Caspases downregulate nickel and hydrogen peroxide-induced IL-8 production via modification of c-Jun N-terminal kinases.

Nickel (Ni) is a typical hapten in allergic contact dermatitis. However, it has been used in various metal materials due to its usefulness. Although Ni ions induce apoptosis of inflammatory cells and the expression of inflammatory cytokines such as interleukin-8 (IL-8), the effects of the apoptotic pathway on the signaling that induces cytokine production have not been sufficiently clarified. Here, we found that NiCl2-induced IL-8 production was enhanced by the pan-caspase inhibitor Z-VAD-FMK in THP-1 cells. Moreover, Z-VAD-FMK enhanced H2O2-induced and NiCl2-induced IL-8 production, but not TNF-α-induced one. The analyses of signaling pathways apparently showed that NiCl2- and H2O2-induced phosphorylation of c-Jun, but not TNF-α-induced one were enhanced by Z-VAD-FMK. The cleavages of p54c-Jun N-terminal kinase (JNK) as well as PARP was induced by NiCl2 and H2O2 but not by TNF-α. Finally, a JNK inhibitor, SP600125, inhibited Z-VAD-FMK-induced enhancement of IL-8 production. In summary, we showed that caspase activation in the apoptotic pathway actively downregulates the JNK-mediated activation of inflammatory cells. This study highlighted the significance of apoptosis in inflammatory diseases, including Ni-induced dermatitis.

The Regulation Pathway of VEGF Gene Expression Is Different between 2D Cells and 3D Spheroids in Human Lung Cancer Cells.

Angiogenesis is involved in the malignant transformation of cancers. Vascular endothelial growth factor (VEGF) is important in inducing angiogenesis. Cultured cells play an important role in analyzing the regulation of VEGF expression, and it is revealed that VEGF expression is induced under hypoxia. However, it has been shown that there are differences in the pathway for gene expression between two-dimensional (2D) cells and in vivo cells. Three-dimensional (3D) spheroids constructed in 3D culture with a gene expression pattern more similar to that of in vivo cells than 2D cells have been used to solve this problem. This study analyzed the VEGF gene expression pathway in 3D spheroids of human lung cancer cells, A549 and H1703. Hypoxia-inducible factor-1α (HIF-1α) and aryl hydrocarbon receptor nuclear translocator (ARNT) regulated VEGF gene expression in 3D spheroids. However, VEGF gene expression was not regulated by HIF-1α in 2D cells. To conclude, we found that the regulatory pathway of VEGF gene expression is different between 2D cells and 3D spheroids in human lung cancer cells. These results suggest the possibility of a new VEGF gene expression regulation pathway in vivo. In addition, they show useful knowledge for the analysis of angiogenesis induction mechanisms and also demonstrate the usefulness of 3D spheroids.

ß-naphthoflavone-induced upregulation of CYP1B1 expression is mediated by the preferential binding of aryl hydrocarbon receptor to unmethylated xenobiotic responsive elements.

Human cytochrome P450 1 (CYP1) enzymes are transcriptionally induced by specific xenobiotics through a mechanism that involves the binding of aryl hydrocarbon receptors (AhR) to target xenobiotic responsive element (XRE) sequences. To examine the effect of DNA methylation on the AhR-mediated pathway, reverse transcription-quantitative PCR analysis was performed. ß-naphthoflavone (ßNF)-induced CYP1B1 expression was found to be potentiated by pre-treatment of human HepG2 liver cancer cells with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, but not HuH7 cells. It was hypothesized that this increase is mediated by the demethylation of CpG sites within XRE2/XRE3 sequences, suggesting that methylation of these sequences inhibits gene expression by interfering with the binding of AhR to the target sequences. To test this hypothesis, a novel method combining the modified chromatin immunoprecipitation of AhR-XRE complexes with subsequent DNA methylation analysis of the XRE regions targeted by activated AhR was applied to both liver cancer cell lines treated with ßNF. XRE2/XRE3 methylation was found to be exclusively observed in the input DNA from HepG2 cells but not in the precipitated AhR-bound DNA. Furthermore, sub-cloning and sequencing analysis revealed that the two XRE sites were unmethylated in the samples from the AhR-bound DNA even though the neighboring CpG sites were frequently methylated. To the best of our knowledge, the present study provides the first direct evidence that ligand-activated AhR preferentially binds to unmethylated XRE sequences in the context of natural chromatin. In addition, this approach can also be applied to assess the effects of DNA methylation on target sequence binding by transcription factors other than AhR.

