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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 cilia are 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 showed that IK positively regulated ofd1 expression required for cilium assembly. Downregulation of ofd1 expression upon depletion of IK led to ciliary defects that was rescued by restoration of 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.

Administration of bisphosphonates following total joint arthroplasty might be beneficial to reduce aseptic loosening. However, their effects on peri-implant bone formation and bone–implant interface strength have not been investigated yet. We used a physiologically loaded mouse implant model to investigate the short-term effects of postoperative systemic alendronate on osseointegration. A titanium implant with a rough surface was inserted in the proximal tibiae of 17-week-old female C57BL/6 mice (n = 44). Postimplantation mice were given alendronate (73 μg/kg/days, n = 22) or vehicle (n = 22) 5 days/week. At 7- and 14-day postimplantation, histology and histomorphometry were conducted. At 28 days, microcomputed tomography and biomechanical testing were performed (n = 10/group). Postoperative alendronate treatment enhanced osseointegration, increasing maximum pullout load by 45% (p < .001) from 19.1 ± 4.5 N in the control mice to 27.6 ± 4.9 N in the treated mice, at day 28 postimplantation. Alendronate treatment increased the bone volume fraction by 139% (p < .001) in the region distal to the implant and 60% (p < .05) in the peri-implant region. At 14-day postimplantation, alendronate treatment decreased the number of osteoclasts per bone perimeter (p < .05) and increased bone volume fraction (p < .01) when compared with the control group. Postimplantation, short-term alendronate treatment enhanced osseointegration as demonstrated by increased bone mass, trabecular bone thickness, and maximum pullout load. Alendronate decreased peri-implant osteoclasts while preserving peri-implant osteoblasts and endothelial cells, in turn, increasing bone volume fraction. This data supports the postoperative clinical use of bisphosphonates, especially in patients with high risks of aseptic loosening.

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.

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.

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).

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., Bone disease: Signaling pathway offers promising drug target Drugs directed at a key signaling receptor involved in breaking down bone tissue could help treat diseases marked by pathological bone loss and destruction. In a review article, Kyung-Hyun Park-Min and colleagues from the Hospital for Special Surgery in New York, USA, discuss the essential roles played by the colony-stimulating factor 1 receptor (CSF1R) protein in the survival, function, proliferation and differentiation of myeloid lineage stem cells in the bone marrow, including bone-resorbing osteoclasts. They explore the links between the CSF1R-mediated signaling pathway and diseases such as cancer and neurodegeneration. The authors largely focus on bone conditions, highlighting mouse studies in which CSF1R-blocking drugs were shown to ameliorate bone loss and inflammatory symptoms in models of arthritis, osteoporosis and metastatic cancer. Clinical trials are ongoing to test therapeutic applications.

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.

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.

Investigations on the therapeutic effects of intravenous immunoglobulin (IVIG) have focused on the suppression of autoantibody and immune complex-mediated inflammatory pathogenesis. Inflammatory diseases such as rheumatoid arthritis are often accompanied by excessive bone erosion but the effect of IVIG on osteoclasts, bone-resorbing cells, has not been studied. Here, we investigate whether IVIG directly regulates osteoclast differentiation and has therapeutic potential for suppressing osteoclast-mediated pathologic bone resorption. IVIG or cross-linking of Fcγ receptors with plate-bound IgG suppressed receptor activator of nuclear factor-κ B ligand (RANKL)-induced osteoclastogenesis and expression of osteoclast-related genes such as integrin β3 and cathepsin K in a dose-dependent manner. Mechanistically, IVIG or plate-bound IgG suppressed osteoclastogenesis by downregulating RANKL-induced expression of NFATC1, the master regulator of osteoclastogenesis. IVIG suppressed NFATC1 expression by attenuating RANKL-induced NF-κB signaling, explained in part by induction of the inflammatory signaling inhibitor A20. IVIG administration attenuated in vivo osteoclastogenesis and suppressed bone resorption in the tumor necrosis factor (TNF)-induced calvarial osteolysis model. Our findings show that, in addition to suppressing inflammation, IVIG directly inhibits osteoclastogenesis through a mechanism involving suppression of RANK signaling. Direct suppression of osteoclast differentiation may provide beneficial effects on preserving bone mass when IVIG is used to treat rheumatic disorders.

