Hirohito Mitsuyama

Tumor Necrosis Factor α Induces Expression of Genes for Matrix Degradation in Human Chondrocyte-like HCS-2/8 Cells Through Activation of NF-κB: Abrogation of the Tumor Necrosis Factor α Effect by Proteasome Inhibitors

Tumor necrosis factor α (TNF‐α) has been suggested to induce chondrocytic chondrolysis in both inflammatory and degenerative joint diseases. However, its intracellular signaling pathway leading to the chondrolysis has not been studied in detail. Thus, we investigated whether TNF‐α activates a transcription factor nuclear factor κB (NF‐κB) in human chondrocyte‐like cells (HCS‐2/8) and induces the expression of genes involved in the degradation of cartilage matrix. Treatment of the cells with TNF‐α markedly increased the levels of matrix metalloproteinase 1 (MMP‐1), MMP‐3, intercellular adhesion molecule 1 (ICAM‐1), and cyclo‐oxygenase 2 (COX‐2) messenger RNAs (mRNAs). The increase in the mRNAs was associated with the activation of p65/p50 heterodimer NF‐κB. IκB‐α and IκB‐β, cytoplasmic molecules preventing the nuclear translocation of NF‐κB, were degraded rapidly by TNF‐α followed by their synthesis to the basal level. Treatment with proteasome inhibitors inhibited the degradation of both IκB‐α and IκB‐β and prevented the TNF‐α‐dependent nuclear translocation of p65. Furthermore, the inhibitors completely prevented the TNF‐α‐dependent induction of MMP‐1, MMP‐3, ICAM‐1, and COX‐2 mRNAs. Thus, it is shown that the activation of p65/p50 NF‐κB by TNF‐α plays a cardinal role in inducing the expression of MMP‐1, MMP‐3, ICAM‐1, and COX‐2 genes, which are involved in matrix degradation and inflammatory reaction in chondrocytes, leading to chondrocytic chondrolysis.

Inhibitory effects of cyclosporin A on calcium mobilization-dependent interleukin-8 expression and invasive potential of human glioblastoma U251MG cells.

Interleukin (IL)-8 produced from glioblastoma is suggested to contribute to its own proliferation and progression. Since various external stimuli have been shown to increase intracellular Ca2+ in glioma cells, we investigated Ca2+ mobilization-dependent IL-8 expression and effect of cyclosporin A (CsA), an inhibitor of calcineurin (Cn), on the expression and invasive potential of human glioblastoma U251MG cells. Combined treatment with Ca2+-ionophore and phorbol-myristate-acetate (A23187/PMA) increased IL-8 mRNA and protein levels. This increase was suppressed by CsA and by another Cn inhibitor FK506. Luciferase reporter gene assay and electrophoretic mobility shift assay revealed that activation of p65-containing nuclear factor-κB was essential for A23187/PMA-dependent activation of IL-8 promoter. CsA suppressed the promoter activity by attenuating IκB-α degradation. U251MG cells expressed IL-8 receptors CXCR-1 and -2, and Matrigel invasion assay revealed that CsA attenuated A23187/PMA-dependent stimulation of invasive potential, probably by inhibiting IL-8 production. In addition, IL-8-dependent proliferation was also suppressed by CsA. Taken together, these results demonstrate the novel inhibitory effects of CsA on glioblastoma cell functions, suggesting CsA as a potential therapeutic adjuvant for glioma treatment.

Cyclosporin A Enhances Interleukin-8 Expression by Inducing Activator Protein-1 in Human Aortic Smooth Muscle Cells

Objective—Cyclosporin A (CsA) and tacrolimus (FK506) are widely used as immunosuppressants. However, their use has been hampered by various adverse effects, such as acceleration of atherosclerosis. Interleukin (IL)-8, a chemotactic cytokine, plays an important role in pathogenesis of atherosclerosis. We thus investigated whether synthesis of IL-8 from primary human aortic smooth muscle cells is influenced by CsA and FK506. Methods and Results—Northern blot analysis revealed that CsA increased IL-8 mRNA level and enhanced its increase by epidermal growth factor or tumor necrosis factor-&agr;. In contrast, FK506 had no effect on the mRNA level. IL-8 accumulation in culture media was also increased by CsA. Stability of IL-8 mRNA was not affected by CsA, whereas luciferase reporter gene assay using the human IL-8 promoter revealed that CsA significantly augmented the promoter activity. Electrophoretic mobility shift assay showed that binding activity of activator protein (AP)-1 was increased by CsA, and introduction of a mutation into the AP-1 site in the promoter abolished its CsA-dependent activation. The increased AP-1 binding activity was accompanied by c-Fos synthesis. Conclusions—CsA stimulates synthesis of IL-8 via activation of AP-1 in human aortic smooth muscle cells, providing a novel aspect of biological effects of CsA on the cells.

Calcium Signaling Pathway Involving Calcineurin Regulates Interleukin‐8 Gene Expression Through Activation of NF‐κB in Human Osteoblast‐Like Cells

Involvement of aberrant IL‐8 production by osteoblasts was demonstrated in pathogenesis of inflammatory joint diseases. We thus investigated intracellular signaling pathways leading to IL‐8 expression in human osteoblast‐like HOS‐TE85 cells. It was demonstrated that Ca2+ signaling pathway involving calcineurin regulates IL‐8 gene expression through activation of a transcription factor, NF‐κB.

Analysis of Interieukin-8 Gene Promoter function in Human Osteoblast-like Cells : Regulation by Ca^ -signaling and Cyclosporin A

We previously reported that an increase in intracellular calcium by calcium ionophore (A23187) and 4β-phorbol-12β-myristate-13a-acetate (PMA) induced the expression of IL-8 mRNA in human osteoblast-like HOS-TE85 cells andthis induction was markedly suppressed by cyclosporin A (CsA). In this study we investigated whether the regulation by A23187, PMA and CsA was occured at a transcriptional level by reporter gene assays. A promoter region spanning from -1460 to +40 of IL-8 gene was PCR-amplified and inserted upstream of a luciferase reporter gene. Various deletion mutants were also constructed. Transfection of the plasmids into HOS-TE85 cells demonstrated that the nucleotides between -133 and -60 base pairs upstream of IL-8 gene are essential and sufficient for its induction by A23187/PMA, and suppression of this induction by CsA.

Effect of Epidermal Growth Factor and Cyclosporin A on InterIeukin-8 Gene Expression in Human Aortic Smooth Muscle Cells

It was reported that accelerated allograft atherosclerosis is associated with usage of cyclosporin A (CsA). Although the immunosuppressive actions of CsA are extensively studied, the mechanisms of this adverse effect remains to be elucidated. We thus studied whether CsA affects the expression of interleukin (IL)-8 in the primary culture of human aortic smooth muscle cells (HASMC) since IL-8 recently has been shown to be a mitogen and a chemoattractant for vascular smooth muscle cells (VSMC). We also studied the effect of epidermal growth factor (EGF) on IL-8 expression because of its potent action of VSMC proliferation in the atherosclerotic lesion. Treatment of HASMC with CsA alone resulted in a significant increase in the IL-8 mRNA level. EGF also increased the mRNA level. CsA together with EGF further increased the IL-8 mRNA level. These results suggested that the CsA-dependent increase in IL-8 in VSMC could be one of the mechanisms for its induced accelerated allograft atherosclerosis.