Fukushi Kambe

TNF-α Increases Expression of IL-6 and ICAM-1 Genes Through Activation of NF-κB in Osteoblast-like ROS17/2.8 Cells

Tumor necrosis factor‐α (TNF‐α) plays a key role in inflammatory diseases such as rheumatoid arthritis and in postmenopausal osteoporosis. In various tissues, TNF‐α action is mediated by a transcription factor, nuclear factor‐kappa B (NF‐κB). However, little is known about how TNF‐α exerts its action in osteoblasts. We thus examined the effect of TNF‐α on the activation of NF‐κB in rat osteoblast‐like osteosarcoma cells (ROS17/2.8). Electrophoretic mobility shift assay revealed that the activation of the p50‐p65 heterodimer NF‐κB was induced by TNF‐α as early as 15 minutes followed by a persistent activation for 48 h. When the binding activity of NF‐κB in cytosol was examined using detergents that dissociate NF‐κB from an inhibitory protein IκB, it decreased during the initial 30 minutes and then increased to the unstimulated level. Northern blot analysis revealed a marked increase in the mRNA levels of p105, a precursor of p50, 6 h after TNF‐α and a gradual increase in p65 mRNA levels during the initial 1 h. Significant increase in both mRNA levels continued until 24 h after TNF‐α. These results suggest that the rapid activation of NF‐κB by TNF‐α is mainly due to the nuclear translocation of NF‐κB pre‐existing in cytosol, and that the subsequent increase in the expression of p50 and p65 may result in the persistent activation of NF‐κB during TNF‐α stimulation. TNF‐α also increased the mRNA levels of interleukin‐6 (IL‐6) and intercellular adhesion molecule‐1 (ICAM‐1). An antioxidant, N‐acetyl‐L‐cysteine, significantly attenuated the TNF‐α–dependent increase in these mRNAs, and simultaneously reduced the activation of NF‐κB by TNF‐α, indicating that NF‐κB mediates the TNF‐α–dependent expression of IL‐6 and ICAM‐1 in ROS17/2.8 cells. These results suggest that the activation of NF‐κB by TNF‐α may play an important role in the production of cytokines and cell adhesion molecules from osteoblasts, leading to the promotion of bone resorption and inflammation.

3β-Hydroxysteroid-Δ24 Reductase Is a Hydrogen Peroxide Scavenger, Protecting Cells from Oxidative Stress-Induced Apoptosis

3beta-Hydroxysteroid-Delta24 reductase (DHCR24) is an endoplasmic reticulum-resident, multifunctional enzyme that possesses antiapoptotic and cholesterol-synthesizing activities. To clarify the molecular basis of the former activity, we investigated the effects of hydrogen peroxide (H(2)O(2)) on embryonic fibroblasts prepared from DHCR24-knockout mice (DHCR24(-/-) mouse embryonic fibroblasts). H(2)O(2) exposure rapidly induced apoptosis, which was associated with sustained activation of apoptosis signal-regulating kinase-1 and stress-activated protein kinases, such as p38 MAPK and c-Jun N-terminal kinase. Complementation of the mouse embryonic fibroblasts by adenovirus expressing DHCR24 attenuated the H(2)O(2)-induced kinase activation and apoptosis. Concomitantly, intracellular generation of reactive oxygen species (ROS) in response to H(2)O(2) was also diminished by the adenovirus, suggesting a ROS-scavenging activity of DHCR24. Such antiapoptotic effects of DHCR24 were duplicated in pheochromocytoma PC12 cells infected with adenovirus. In addition, it was found that DHCR24 exerted cytoprotective effects in the tunicamycin-induced endoplasmic reticulum stress by eliminating ROS. Finally, using in vitro-synthesized and purified proteins, DHCR24 and its C-terminal deletion mutant were found to exhibit high H(2)O(2)-scavenging activity, whereas the N-terminal deletion mutant lost such activity. These results demonstrate that DHCR24 can directly scavenge H(2)O(2), thereby protecting cells from oxidative stress-induced apoptosis.

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.