While the prevalence of osteoporosis is growing rapidly with population aging, therapeutic options remain limited. Here, we identify potentially novel roles for CaV1.2 L-type voltage-gated Ca2+ channels in osteogenesis and exploit a transgenic gain-of-function mutant CaV1.2 to stem bone loss in ovariectomized female mice. We show that endogenous CaV1.2 is expressed in developing bone within proliferating chondrocytes and osteoblasts. Using primary BM stromal cell (BMSC) cultures, we found that Ca2+ influx through CaV1.2 activates osteogenic transcriptional programs and promotes mineralization. We used Prx1-, Col2a1-, or Col1a1-Cre drivers to express an inactivation-deficient CaV1.2 mutant in chondrogenic and/or osteogenic precursors in vivo and found that the resulting increased Ca2+ influx markedly thickened bone not only by promoting osteogenesis, but also by inhibiting osteoclast activity through increased osteoprotegerin secretion from osteoblasts. Activating the CaV1.2 mutant in osteoblasts at the time of ovariectomy stemmed bone loss. Together, these data highlight roles for CaV1.2 in bone and demonstrate the potential dual anabolic and anticatabolic therapeutic actions of tissue-specific CaV1.2 activation in osteoblasts.

Osteoporosis is a metabolic bone disorder associated with compromised bone strength and an increased risk of fracture. Inhibition of the differentiation of bone-resorbing osteoclasts is an effective strategy for the treatment of osteoporosis. Prior work by our laboratory and others has shown that MYC promotes osteoclastogenesis in vitro, but the underlying mechanisms are not well understood. In addition, the in vivo importance of osteoclast-expressed MYC in physiological and pathological bone loss is not known. Here, we have demonstrated that deletion of Myc in osteoclasts increases bone mass and protects mice from ovariectomy-induced (OVX-induced) osteoporosis. Transcriptomic analysis revealed that MYC drives metabolic reprogramming during osteoclast differentiation and functions as a metabolic switch to an oxidative state. We identified a role for MYC action in the transcriptional induction of estrogen receptor-related receptor α (ERRα), a nuclear receptor that cooperates with the transcription factor nuclear factor of activated T cells, c1 (NFATc1) to drive osteoclastogenesis. Accordingly, pharmacological inhibition of ERRα attenuated OVX-induced bone loss in mice. Our findings highlight a MYC/ERRα pathway that contributes to physiological and pathological bone loss by integrating the MYC/ERRα axis to drive metabolic reprogramming during osteoclast differentiation.

Objective Macrophage migration inhibitory factor (MIF) is an important modulator of innate and adaptive immunity as well as local inflammatory responses. We previously reported that MIF down-regulated osteoclastogenesis through a mechanism that requires CD74. The aim of the current study was to examine whether MIF modulates osteoclastogenesis through Lyn phosphorylation, and whether down-regulation of RANKL-mediated signaling requires the association of CD74, CD44, and Lyn. Methods CD74-knockout (CD74-KO), CD44-KO, and Lyn-KO mouse models were used to investigate whether Lyn requires these receptors and coreceptors. The effects of MIF on osteoclastogenesis were assessed using Western blot analysis, small interfering RNA (siRNA)–targeted down-regulation of Lyn, Lyn-KO mice, and real-time imaging of Lyn molecules to surface proteins. Results MIF treatment induced Lyn expression, and MIF down-regulated RANKL-induced activator protein 1 (AP-1) and the Syk/phospholipase Cγ cascade during osteoclastogenesis through activated Lyn tyrosine kinase. The results of immunoprecipitation studies revealed that MIF receptors associated with Lyn in response to MIF treatment. Studies using Lyn-specific siRNA and Lyn-KO mice confirmed our findings. Conclusion Our findings indicate that the tyrosine kinase Lyn is activated when MIF binds to its receptor CD74 and its coreceptor CD44 and, in turn, down-regulates the RANKL-mediated signaling cascade by suppressing NF-ATc1 protein expression through down-regulation of AP-1 and calcium signaling components.

