-marrow macrophage (BMM) osteoclast differentiation cultures, whereas late addition did not suppress osteoclast formation. Early Wnt3a treatment inactivated the crucial transcription factor NFATc1 in osteoclast progenitors. Wnt3a led to the accumulation of nuclear ß-catenin, confirming activation of canonical Wnt signaling. Reducing low-density lipoprotein receptor-related proteins (Lrp) 5 and Lrp6 protein expression prevented Wnt3a-induced inactivation of NFATc1; however, deletion of ß-catenin did not block Wnt3a inactivation of NFATc1, suggesting that this effect was mediated by a noncanonical pathway. Wnt3a rapidly activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and pharmacological stimulation of cAMP/PKA signaling suppressed osteoclast differentiation; Wnt3a-induced NFATc1 phosphorylation was blocked by inhibiting interactions between PKA and A-kinase anchoring proteins (AKAPs). These data indicate that Wnt3a directly suppresses osteoclast differentiation through both canonical (ß-catenin) and noncanonical (cAMP/PKA) pathways in osteoclast precursors. In vivo reduction of Lrp5 and Lrp6 expressions in the early osteoclast lineage via Rank promoter Cre recombination reduced trabecular bone mass, whereas disruption of Lrp5/6 expression in late osteoclast precursors via cathepsin K (Ctsk) promoter Cre recombination did not alter the skeletal phenotype. Surprisingly, reduction of Lrp5/6 in the early osteoclast lineage decreased osteoclast numbers, as well as osteoblast numbers. Published studies have previously noted that ß-catenin signaling is required for osteoclast progenitor proliferation. Our in vivo data suggest that Rank promoter Cre-mediated deletion of Lrp5/6 may similarly impair osteoclast progenitor proliferation.

Osteoclast TGF-ß Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation.

Osteoblast-mediated bone formation is coupled to osteoclast-mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age-related bone loss. Osteoclasts release and activate TGF-ß from the bone matrix. Here we show that osteoclast-specific inhibition of TGF-ß receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF-ß induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF-ß receptor signaling. Osteoclasts in aged murine bones had lower TGF-ß signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF-ß-induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF-ß availability with age. Therefore, osteoclast responses to TGF-ß are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age-related bone loss.

3-Deoxyschweinfurthin B Lowers Cholesterol Levels by Decreasing Synthesis and Increasing Export in Cultured Cancer Cell Lines.

The schweinfurthins have potent antiproliferative activity in multiple glioblastoma multiforme (GBM) cell lines; however, the mechanism by which growth is impeded is not fully understood. Previously, we demonstrated that the schweinfurthins reduce the level of key isoprenoid intermediates in the cholesterol biosynthetic pathway. Herein, we describe the effects of the schweinfurthins on cholesterol homeostasis. Intracellular cholesterol levels are greatly reduced in cells incubated with 3-deoxyschweinfurthin B (3dSB), an analog of the natural product schweinfurthin B. Decreased cholesterol levels are due to decreased cholesterol synthesis and increased cholesterol efflux; both of these cellular actions can be influenced by liver X-receptor (LXR) activation. The effects of 3dSB on ATP-binding cassette transporter 1 levels and other LXR targets are similar to that of 25-hydroxycholesterol, an LXR agonist. Unlike 25-hydroxycholesterol, 3dSB does not act as a direct agonist for LXR α or ß. These data suggest that cholesterol homeostasis plays a significant role in the growth inhibitory activity of the schweinfurthins and may elucidate a mechanism that can be targeted in human cancers such as GBM.

Transforming growth factor beta 1 induces CXCL16 and leukemia inhibitory factor expression in osteoclasts to modulate migration of osteoblast progenitors.

The processes of bone resorption and bone formation are tightly coupled in young adults, which is crucial to maintenance of bone integrity. We have documented that osteoclasts secrete chemotactic agents to recruit osteoblast lineage cells, contributing to coupling. Bone formation subsequent to bone resorption becomes uncoupled with aging, resulting in significant bone loss. During bone resorption, osteoclasts release and activate transforming growth factor beta 1 (TGF-ß1) from the bone matrix; thus, elevated bone resorption increases the level of active TGF-ß in the local environment during aging. In this study, we examined the influences of TGF-ß1 on the ability of osteoclasts to recruit osteoblasts. TGF-ß1 increased osteoclast expression of the chemokine CXCL16 to promote osteoblast migration. TGF-ß1 also directly stimulated osteoblast migration; however, this direct response was blocked by conditioned medium from TGF-ß1-treated osteoclasts due to the presence of leukemia inhibitory factor (LIF) in the medium. CXCL16 and LIF expression was dependent on TGF-ß1 activation of Smad2 and Smad3. These results establish that TGF-ß1 induces CXCL16 and LIF production in osteoclasts, which modulate recruitment of osteoblasts to restore the bone lost during the resorptive phase of bone turnover.