Hypoxia-Sensitive COMMD1 Integrates Signaling and Cellular Metabolism in Human Macrophages and Suppresses Osteoclastogenesis.

Hypoxia augments inflammatory responses and osteoclastogenesis by incompletely understood mechanisms. We identified COMMD1 as a cell-intrinsic negative regulator of osteoclastogenesis that is suppressed by hypoxia. In human macrophages, COMMD1 restrained induction of NF-κB signaling and a transcription factor E2F1-dependent metabolic pathway by the cytokine RANKL. Downregulation of COMMD1 protein expression by hypoxia augmented RANKL-induced expression of inflammatory and E2F1 target genes and downstream osteoclastogenesis. E2F1 targets included glycolysis and metabolic genes including CKB that enabled cells to meet metabolic demands in challenging environments, as well as inflammatory cytokine-driven target genes. Expression quantitative trait locus analysis linked increased COMMD1 expression with decreased bone erosion in rheumatoid arthritis. Myeloid deletion of Commd1 resulted in increased osteoclastogenesis in arthritis and inflammatory osteolysis models. These results identify COMMD1 and an E2F-metabolic pathway as key regulators of osteoclastogenic responses under pathological inflammatory conditions and provide a mechanism by which hypoxia augments inflammation and bone destruction.

Adiponectin affects the migration ability of bone marrow-derived mesenchymal stem cells via the regulation of hypoxia inducible factor 1α.

BACKGROUND: Bone marrow (BM) is progressively filled with adipocytes during aging process. Thus, BM adipocytes-derived adiponectin (APN) affects the function of bone marrow-derived mesenchymal stem cells (BMSCs). However, little is known about the effect of APN on migration ability of BMSCs cultured under hypoxic conditions, which is similar to the BM microenvironment. RESULTS: We found that the population and migration ability of BMSCs from APN KO mice was higher than that of WT mice due to increased stability of hypoxia inducible factor 1α (HIF1α). Stem cell factor (SCF)-activated STAT3 stimulated the induction of HIF1α which further stimulated SCF production, indicating that the SCF/STAT3/HIF1α positive loop was highly activated in the absence of APN. It implies that APN negatively regulated this positive loop by stimulating HIF1α degradation via the inactivation of GSK3ß. Furthermore, APN KO BMSCs were highly migratory toward EL-4 lymphoma, and the interaction between CD44 in BMSCs and hyaluronic acid (HA) from EL-4 enhanced the migration of BMSCs. On the other hand, the migrated BMSCs recruited CD8+ T cells into the EL-4 tumor tissue, resulting in the retardation of tumor growth. Additionally, gradually increased APN in BM on the aging process affects migration and related functions of BMSCs, thus aged APN KO mice showed more significant suppression of EL-4 growth than young APN KO mice due to higher migration and recruitment of CD8+ T cells. CONCLUSION: APN deficiency enhances CD44-mediated migration ability of BMSCs in the hypoxic conditions by the SCF/STAT3/HIF1α positive loop and influences the migration ability of BMSCs for a longer time depending on the aging process. Video Abstract.

Cutting Edge: EZH2 Promotes Osteoclastogenesis by Epigenetic Silencing of the Negative Regulator IRF8.

Osteoclasts are resorptive cells that are important for homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role for epigenetic mechanisms in osteoclastogenesis. A recent study showed that epigenetic silencing of the negative regulator of osteoclastogenesis Irf8 by DNA methylation is required for osteoclast differentiation. In this study, we investigated the role of EZH2, which epigenetically silences gene expression by histone methylation, in osteoclastogenesis. Inhibition of EZH2 by the small molecule GSK126, or decreasing its expression using antisense oligonucleotides, impeded osteoclast differentiation. Mechanistically, EZH2 was recruited to the IRF8 promoter after RANKL stimulation to deposit the negative histone mark H3K27me3 and downregulate IRF8 expression. GSK126 attenuated bone loss in the ovariectomy mouse model of postmenopausal osteoporosis. Our findings provide evidence for an additional mechanism of epigenetic IRF8 silencing during osteoclastogenesis that likely works cooperatively with DNA methylation, further emphasizing the importance of IRF8 as a negative regulator of osteoclastogenesis.