CD74 is a type II transmembrane protein that can act as a receptor for macrophage migration inhibitory factor (MIF) and plays a role in MIF-regulated responses. We reported that MIF inhibited osteoclast formation and MIF knockout (KO) mice had decreased bone mass. We therefore examined if CD74 was involved in the ability of MIF to alter osteoclastogenesis in cultured bone marrow (BM) from wild-type (WT) and CD74-deficient (KO) male mice. We also measured the bone phenotype of CD74 KO male mice. Bone mass in the femur of 8-week-old mice was measured by micro–computed tomography and histomorphometry. Bone marrow cells from CD74 KO mice formed 15% more osteoclast-like cells (OCLs) with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) (both at 30 ng/mL) compared to WT. Addition of MIF to WT cultures inhibited OCL formation by 16% but had no effect on CD74KO cultures. The number of colony forming unit granulocyte-macrophage (CFU-GM) in the bone marrow of CD74 KO mice was 26% greater than in WT controls. Trabecular bone volume (TBV) in the femurs of CD74 KO male mice was decreased by 26% compared to WT. In addition, cortical area and thickness were decreased by 14% and 11%, respectively. Histomorphometric analysis demonstrated that tartrate-resistant acid phosphatase (TRAP)(+) osteoclast number and area were significantly increased in CD74 KO by 35% and 43%, respectively compared to WT. Finally, we examined the effect of MIF on RANKL-induced-signaling pathways in bone marrow macrophage (BMM) cultures. MIF treatment decreased RANKL-induced nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and c-Fos protein in BMM cultures by 70% and 41%, respectively. Our data demonstrate that CD74 is required for MIF to affect in vitro osteoclastogenesis. Further, the bone phenotype of CD74 KO mice is similar to that of MIF KO mice. MIF treatment of WT cultures suppressed RANKL-induced activator protein 1 (AP-1) expression, which resulted in decreased osteoclast differentiation in vitro. We propose that CD74 plays a critical role in the MIF inhibition of osteoclastogenesis. © 2013 American Society for Bone and Mineral Research.

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.

Osteoclasts (OCs) are the only bone-resorbing cells and are critically involved in various bone-associated diseases, including osteoporosis and rheumatoid arthritis. Differentiation of OCs from bone marrow macrophage cells (BMMs) is regulated by RANK and the adaptor protein (DAP12/FcRγ)-mediated costimulatory signals. However, it is unknown how RANKL/RANK signal stimulates phosphorylation of DAP12/FcRγ to initiate the costimulatory signals. As reported here, we found that OC differentiation and acquisition of bone resorption capacity were suppressed in RANKL-stimulated Fyn(-/-) or Fyn-siRNA-transfected BMMs, but could be restored by overexpression of Fyn kinase in Fyn(-/-) BMMs. However, the RANKL-stimulated proliferation of BMMs was unaffected by the absence of Fyn. In addition, RANKL-stimulated Fyn(-/-) BMMs no longer exhibited the optimal induction of typical OC markers such as NFATc1, c-Fos, c-Src, TRAF6, and cathepsin K or costimulatory signals such as the activating phosphorylations of Syk, PLCγ2, and Gab2. These were restored by overexpression of Fyn in Fyn(-/-) BMMs. Immunoprecipitation studies also indicated that the adaptor proteins DAP12/FcRγ and Syk interacted with RANK during RANKL stimulation in BMMs in a Fyn-dependent manner. Phosphorylation of the DAP12/FcRγ and the recruitment of Syk by DAP12/FcRγ were suppressed in Fyn(-/-) BMMs. This is the first demonstration that Fyn relays the initial RANK/RANKL signal to the ITAM-containing adaptors DAP12/FcRγ for OC differentiation.

