Nickel ions bind to HSP90ß and enhance HIF-1α-mediated IL-8 expression.

Nickel ions (Ni2+) eluted from biomedical devices cause inflammation and Ni allergy. Although Ni2+ and Co2+ elicit common effects, Ni2+ induces a generally stronger inflammatory reaction. However, the molecular mechanism by which Ni2+ and Co2+ induce such different responses remains to be elucidated. In the present study, we compared the effects of Ni2+ and Co2+ on the expression of interleukin (IL)-8 in human monocyte THP-1 cells. We report that NiCl2 but not CoCl2 induced the expression of IL-8; in contrast, CoCl2 elicited a higher expression of hypoxia-inducible factor-1α (HIF-1α). The NiCl2-induced expression of IL-8 in late phase was blocked by a HIF-1α inhibitor, PX-478, indicating that NiCl2 targets additional factors responsible for activating HIF-1α. To identify such targets, proteins that bound preferentially to Ni-NTA beads were analyzed by LC/MS/MS. The analysis yielded heat shock protein 90ß (HSP90ß) as a possible candidate. Furthermore, Ni2+ reduced the interaction of HSP90ß with HIF-1α, and instead promoted the interaction between HIF-1α and HIF-1ß, as well as the nuclear localization of HIF-1α. Using various deletion variants, we showed that Ni2+ could bind to the linker domain on HSP90ß. These results suggest that HSP90ß plays important roles in Ni2+-induced production of IL-8 and could be a potential target for the regulation of Ni2+-induced inflammation.

Induced histamine regulates Ni elution from an implanted Ni wire in mice by downregulating neutrophil migration.

Histamine regulates various inflammatory reactions. We have reported that the expression of histidine decarboxylase (HDC) was induced by subcutaneous implantation of nickel (Ni) wire. However, the source and functions of histamine in Ni elution and Ni wire-induced inflammation have not been completely studied. We aimed to elucidate the effects of de novo synthesized histamine on leucocyte infiltration and Ni elution. Implantation of Ni wire induced an increase in the Ni ion content of the surrounding tissues and serum and in the mRNA levels of HDC, a histamine-producing enzyme, macrophage inflammatory protein-2 (MIP-2), a chemoattractant for neutrophils, and monocyte chemoattractant protein-1 (MCP-1), a chemoattractant for monocytes. The Ni wire induced HDC expression even in mast cell-deficient WBB6F1-W/WV mice. In HDC knockout (HDC KO) mice, the Ni wire-induced increase in MIP-2 mRNA expression was significantly higher than that in wild-type mice but not MCP-1. MIP-2 expression was enhanced in histamine H2 receptor knockout (H2R KO) mice but not in WBB6F1-W/WV mice. Histamine inhibited NiCl2 -induced MIP-2 mRNA expression in mouse bone marrow-derived macrophages (BMDMs) obtained from wild-type mice; this inhibition was not observed in BMDMs from H2R KO mice. Ni elution increased in HDC KO mice, in which leucocyte infiltration also increased, and was suppressed in mice treated with neutrophil-specific antibody. These results suggest that the Ni wire induced HDC expression in non-mast cells and that, in the chronic phase of inflammation, endogenous histamine reduced Ni elution, probably through regulation of MIP-2 expression and neutrophil migration.

Involvement of COX-2 in nickel elution from a wire implanted subcutaneously in mice.