New Horizons: Translational Aspects of Osteomorphs.

Osteomorphs are a newly described osteoclast lineage cell in mice, which are suggested to play a significant role in the maintenance of bone resorption. Preclinical investigations revealed that osteomorphs are generated through the fission of multinucleated bone-resorbing osteoclasts and can also re-fuse with existing osteoclasts. Modifications to RANKL signaling have been shown to alter cycles of fission and re-fusion of osteomorphs in mice. These novel findings were also shown to contribute to the rebound phenomenon after cessation of anti-RANKL therapy in mice. Moreover, the absence of osteomorph-specific genes in mice exhibits bone structural and quality phenotypes. Given these insights, it could be speculated that osteomorphs play a significant role in bone homeostasis, bone metabolic diseases, and response to therapeutics. In this review, we discuss these potential translational roles for osteomorphs. Importantly, we highlight the need for future preclinical and clinical studies to verify the presence of osteomorphs in humans and explore further the translational implications of this discovery.

IK is essentially involved in ciliogenesis as an upstream regulator of oral-facial-digital syndrome ciliopathy gene, ofd1.

BACKGROUND: The cilia are microtubule-based organelles that protrude from the cell surface. Abnormalities in cilia result in various ciliopathies, including polycystic kidney disease (PKD), Bardet-Biedl syndrome (BBS), and oral-facial-digital syndrome type I (OFD1), which show genetic defects associated with cilia formation. Although an increasing number of human diseases is attributed to ciliary defects, the functions or regulatory mechanisms of several ciliopathy genes remain unclear. Because multi ciliated cells (MCCs) are especially deep in vivo, studying ciliogenesis is challenging. Here, we demonstrate that ik is essential for ciliogenesis in vivo. RESULTS: In the absence of ik, zebrafish embryos showed various ciliopathy phenotypes, such as body curvature, abnormal otoliths, and cyst formation in the kidney. RNA sequencing analysis revealed that ik positively regulated ofd1 expression required for cilium assembly. In fact, depletion of ik resulted in the downregulation of ofd1 expression with ciliary defects, and these ciliary defects in ik mutants were rescued by restoring ofd1 expression. Interestingly, ik affected ciliogenesis particularly in the proximal tubule but not in the distal tubule in the kidney. CONCLUSIONS: This study demonstrates the role of ik in ciliogenesis in vivo for the first time. Loss of ik in zebrafish embryos displays various ciliopathy phenotypes with abnormal ciliary morphology in ciliary tissues. Our findings on the ik-ofd1 axis provide new insights into the biological function of ik in clinical ciliopathy studies in humans.

THOC5 regulates human osteoclastogenesis.

Osteoclasts are bone resorbing cells that are responsible for physiological and pathological bone resorption. Macrophage colony stimulating factor (M-CSF) binds to the M-CSF receptor (c-FMS) and plays a key role in the differentiation and survival of macrophages and osteoclasts. THOC5, a member of the THO complex, has been shown to regulate hematopoiesis and M-CSF-induced macrophage differentiation. However, the role of THOC5 in osteoclasts remains unclear. Here, our study reveals a new role of THOC5 in osteoclast formation. We found that THOC5 shuttles between nucleus and cytoplasm in an M-CSF signaling dependent manner. THOC5 bound to FICD, a proteolytic cleavage product of c-FMS, and THOC5 facilitates the nuclear translocations of FICD. Decreased expression of THOC5 by siRNA-mediated knock down suppressed osteoclast differentiation, in part, by regulating RANK, a key receptor of osteoclasts. Mechanistically, knock down of THOC5 inhibited the expression of RANKL-induced FOS and NFATc1. Our findings highlight THOC5's function as a positive regulator of osteoclasts.

Interleukin-33 stimulates formation of functional osteoclasts from human CD14(+) monocytes.