Osteoclasts, multinucleated bone-resorbing cells, are closely associated with bone diseases such as rheumatoid arthritis and osteoporosis. Osteoclasts are derived from hematopoietic precursor cells, and their differentiation is mediated by two cytokines, including macrophage colony stimulating factor and receptor activator of nuclear factor κB ligand (RANKL). Previous studies have shown that arctigenin exhibits an anti-inflammatory effect. However, the effect of arctigenin on osteoclast differentiation is yet to be elucidated. In this study, we found that arctigenin inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages in a dose-dependent manner and suppressed RANKL-mediated bone resorption. Additionally, the expression of typical marker proteins, such as NFATc1, c-Fos, TRAF6, c-Src, and cathepsin K, were significantly inhibited. Arctigenin inhibited the phosphorylation of Erk1/2, but not p38 and JNK, in a dose-dependent manner. Arctigenin also dramatically suppressed immunoreceptor tyrosine-based activation motif-mediated costimulatory signaling molecules, including Syk and PLCγ2, and Gab2. Notably, arctigenin inhibited the activation of Syk through RANKL stimulation. Furthermore, arctigenin prevented osteoclast differentiation in the calvarial bone of mice following stimulation with lipopolysaccharide. Our results show that arctigenin inhibits osteoclast differentiation in vitro and in vivo. Therefore, arctigenin may be useful for treating rheumatoid arthritis and osteoporosis.

Interleukin-7 is a critical cytokine for lymphoid development and a direct inhibitor of in vitro osteoclastogenesis in murine bone marrow cultures. To explore the role of IL-7 in bone, we generated transgenic mouse lines bearing the 2.3-kb rat collagen 1α1 promoter driving the expression of human IL-7 specifically in osteoblasts. In addition, we crossed these mice with IL-7-deficient mice to determine if the alterations in lymphopoiesis, bone mass, and osteoclast formation observed in the IL-7 knockout (KO) mice could be rescued by osteoblast-specific overexpression of IL-7. Here, we show that mice overexpressing human IL-7 in the osteoblast lineage showed increased trabecular bone volume in vivo by µCT and decreased osteoclast formation in vitro. Furthermore, targeted overexpression of IL-7 in osteoblasts rescued the osteopenic bone phenotype and B-cell development of IL-7 KO mice but did not have an effect on T lymphopoiesis, which occurs in the periphery. The bone phenotypes in IL-7 KO mice and targeted IL-7-overexpressing mouse models were observed only in females. These results likely reflect both direct inhibitory effects of IL-7 on osteoclastogenesis in vivo and sex-specific differences in responses to IL-7.

Interleukin (IL)-32 is a recently described cytokine that appears to play a critical role in a variety of inflammatory diseases including chronic obstructive pulmonary disease (COPD). However, thus far, the regulation of IL-32 production has not been fully established. Here, we report on signaling pathways that regulate the production of IL-32α, the most abundant isoform, in the human alveolar epithelial cell line, A549. IL-32α was expressed and secreted by IL-1β. The IL-32 expression was attenuated by PP2 (a Src-family kinase [SFK] inhibitor), rottlerin (a protein kinase [PK] Cδ inhibitor), and LY294002 (a phosphatidylinositol 3-kinase [PI3K] inhibitor). Furthermore, the overexpression of Fgr rather than other SFKs upregulated IL-32α expression, while Fgr small interfering RNA (siRNA) transfection downregulated it. The suppression of Fgr with PP2 and Fgr siRNA inhibited activating phosphorylation of PKCδ and PI3K/Akt, but not IL-1 receptor-associated kinase (IRAK)1, a well-known MyD88-dependent signaling molecule, and Erk1/2, p38, and JNK. Rottlerin and PKCδ siRNA also inhibited expression of IL-32α and activation of PI3K/Akt, but not of IRAK1 and mitogen activation protein (MAP) kinases. MyD88 siRNA suppressed the expression of IL-32α and the phosphorylation of IRAK1, PI3K, and MAP kinases, but not of PKCδ. Of interest, both Fgr/PKCδ and MyD88-dependent signals regulated PI3K/Akt, suggesting that it is a crosstalk molecule. Among MyD88-dependent MAP kinases, only p38 regulated IL-32α expression and PI3K/Akt activation. With these results, we demonstrated that the expression and secretion of IL-32α are regulated by MyD88-dependent IRAK1/p38/PI3K and independent Fgr/PKCδ/PI3K pathways, and that Fgr and PKCδ are critical for the MyD88-independent IL-32α production.