Lactate released from human fibroblasts enhances Ni elution from Ni plate.

Elution of Ni ions from medical devices induces inflammation and toxicity. We previously reported that elution of Ni ions from Ni wires induced COX-2 expression and increased lactate production, but whether lactate is involved in the further elution of Ni ions remains unclear. In this study, using KMST-6, a human fibroblast cell line, we examined the molecular mechanisms by which Ni ions increase lactate release and the role of lactate in enhancing the elution of Ni ions. When KMST-6 cells were incubated on a Ni plate or stimulated with NiCl2 (1 mM), the expression of glucose transporter 1 (GLUT1), hexokinase 2 (HK2), and lactate dehydrogenase A (LDHA), and the release of lactate were enhanced. The NiCl2 (1 mM)-induced expression of these genes was inhibited by a hypoxia-inducible factor-1α (HIF-1α) inhibitor, PX-478 (10-25 µM). Stimulation of cells with a prolyl hydroxylase domain (PHD) inhibitor, roxadustat, increased the expression of these genes, lactate release, and elution of Ni ions at 10 µM. A monocarboxylate transporter-4 (MCT4) inhibitor, syrosingopine, inhibited lactate release from roxadustat-treated cells and reduced the elution of Ni ions by the cells at 10 µM. Finally, syrosingopine (10 µM) reduced the elution of Ni ions by the cells from the Ni plate. These results suggest that elution of Ni ions from metals promotes the production of lactate via HIF-1α-mediated gene expression and causes further Ni elution. Thus, Ni ions show a positive feedback mechanism of Ni elution, and this step may be potentially targeted to protect against metal elution from metal devices.

[Analysis of the Distribution of Community Pharmacies and Pharmacists in Miyagi Prefecture].

In Japan, the aging of the population is serious problem. The Ministry of Health, Labour and Welfare is constructing a new support system for elderly people called "Community-based integrated care system". In this system, community pharmacists are expected to play an important role as healthcare professionals for the whole community, including elderly people. Since pharmacists will be needed to manage community health in addition to their daily tasks, it is required to reassess the distribution of community pharmacies and pharmacists. In this study, we surveyed their distribution in Miyagi prefecture by using statistical data from public institutions and reevaluated the distribution to raise problems. Based on the numbers of community pharmacies and pharmacists per 1000 population in the whole Miyagi prefecture, each area was ranked to 2 categories and analyzed regarding population, aging rate and inhabitable land area. It was disclosed that the higher aging rate areas had the higher rate of category below the average of whole Miyagi prefecture, especially in the number of pharmacists. When the numbers of pharmacies and pharmacists per the inhabitable land area were used, the uneven distribution became clearer than when those per population were used. These findings suggested that it was important to characterize the areas by not only the ratios of community pharmacies and pharmacists to population numbers but also by the aging rates and inhabitable land area, which were related to the work efficiency of pharmacists and accessibility for resident to pharmacies.

Zinc ions have a potential to attenuate both Ni ion uptake and Ni ion-induced inflammation.

Nickel ions (Ni2+) are eluted from various metallic materials, such as medical devices implanted in human tissues. Previous studies have shown that Ni2+ enters inflammatory cells inducing inflammation. However, the regulation of Ni2+ uptake in cells has not yet been reported in detail. In the present study, we investigated the effects of various divalent cations on Ni2+ uptake and Ni2+-induced interleukin (IL)-8 production in the human monocytic cell line, THP-1. We demonstrated that ZnCl2, MnCl2, and CoCl2 inhibited the Ni2+ uptake, while CuCl2, FeCl2, MgCl2, and divalent metal transporter (DMT)-1 inhibitor, Chlorazol Black, did not. Furthermore, ZnCl2 inhibited Ni2+-induced IL-8 production, correlating with the inhibition of Ni2+ uptake. These results suggested that Ni2+ uptake occurred through Zn2+, Mn2+, and Co2+-sensitive transporters and that the inhibition of Ni2+ uptake resulted in the inhibition of IL-8 production. Furthermore, using an Ni wire-implanted mouse model, we found that Ni wire-induced expression of mouse macrophage inflammatory protein-2 (MIP-2) and cyclooxygenase-2 (COX-2) mRNA in the skin tissue surrounding the wire were enhanced by low Zn conditions. These results suggested that the physiological concentration of Zn2+ modulates Ni2+ uptake by inflammatory cells, and a Zn deficient state might increase sensitivity to Ni.

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.