Interleukin (IL)-33 is a recently described pro-inflammatory cytokine. Here we demonstrate IL-33 as a regulator of functional osteoclasts (OCs) from human CD14(+) monocytes. IL-33 stimulates formation of tartrate-resistant acid phosphatase (TRAP)(+) multinuclear OCs from monocytes. This action was suppressed by anti-ST2 antibody, suggesting that IL-33 acts through its receptor ST2, but not by the receptor activator of NF-κB ligand (RANKL) decoy, osteoprotegerin, or anti-RANKL antibody. IL-33 stimulated activating phosphorylations of signaling molecules in monocytes that are critical for OC development. These included Syk, phospholipase Cγ2, Gab2, MAP kinases, TAK-1, and NF-κB. IL-33 also enhanced expression of OC differentiation factors including TNF-α receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells cytoplasmic 1, c-Fos, c-Src, cathepsin K, and calcitonin receptor. IL-33 eventually induced bone resorption. This study suggests that the osteoclastogenic property of IL-33 is mediated through TRAF6 as well as the immunoreceptor tyrosine-based activation motif-dependent Syk/PLCγ pathway in human CD14(+) monocytes.

MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS.

Osteoporosis is a metabolic bone disease with dysregulated coupling between bone resorption and bone formation, which results in decreased bone mineral density. The MEF2C locus, which encodes the transcription factor MADS box transcription enhancer factor 2, polypeptide C (MEF2C), is strongly associated with adult osteoporosis and osteoporotic fractures. Although the role of MEF2C in bone and cartilage formation by osteoblasts, osteocytes, and chondrocytes has been studied, the role of MEF2C in osteoclasts, which mediate bone resorption, remains unclear. In this study, we identified MEF2C as a positive regulator of human and mouse osteoclast differentiation. While decreased MEF2C expression resulted in diminished osteoclastogenesis, ectopic expression of MEF2C enhanced osteoclast generation. Using transcriptomic and bioinformatic approaches, we found that MEF2C promotes the RANKL-mediated induction of the transcription factors c-FOS and NFATc1, which play a key role in osteoclastogenesis. Mechanistically, MEF2C binds to FOS regulatory regions to induce c-FOS expression, leading to the activation of NFATC1 and downstream osteoclastogenesis. Inducible deletion of Mef2c in mice resulted in increased bone mass under physiological conditions and protected mice from bone erosion by diminishing osteoclast formation in K/BxN serum induced arthritis, a murine model of inflammatory arthritis. Our findings reveal direct regulation of osteoclasts by MEF2C, thus adding osteoclasts as a cell type in which altered MEF2C expression or function can contribute to pathological bone remodeling.

Augmenting MNK1/2 activation by c-FMS proteolysis promotes osteoclastogenesis and arthritic bone erosion.

Osteoclasts are bone-resorbing cells that play an essential role in homeostatic bone remodeling and pathological bone erosion. Macrophage colony stimulating factor (M-CSF) is abundant in rheumatoid arthritis (RA). However, the role of M-CSF in arthritic bone erosion is not completely understood. Here, we show that M-CSF can promote osteoclastogenesis by triggering the proteolysis of c-FMS, a receptor for M-CSF, leading to the generation of FMS intracellular domain (FICD) fragments. Increased levels of FICD fragments positively regulated osteoclastogenesis but had no effect on inflammatory responses. Moreover, myeloid cell-specific FICD expression in mice resulted in significantly increased osteoclast-mediated bone resorption in an inflammatory arthritis model. The FICD formed a complex with DAP5, and the FICD/DAP5 axis promoted osteoclast differentiation by activating the MNK1/2/EIF4E pathway and enhancing NFATc1 protein expression. Moreover, targeting the MNK1/2 pathway diminished arthritic bone erosion. These results identified a novel role of c-FMS proteolysis in osteoclastogenesis and the pathogenesis of arthritic bone erosion.

MYC-mediated early glycolysis negatively regulates proinflammatory responses by controlling IRF4 in inflammatory macrophages.