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.

Background Activation of mast cells through the high-affinity receptor for IgE (FcɛRI) 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-α and IL-4 was apparent at concentrations as low as 3 μmol/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.

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.

Bee venom (BV) has been used in patients with rheumatoid arthritis, a condition characterized by rheumatoid joint destruction mediated, in large part, by matrix metalloproteinases (MMPs). We investigated the effects of melittin, a major component of bee venom, on the production of MMPs in human rheumatoid arthritic fibroblast-like synoviocytes (FLS). Lipopolysaccharide (LPS)-stimulated MMP3 production was significantly inhibited by melittin, which also inhibited LPS-induced DNA binding by nuclear factor κ B (NF-κ B). Mellitin had no effect on IL-1β –or TNF-α –induced MMP1 or MMP3 production and did not decrease LPS-induced secretion of MMP1. Taken together, these findings suggest that melittin may exert its anti-rheumatoid effects, at least in part, by inhibiting MMP3 production, most likely through inhibition of NF-κ B activity. Keywords:: rheumatoid arthritis, melittin, matrix metalloproteinase-1 and -3

The antiallergic activity of Polygoni cuspidati radix (PR) and the mechanism of action by which it functions were investigated in this study. The extract of PR exhibited potent inhibitory activity in mast cells; its IC50 values were 62 ± 2.1 μg/ml for RBL-2H3 mast cells and 46 ± 3.2 μg/m for bone marrow–derived mast cells by antigen stimulation, and it also suppressed the expression of tumor necrosis factor-α and interleukin-4 in RBL-2H3 cells. According to the in vivo animal allergy model, it inhibited a local allergic reaction, passive cutaneous anaphylaxis, in a dose-dependent manner. With regard to its mechanism of action, PR inhibited the activating phosphorylation of Syk, a key signaling protein for the activation of mast cells. It also suppressed Akt and the mitogen-activated protein kinases ERK1/2, p38, and JNK, which are critical for the production of various inflammatory cytokines in mast cells. The results of the study indicate that the antiallergic activity of PR is mediated through the inhibition of histamine release and allergic cytokine production by the inhibition of Syk activating phosphorylation in mast cells.

The anti-allergic action of various Oriental medicinal herbs was investigated using in vitro and in vivo experimental models. Of these extracts, the ethanol extract of Meliae cortex (MC) exhibited the most potent activity in mast cells; its IC(50) values were 29+/-1.5 microg/ml for antigen stimulation and 57+/-3.4 microg/ml for thapsigargin stimulation. It inhibited compound-48/80-induced systemic anaphylaxis by 52.9% at a dose of 300 mg/kg in mice; it also inhibited the expression of the proinflammatory mediator TNF-alpha. With regard to its mechanism of action, MC suppressed the activating phosphorylation of Syk, a key enzyme in mast-cell signaling processes and that of Akt in a dose-dependent manner. It also inhibited the MAP kinase ERK1/2, which is critical for the production of inflammatory cytokines in mast cells, as indicated by the suppression of the activating phosphorylation of ERK1/2. Taken together, these results suggest that the anti-allergic activity of MC may be due to the inhibition of histamine secretion and cytokine expression through the Syk inhibition in mast cells.