Many types of medical alloys include nickel (Ni), and the elution of Ni ions from these materials causes toxicities and inflammation. We have previously reported that inflammation enhances Ni elution, although the molecular mechanisms underlying this effect remain unclear. In this study, we investigated how inflammatory responses enhanced Ni elution in a wire-implantation mouse model. Subcutaneous implantation of Ni wire induced the expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) mRNA in the surrounding tissues. Immunostaining analysis showed that cells expressing COX-2 were mainly fibroblast-like cells 8h after implantation of a Ni wire, but were mainly infiltrated leukocytes at 24h. NiCl2 induced the expression of COX-2 mRNA in primary fibroblasts, neutrophils, RAW 264 cells, and THP-1 cells, indicating that Ni ions can induce COX-2 expression in various types of cells. The elution of Ni ions from the implanted Ni wire at 8h was reduced by dexamethasone (Dex), indomethacin (Ind), or celecoxib (Cel) treatment. Ni wire implantation induced an increase in mRNA levels for anaerobic glycolytic pathway components glucose transporter 1 (GLUT1), hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 4 (MCT4); the expression of these genes was also inhibited by Dex, Ind, and Cel. In primary fibroblasts, the expression of these mRNAs and the production of lactate were induced by NiCl2 and further potentiated by PGE2. Furthermore, Ni wire-induced infiltration of inflammatory leukocytes was significantly reduced by Dex, Ind, or Cel. Depletion of neutrophils with a specific antibody caused reduction of both leukocyte infiltration and Ni elution. These results indicate that Ni ions eluted from wire induced COX-2 expression, which further promoted elution of Ni ions by increasing lactate production and leukocyte infiltration. Since COX inhibitors and Dex reduced the elution of Ni ions, these drugs may be useful for prevention of metal-related inflammation and allergy.

Nickel ions selectively inhibit lipopolysaccharide-induced interleukin-6 production by decreasing its mRNA stability.

Nickel (Ni) ions easily elute from many alloys and elicit inflammation and allergies. Previous studies have shown that infections due to the implantation of medical devices cause inflammation and enhance the elution of Ni ions (Ni²âº). However, cross-talk between infection- and Ni²âº-induced signaling pathways has not yet been elucidated in detail. In the present study, we investigated the effects of Ni2+ on the lipopolysaccharide (LPS)-induced production of cytokines in a LPS-induced air pouch-type inflammation model in BALB/c mice and the murine macrophage cell line RAW264. We demonstrated that Ni²âº inhibited the LPS-induced production of interleukin (IL)-6, but not that of tumor necrosis factor (TNF)-α both in vivo and in vitro. This inhibitory effect was also observed with cobalt ion (Co²âº), but not with chloride ion (Cl⁻), zinc ion (Zn²âº), or palladium ion (Pd²âº), and was highly selective to the production of IL-6. Ni²âº did not inhibit the activation of ERK1/2, p38 MAPK, or JNK. Although Ni²âº decreased IL-6 mRNA levels, it failed to inhibit the LPS-induced activation of the IL-6 promoter. An experiment using actinomycin D, a transcription inhibitor, revealed that Ni²âº decreased the stability of IL-6 mRNA. Moreover, Ni²âº inhibited the LPS-induced expression of Arid5a, but not regnase-1. These results demonstrated that Ni²âº may have selectively inhibited the LPS-induced production of IL-6 by decreasing the Arid5a-dependent stabilization of IL-6 mRNA.

Biochemical assay of G protein-coupled receptor oligomerization: adenosine A1 and thromboxane A2 receptors form the novel functional hetero-oligomer.

G protein-coupled receptors (GPCRs) are classified into a family of seven transmembrane receptors. Receptor oligomerization may be the key to the expression and function of these receptors, for example, ligand binding, desensitization, membrane trafficking, and signaling. The accumulation of evidence that GPCRs form an oligomerization with a functional alternation may change the strategy for the discovery of novel drugs targeting GPCRs. Identification of the oligomer is essential to elucidate GPCR oligomerization. GPCR oligomerizations have been demonstrated using various biochemical approaches, which include the coimmunoprecipitation method, fluorescence resonance energy transfer assay, and bioluminescence RET assay. Thus, various assays are useful for the study of GPCR oligomerization, and we should choose the best method to match the purpose. We previously targeted adenosine A1 and thromboxane A2 (TP) receptors to form a functionally novel hetero-oligomer, since both receptors function in the same cells. This chapter describes the methods used to detect GPCR oligomerization and alterations in the signaling pathways, principally according to our findings on oligomerization between adenosine A1 and TPα receptors.