MYC activates different metabolic programs in a cell-type- and cell-status-dependent manner. However, the role of MYC in inflammatory macrophages has not yet been determined. Metabolic and molecular analyses reveal that MYC, but not hypoxia inducible factor 1 (HIF1), is involved in enhancing early glycolytic flux during inflammatory macrophage polarization. Ablation of MYC decreases lactate production by regulating lactate dehydrogenase (LDH) activity and causes increased inflammatory cytokines by regulating interferon regulatory factor 4 (IRF4) in response to lipopolysaccharide. Moreover, myeloid-specific deletion of MYC and pharmacological inhibition of the MYC/LDH axis enhance inflammation and the bacterial clearance in vivo. These results elucidate the potential role of the MYC/LDH/IRF4 axis in inflammatory macrophages by connecting early glycolysis with inflammatory responses and suggest that modulating early glycolytic flux mediated by the MYC/LDH axis can be used to open avenues for the therapeutic modulation of macrophage polarization to fight against bacterial infection.

Sexual Dimorphism in Differentiating Osteoclast Precursors Demonstrates Enhanced Inflammatory Pathway Activation in Female Cells.

Sexual dimorphism of the skeleton is well documented. At maturity, the male skeleton is typically larger and has a higher bone density than the female skeleton. However, the underlying mechanisms for these differences are not completely understood. In this study, we examined sexual dimorphism in the formation of osteoclasts between cells from female and male mice. We found that the number of osteoclasts in bones was greater in females. Similarly, in vitro osteoclast differentiation was accelerated in female osteoclast precursor (OCP) cells. To further characterize sex differences between female and male osteoclasts, we performed gene expression profiling of cultured, highly purified, murine bone marrow OCPs that had been treated for 3 days with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). We found that 125 genes were differentially regulated in a sex-dependent manner. In addition to genes that are contained on sex chromosomes, transcriptional sexual dimorphism was found to be mediated by genes involved in innate immune and inflammatory response pathways. Furthermore, the NF-κB-NFATc1 axis was activated earlier in female differentiating OCPs, which partially explains the differences in transcriptomic sexual dimorphism in these cells. Collectively, these findings identify multigenic sex-dependent intrinsic difference in differentiating OCPs, which results from an altered response to osteoclastogenic stimulation. In humans, these differences could contribute to the lower peak bone mass and increased risk of osteoporosis that females demonstrate relative to males. © 2021 American Society for Bone and Mineral Research (ASBMR).

Interleukin-32gamma enhances the production of IL-6 and IL-8 in fibroblast-like synoviocytes via Erk1/2 activation.

INTRODUCTION: In the present study, we examined the effect of the pro-inflammatory cytokine IL-32gamma, the most biologically active isoform, and its related molecules in fibroblast-like synoviocytes (FLS). MATERIALS AND METHODS: FLS were isolated from synovial tissues of rheumatoid arthritis (RA) patients. The secretion and expression of IL-6 and IL-8 were examined by ELISA and real-time PCR, and the activation of signaling molecules was evaluated by Western blot, electrophoretic mobility shift assay (EMSA), real-time PCR, and siRNA transfection. RESULTS: By IL-32gamma stimulation in RA FLS, the expressions of IL-6 and IL-8 were increased significantly, and the phosphorylated Erk1/2 and AP-1 were expressed prominently in Western blot and EMSA. In the Erk1/2 inhibited cells, IL-32gamma stimulation did not increase the mRNA expression of IL-6 and IL-8. CONCLUSION: Our results suggest that IL-32gamma stimulation can induce the production of IL-6 and IL-8 from RA FLS via Erk1/2 activation.

Allergen-specific B cell subset responses in cow's milk allergy of late eczematous reactions in atopic dermatitis.