The rat RBL-2H3 mast cells contain various Src-family kinases. Previous reports with this cell line indicated that Lyn activation is an important initial signaling for the activation of the cells. However, the role and location of other Src-family kinase isoforms which are expressed in the cells are not clear. In this study, we now show that isoforms of Src-family kinases, Lyn, Fyn, Fgr, c-Src, and Yes are differentially expressed and located differently in the cells as indicated by RT-PCR, immunoblotting analysis, and confocal microscopy. Lyn and Fgr were located on plasma membrane but on the other hand c-Src and Yes were located on intracellular organelle. All of Src-family kinases were cloned and overexpressed for investigating the roles of the isoforms. Overexpression of Fyn and Fgr, not Lyn and c-Src, stimulated Ag-induced degranulation in the cells. Our findings strongly suggest for the first time that each of Src-family kinase isoform can regulate differentially FceRI-mediated signaling in RBL-2H3 mast cells.

In this study, we aimed to investigate the anti-inflammatory activity, antinociceptive activity and the action mechanism of Trachelospermi caulis extract. The anti-inflammatory effects were investigated using arachidonic acid, 12-O-tetradecanoylphorbol 13-acetate or carrageenan-induced oedema assays. Antinociceptive activity, using the acetic acid-induced writhing model, was also tested in mice. The extract exhibited dose-dependent and significant (P & 0.05 at 100–400 mg kg−1) anti-inflammatory and antinociceptive activity in the animals. To further understand the mechanism of activity, we investigated whether the extract inhibited the expression of inducible nitric oxide synthase (iNOS), the production of nitric oxide (NO) and the expression of TNF-α from murine macrophage RAW 264.7 cells. Similar to the in-vivo activity, the iNOS expression, NO production and TNF-α expression were found to be dose dependent and significantly suppressed by the extract through the inhibition of the p38 MAP kinase/NF-κB pathway. Taken together, the results presented here suggest that T. caulis extract may be useful for the treatment of various inflammatory diseases.

Objective CD97, a member of the 7-transmembrane epidermal growth factor family of adhesion G protein–coupled receptors, is expressed on various cell types. This study was undertaken to elucidate the functions of CD97 in bone and inflammation in an experimental mouse model, by examining the effect of CD97 on osteoclastogenesis in vitro, characterizing the skeletal phenotype of CD97-deficient (CD97-knockout [KO]) mice, and assessing the responses to tumor necrosis factor (TNF) treatment. Methods Femoral tissue and bone marrow (BM)–derived cells from CD97-KO and wild-type (WT) mice were assessed using histomorphometric analyses, in vitro cultures, and reverse transcription–polymerase chain reaction. Serum cytokine and chemokine levels in the presence or absence of TNF challenge were analyzed by multiplex assay. Results In cultures of mouse BM-derived macrophages in vitro, RANKL induced the expression of CD97. In vivo, the trabecular bone volume of the femurs of female CD97-KO mice was increased, and this was associated with a decrease in the number of osteoclasts. Compared to WT mice, CD97-KO mice had a reduced potential to form osteoclast-like cells in vitro. Furthermore, TNF treatment augmented the formation of osteoclasts in the calvaria of CD97-KO mice in vivo, by increasing the production of RANKL and other cytokines and chemokines and by reducing the production of osteoprotegerin by calvarial cells. Conclusion These findings demonstrate that CD97 is a positive regulator of osteoclast-like cell differentiation, a mechanism that influences bone homeostasis. However, the presence of CD97 may be essential to suppress the initial osteoclastogenesis that occurs in response to acute and local inflammatory stimuli.

