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.

Receptor activator of NF-kB ligand (RANKL) generates intracellular reactive oxygen species (ROS), which increase RANKL-mediated signaling in osteoclast (OC) precursor bone marrow macrophages (BMMs). Here we show that a ROS scavenging protein DJ-1 negatively regulates RANKL-driven OC differentiation, also called osteoclastogenesis. DJ-1 ablation in mice leads to a decreased bone volume and an increase in OC numbers. In vitro, the activation of RANK-dependent signals is enhanced in DJ-1-deficient BMMs as compared to wild-type BMMs. DJ-1 suppresses the activation of both RANK-TRAF6 and RANK-FcRγ/Syk signaling pathways because of activation of Src homology region 2 domain-containing phosphatase-1, which is inhibited by ROS. Ablation of DJ-1 in mouse models of arthritis and RANKL-induced bone disease leads to an increase in the number of OCs, and exacerbation of bone damage. Overall, our results suggest that DJ-1 plays a role in bone homeostasis in normal physiology and in bone-associated pathology by negatively regulating osteoclastogenesis.

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 transcriptasepolymerase 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 ␣ (TNF␣). TNF␣-induced expression of IL-32 was significantly suppressed, in a dose-dependent manner, by inhibitors of Syk, protein kinase C␦ (PKC␦), and JNK and by knockdown of these kinases and c-Jun with siRNA. We also observed that PKC␦ 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 PKC␦.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/PKC␦-mediated signaling events.

Abstract. 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.

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Background DJ-1 is an anti-oxidant protein, known to reduce levels of reactive oxygen species (ROS), but its presence or function in mast cells and allergic diseases is unknown. Objectives To determine the role and mechanism of DJ-1 in allergic responses in vitro and in vivo. Methods The level of ROS and DJ-1 in serum or culture medium was measured with ELISA kits. The role of DJ-1 was evaluated in mast cell cultures and passive cutaneous anaphylaxis (PCA) in normal or DJ-1 knockout (KO) mice. The mechanism of DJ-1 action was examined by immunoblotting, immunoprecipitation, RT-PCR, and other molecular biological approaches. Results Patients with atopic dermatitis had elevated levels of ROS and diminished levels of DJ-1. DJ-1 KO mice exhibited enhanced PCA and augmented ROS levels in serum and in bone marrow-derived mast cells (BMMCs) from these mice. Furthermore, antigen-induced degranulation as well as production of TNF-α and IL-4 were significantly amplified in DJ-1 KO and anti-DJ-1 siRNA-transfected BMMCs compared to wild type BMMCs. Studies with these and BMMCs transfected with siRNAs against the phosphatases SHP-1 or SHP-2 revealed that the DJ-1 KO phenotype could be attributed to suppression of SHP-1 activity and enhancement of SHP-2 activity leading to strengthened signaling through LAT, phospholipase Cγ, and MAP kinases. Conclusions A deficiency or constitutive activation of DJ-1 can have implications in mast cell-driven allergic diseases such as asthma and anaphylaxis.

Mast cells are critical for various allergic disorders. Mast cells express Src-family kinases which relay positive and negative regulatory signals by antigen. Lyn, for example, initiates activating signaling events but it also induces inhibitory signals. Fyn and Hck are reported to be positive regulators but little is known about the roles of other Src kinases, including Fgr, in mast cells. In this study, we define the role of Fgr. Endogenous Fgr associates with FcεRI and promotes phosphorylation of Syk, Syk substrates which include LAT, SLP76, and Gab2, and downstream targets such as Akt and the MAP kinases in antigen-stimulated mast cells. As a consequence, Fgr positively regulates degranulation, production of eicosanoids, and cytokines. Fgr and Fyn appeared to act in concert as phosphorylation of Syk and degranulation are enhanced by overexpression of Fgr and further augmented by overexpression of Fyn but is suppressed by overexpression of Lyn. Moreover, knockdown of Fgr by siRNAs further suppressed degranulation in Fyn-deficient BMMCs. Overexpression of Fyn or Fgr restored phosphorylation of Syk and partially restored degranulation in Fyn-deficient cells. Additionally, knockdown of Fgr by siRNAs inhibited association of Syk with FcεRIγ as well as the tyrosine phosphorylation of FcεRIγ. Of note, the injection of Fgr siRNAs diminished the protein level of Fgr in mice and simultaneously inhibited IgE-mediated anaphylaxis. In conclusion, Fgr positively regulates mast cell through activation of Syk. These findings help clarify the interplay among Src-family kinases and identify Fgr as a potential therapeutic target for allergic diseases.

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