B cells have regulatory functions in immune responses. Antigen-specific responses of B cell subsets by allergen stimulation ex vivo were examined in milk allergy of late eczematous reactions. Eight milk allergy subjects and 13 milk tolerant subjects were selected by DBPCFC. PBMCs were stimulated by casein ex vivo and stained for B cell subsets using monoclonal antibodies. CD19+ B cells unchanged from 8.7+/-3.8% to 8.0+/-5.1% (p=0.504, n=8) in the milk allergy group and decreased in the milk tolerant group from 8.5+/-3.2% to 5.0+/-1.6% (p=0.001, n=13). The fraction of apoptotic B cells in B cells significantly decreased 4.4+/-3.1% to 1.3+/-0.4% (p=0.027, n=4) in the allergy group and insignificantly increased from 2.8+/-0.6% to 5.4+/-2.6% (p=0.059, n=6) in the milk tolerant group. CD5+ regulatory B1 cell% in B cells decreased in milk allergy subjects from 36.2+/-5.0% to 31.0+/-5.7% (p=0.010) and unchanged in milk tolerant subjects from 41.6+/-10.2% to 43.8+/-10.0% (p=0.413). IL-10 producing CD19+CD5+ regulatory B cell% in CD19+CD5+ regulatory B cells significantly decreased from 24.9+/-6.5% to 13.8+/-5.6% (p=0.002, n=5) by casein stimulation in milk allergy group and unchanged from 44.8+/-11.3% to 43.9+/-10.0% (p=0.297, n=5) in the milk tolerant group. B cell subset responses to IL-4 and IL-5 were also similar in both groups. B cell subset changes seemed to have diagnostic value. Exact immunologic roles of regulatory CD5+ B1 cells need further investigation.

The M-CSF receptor in osteoclasts and beyond.

Colony-stimulating factor 1 receptor (CSF1R, also known as c-FMS) is a receptor tyrosine kinase. Macrophage colony-stimulating factor (M-CSF) and IL-34 are ligands of CSF1R. CSF1R-mediated signaling is crucial for the survival, function, proliferation, and differentiation of myeloid lineage cells, including osteoclasts, monocytes/macrophages, microglia, Langerhans cells in the skin, and Paneth cells in the intestine. CSF1R also plays an important role in oocytes and trophoblastic cells in the female reproductive tract and in the maintenance and maturation of neural progenitor cells. Given that CSF1R is expressed in a wide range of myeloid cells, altered CSF1R signaling is implicated in inflammatory, neoplastic, and neurodegenerative diseases. Inhibiting CSF1R signaling through an inhibitory anti-CSF1R antibody or small molecule inhibitors that target the kinase activity of CSF1R has thus been a promising therapeutic strategy for those diseases. In this review, we cover the recent progress in our understanding of the various roles of CSF1R in osteoclasts and other myeloid cells, highlighting the therapeutic applications of CSF1R inhibitors in disease conditions.

NRF2 Is an Upstream Regulator of MYC-Mediated Osteoclastogenesis and Pathological Bone Erosion.

Osteoclasts are the sole bone-resorbing cells that play an essential role in homeostatic bone remodeling and pathogenic bone destruction such as inflammatory arthritis. Pharmacologically targeting osteoclasts has been a promising approach to alleviating bone disease, but there remains room for improvement in mitigating drug side effects and enhancing cell specificity. Recently, we demonstrated the crucial role of MYC and its downstream effectors in driving osteoclast differentiation. Despite these advances, upstream regulators of MYC have not been well defined. In this study, we identify nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor known to regulate the expression of phase II antioxidant enzymes, as a novel upstream regulator of MYC. NRF2 negatively regulates receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis through the ERK and p38 signaling-mediated suppression of MYC transcription. Furthermore, the ablation of MYC in osteoclasts reverses the enhanced osteoclast differentiation and activity in NRF2 deficiency in vivo and in vitro in addition to protecting NRF2-deficient mice from pathological bone loss in a murine model of inflammatory arthritis. Our findings indicate that this novel NRF2-MYC axis could be instrumental for the fine-tuning of osteoclast formation and provides additional ways in which osteoclasts could be therapeutically targeted to prevent pathological bone erosion.

Curcumin, a constituent of curry, suppresses IgE-mediated allergic response and mast cell activation at the level of Syk.