Tumor necrosis factor-alpha (TNF-alpha) is a major cytokine of host immune reaction by foreign agents. Phytohemagglutinin (PHA) is a dynamic contributor to mitogenic stimulation and augmentation of host immune defense. Interferon-gamma (IFN-gamma) induces induction of cytokines in macrophages and lymphocytes. The aim of this study was to examine the synergistic effects of PHA plus low dose IFN-gamma on TNF-alpha mRNA production, cytosolic levels, and secretion in RAW 264.7 cells. The cells were stimulated with PHA or IFN-gamma using various concentrations for various times. The effects of PHA on TNF-alpha expression appeared in dose- and time-dependent manners. The maximum doses of PHA and IFN-gamma to produce them were 300 microg/ml PHA and 10 ng/ml IFN-gamma. The optimum time of PHA for the TNF-alpha mRNA production and release were 6 and 7 h after stimulation, respectively, whereas the time of IFN-gamma on them was achieved at 3 and 8 h. Although the TNF-alpha mRNA production, cytosolic levels, and secretion from the cells were slightly detected under 10 microg/ml PHA and 1 ng/ml IFN-gamma, the combination of PHA (10 microg/ml) and IFN-gamma (1 ng/ml) greatly increased them, indicating the synergistic effect of PHA plus low dose IFN-gamma on TNF-alpha expression.

Objective To demonstrate the effects of disease-modifying antirheumatic drugs and antiinflammatory cytokines on human osteoclastogenesis through their effects on receptor activator of nuclear factor κB (RANK), osteoprotegerin (OPG), and RANK ligand (RANKL). Methods Peripheral blood mononuclear cells (PBMCs) and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were cocultured in the presence of macrophage colony-stimulating factor, 1,25-dihydroxyvitamin D3, and various concentrations of methotrexate (MTX), sulfasalazine (SSZ), hydroxychloroquine (HCQ), anti–tumor necrosis factor α monoclonal antibody (infliximab), interleukin-4 (IL-4), and IL-10. Osteoclast formation was assayed by counting cells after staining for tartrate-resistant acid phosphatase. RANKL expression in RA FLS and RANK expression in PBMCs were assayed by Western blotting, reverse transcription–polymerase chain reaction (RT-PCR), and real-time PCR. OPG expression was measured by enzyme-linked immunosorbent assay, RT-PCR, and real-time PCR in cultures of RA FLS. Results MTX, SSZ, infliximab, and IL-4, but not IL-10 and HCQ, each inhibited osteoclast formation in a dose-dependent manner. We observed no evidence of synergistic inhibition of osteoclast formation by IL-4 and IL-10. High doses of infliximab suppressed the expression of RANK in PBMCs. MTX, SSZ, infliximab, and IL-4 each inhibited the expression of RANKL in RA FLS in a dose-dependent manner, and also increased the secretion of OPG in RA FLS supernatants. Conclusion MTX, SSZ, infliximab, and IL-4 inhibit human osteoclastogenesis by modulating the interaction of RANKL, RANK, and OPG. These results are indicative of the underlying mechanisms of the antiresorptive effects of these 4 agents.

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).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.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.Our results suggest that IL-32gamma stimulation can induce the production of IL-6 and IL-8 from RA FLS via Erk1/2 activation.

We investigated whether oral administration of curcumin suppressed type II collagen–induced arthritis (CIA) in mice and its effect and mechanism on matrix metalloproteinase (MMP)-1 and MMP-3 production in CIA mice, RA fibroblast-like synoviocytes (FLS), and chondrocytes. CIA in mice was suppressed by oral administration of curcumin in a dose-dependent manner. Macroscopic observations were confirmed by histological examinations. Histological changes including infiltration of immune cells, synovial hyperplasia, cartilage destruction, and bone erosion in the hind paw sections were extensively suppressed by curcumin. The histological scores were consistent with clinical arthritis indexes. Production of MMP-1 and MMP-3 were inhibited by curcumin in CIA hind paw sections and tumor necrosis factor (TNF)-α–stimulated FLS and chondrocytes in a dose-dependent manner. As for the mechanism, curcumin inhibited activating phosphorylation of protein kinase Cδ (PKCδ) in CIA, FLS, and chondrocytes. Curcumin also suppressed the JNK and c-Jun activation in those cells. This study suggests that the suppression of MMP-1 and MMP-3 production by curcumin in CIA is mediated through the inhibition of PKCδ and the JNK/c-Jun signaling pathway. Keywords:: curcumin, matrix metalloproteinase (MMP)-1, MMP-3, protein kinase Cδ (PKCδ), collagen-induced arthritis

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.

Objective Interleukin-32 (IL-32) is a recently discovered cytokine that appears to play a critical role in human rheumatoid arthritis (RA). It is highly expressed in synovium and fibroblast-like synoviocytes (FLS) from RA patients, but not in patients with osteoarthritis (OA). This study was undertaken to assess IL-32 levels in RA synovial fluid (SF) and to investigate the secretion and regulation of IL-32 in RA FLS. Methods FLS and SF were obtained from the joints of RA patients. The secretion and expression of IL-32 and activation of signaling molecules were examined by enzyme-linked immunosorbent assay, immunoblotting, immunoprecipitation, reverse transcriptase–polymerase chain reaction, and small interfering RNA (siRNA) transfection. Results IL-32 levels were high in RA SF compared with OA SF. Furthermore, RA FLS expressed and secreted IL-32 when stimulated with tumor necrosis factor α (TNFα). TNFα-induced expression of IL-32 was significantly suppressed, in a dose-dependent manner, by inhibitors of Syk, protein kinase Cδ (PKCδ), and JNK and by knockdown of these kinases and c-Jun with siRNA. We also observed that PKCδ mediated the activation of JNK and c-Jun, and experiments using specific inhibitors and siRNA demonstrated that Syk was the upstream kinase for the activation of PKCδ. Conclusion The present findings suggest that IL-32 may be a newly identified prognostic biomarker in RA, thereby adding valuable knowledge to the understanding of this disease. The results also demonstrate that the production of IL-32 in RA FLS is regulated by Syk/PKCδ-mediated signaling events.

Mast cells are responsible for IgE-mediated allergic responses. Although dietary flavonoid morin has been known to suppress mast cell activation, its in vivo anti-allergic activity and the underlying mechanisms remain are largely unknown. In this study, we determine whether morin suppresses IgE-mediated allergic responses in an animal model and its mechanism of action. Morin suppressed IgE-mediated PCA in mice (ED50 23.9 mg/kg) and inhibited degranulation and production of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-4 in antigen (Ag)-stimulated mast cells. The mechanism of action was a follows. Morin inhibited the activating phosphorylation of spleen tyrosine kinase (Syk) and linker for activation of T cells (LAT) in rat basophilic leukemia (RBL)-2H3 cells and bone marrow-derived mast cells (BMMCs). Akt and the mitogen-activated protein (MAP) kinases, p38, extracellular signal-regulated kinase (ERK)1/2, and c-Jun N-terminal kinase (JNK) were inhibited as well. In vitro kinase assay indicated that Fyn kinase, not Lyn and Syk, was inhibited by morin in a dose-dependent manner (IC50 5.7 μM). In conclusion, the results suggest that morin suppresses the IgE-mediated allergic response by primarily inhibiting Fyn kinase in mast cells.

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 IC50 values of ~31.6 μg/mL (RBL-2H3 mast cells) and ~47.8 μg/mL (bone marrow-derived mast cells). Sophorae flos also suppressed the expression and secretion of TNF-α 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.

Regulatory T cell (Treg) is a subset of CD4+ T lymphocytes expressing CD25 with immunosuppressive activity. However the function of Tregs onto osteoclastogenesis remains unknown. We investigated the effect and regulatory mechanism of Treg focusing on osteoclastogenesis from PBMCs. Tregs were isolated from PBMCs by magnetic cell sorting-column and analyzed by flow cytometry. RT-PCR was performed to identify Foxp3 mRNA. Using PBMCs and Tregs coculture system, we could find that Tregs inhibited osteoclasts differentiation from PBMCs and reduced the resorbed areas on pit assay (p

The extract of Pyrolae herba (PH), which has been used as an anti-inflammatory folk remedy in Korea and China, was investigated for its anti-inflammatory action using arachidonic acid, 12-O-tetradecanoylphorbol 13-acetate or carrageenan-induced edema assays. The anti-nociceptive activity of PH was also tested in mice using the acetic acid-induced writhing model. PH showed dose-dependent and significant (P