BACKGROUND: Activation of mast cells through the high-affinity receptor for IgE (FcepsilonRI) underlies atopic allergic reactions. Curcumin can block this activation, but the mechanism and the effects of curcumin on IgE-mediated allergic reactions are unknown. OBJECTIVES: We sought to determine the antiallergic activity of curcumin in vivo and its mechanism of action in mast cells. METHODS: The antiallergic activity of curcumin was evaluated in mast cell cultures and the passive cutaneous anaphylaxis model. The effects of curcumin on mast cell signaling events were examined by using immunoblotting, immunoprecipitation, RT-PCR, and other molecular biologic approaches. RESULTS: Curcumin inhibited antigen-mediated activation of mast cells and passive cutaneous anaphylaxis in mice. Suppression of degranulation and secretion of TNF-alpha and IL-4 was apparent at concentrations as low as 3 micromol/L curcumin in activated mast cells. Similar concentrations of curcumin suppressed Syk-dependent phosphorylations of the adaptor proteins linker of activated T cells and Grb2-associated binder 2, which are critical for mast cell activation. Although curcumin did not inhibit the phosphorylation of Syk itself, it directly inhibited Syk kinase activity in vitro. Further downstream, activating phosphorylations of Akt and the mitogen-activated protein kinases p38, p44/42 (extracellular signal-regulated kinase 1/2), and c-Jun N-terminal kinase, which are critical for the production of inflammatory cytokines, were also inhibited. CONCLUSIONS: Curcumin inhibits Syk kinase-dependent signaling events in mast cells and might thus contribute to its antiallergic activity. Therefore curcumin might be useful for the treatment of mast cell-related immediate and delayed allergic diseases.

Mast cell-mediated allergic response is suppressed by Sophorae flos: inhibition of SRC-family kinase.

Complementary and alternative medicines are considered as a promising direction for the development of anti-allergic therapies in oriental countries. We screened approximately 100 oriental herbal medicines for anti-allergic activity. Sophorae flos exhibited the most potent effect on degranulation in antigen-stimulated mast cells. We further investigated the effect of Sophorae flos on the IgE-mediated allergic response in vivo and its mechanism of action in mast cells. Sophorae flos exhibited a significant inhibitory effect on degranulation in antigen-stimulated mast cells with IC(50) values of approximately 31.6 microg/mL (RBL-2H3 mast cells) and approximately 47.8 microg/mL (bone marrow-derived mast cells). Sophorae flos also suppressed the expression and secretion of TNF-alpha and IL-4 in the cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Sophorae flos inhibited the activating phosphorylation of Syk and LAT in mast cells. Further downstream, activating phosphorylation of Akt and the prototypic MAP kinases, namely, p38, ERK1/2, and JNK, were also inhibited. These results suggest that Sophorae flos inhibits the Src family kinase-dependent signaling cascades in mast cells and may thus exert anti-allergic activity.

Alpha-lipoic acid inhibits TNF-alpha induced NF-kappa B activation through blocking of MEKK1-MKK4-IKK signaling cascades.

The therapeutic effects of alpha-lipoic acid (alpha-LA) via NF-kappa B down regulation were demonstrated on joint inflammation and erosion in an animal model. In this study, we investigated how alpha-LA inhibits the pathway of NF-kappa B activation by TNF-alpha via the mitogen-activated protein kinase (MAPK) pathway in rheumatoid arthritis (RA) fibroblast-like synovial cells (FLS). FLS were stimulated with TNF-alpha following pre-treatment with or without alpha-LA. Electrophoretic mobility shift assays (EMSA) revealed that TNF-alpha activates NF-kappa B in FLS. This was inhibited by alpha-LA at concentrations of 1 mM. TNF-alpha induced IKK mediated phosphorylation of GST-I kappa B and pre-treatment with alpha-LA inhibited this pathway. FLS constitutively express MEKK1, MEKK2, MEKK3, and TAK1 and that their levels are unaffected by TNF-alpha or alpha-LA. Immunoprecipitation using anti-MEKK1 antibody phosphorylated GST-I kappa B and pre-treating the cells with alpha-LA could abolish the reaction. FLS were immunoprecipitated using an antibody to MEKK1, and MKK4 was coprecipitated with MEKK1. In addition, immune complexes precipitated with anti-MKK4 antibody phosphorylated GST-I kappa B, and pre-treatment with alpha-LA inhibited the phosphorylation. Immunoprecipitation assay showed that MEKK1, MKK4, IKK-alpha, IKK-beta, I kappa B, and NF-kappa B comprised immunocomplex. It can be concluded that TNF-alpha activates NF-kappa B in FLS through MEKK1-MKK4-IKK signaling complex, and alpha-LA inhibits this signaling at the level of or upstream of IKK-alpha and IKK-beta.