Activation of Transcriptionally Active Nuclear Factor-κB by Tumor Necrosis Factor-α and Its Inhibition by Antioxidants in Rat Thyroid FRTL-5 Cells1

Tumor necrosis factor-α (TNF-α) exerts pleiotropic effects on thyroid follicular cells. However, the intracellular signaling pathway for the TNF-α action has not been well elucidated. The present study examined the effects of TNF-α on the activation of nuclear factor-κ B (NF-κB) and on the expression of interleukin (IL)-6 gene in rat thyroid FRTL-5 cells. The treatment of the cells with TNF-α resulted in the nuclear translocation of p65-p50 heterodimer as well as p50-p50 homodimer NF-κBs. The treatment with the antioxidants 20 mm N-acetyl-L-cysteine (NAC) and 10 μm pyrrolidine dithiocarbamate (PDTC) inhibited the TNF-α-dependent activation of p65-p50 heterodimer but not the p50-p50 homodimer, indicating that generation of oxidants is required for the activation of the heterodimer NF-κB. When the plasmid containing the multimerized NF-κB sites upstream of a luciferase reporter gene was transfected into FRTL-5 cells, the treatment with NAC or PDTC prevented the TNF-α-dependent increase in the luciferase act...

Spatiotemporal expression of BDNF in the hippocampus induced by the continuous intracerebroventricular infusion of β-amyloid in rats

The beta-amyloid protein (Abeta) is the major component of senile plaques found in the brain in Alzheimers disease (AD). Its neurotoxic properties in vivo, however, are not well defined. Since the expression of neurotrophin genes is considered an important component of the intrinsic neuroprotective response to insults, we analyzed the gene expression of neurotrophins in the brains of rats which received a continuous infusion of Abeta-(1-42) into the cerebroventricle. Northern blot analysis revealed a significant increase in brain-derived neurotrophic factor (BDNF) expression in the hippocampus but no change in the cerebral cortices. The alteration peaked on days 3-7 and returned to the basal level on day 14 after the start of Abeta-(1-42) infusion. No significant changes in nerve growth factor or neurotrophin-3 mRNA expression were observed. The infusion of Abeta-(1-40) and (25-35) also triggered the expression of BDNF mRNA, whereas neither Abeta-(40-1) nor (1-16) had any effect. In situ hybridization histochemistry revealed that the expression mainly occurred in the hilus and granular layer of the dentate gyrus and to a lesser extent in the pyramidal neurons of the CA1 region. These results demonstrate that the continuous intracerebroventricular infusion of Abeta induces selective and spatiotemporal expression of BDNF mRNA in the hippocampus.

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.

Increased expression of aquaporin-3 in the epidermis of DHCR24 knockout mice.

Background  The DHCR24 (3β‐hydroxysterol‐Δ24 reductase) gene encodes an enzyme catalysing conversion of desmosterol to cholesterol. Desmosterolosis is an autosomal recessive disease due to mutation in the DHCR24 gene, with low cholesterol and high desmosterol levels. To understand the pathophysiology of this disease, we utilized DHCR24 knockout mice and reported that DHCR24−/− mice die soon after birth. Their skin was less wrinkled, shiny, and revealed features of lethal restrictive dermopathy associated with severe defects in epidermal maturation and barrier function.

Molecular cloning of prostaglandin EP3 receptors from canine sensory ganglia and their facilitatory action on bradykinin-induced mobilization of intracellular calcium

We previously demonstrated that the activation of prostaglandin E‐prostanoid‐3 (EP3) receptor sensitized the canine nociceptor response to bradykinin (BK). To elucidate the molecular mechanism for this sensitization, we cloned two cDNAs encoding EP3s with different C‐terminals, from canine dorsal root ganglia, and established the transformed cell lines stably expressing them. In both transformants, EP3 agonist did not increase intracellular cAMP levels, but it attenuated forskolin‐dependent cAMP accumulation in a pertussis toxin (PTX)‐sensitive manner and increased intracellular calcium levels in a PTX‐resistant manner, indicating that both EP3s can couple with Gi and Gq, but not with Gs proteins. As the nociceptor response to BK is mediated by BK B2 receptor, it was transfected into the transformants and the effects of EP3 agonist on BK‐dependent calcium mobilization were investigated. When BK was applied twice with a 6‐min interval, the second response was markedly attenuated. Pre‐treatment with EP3 agonist had no effect on the initial response, but restored the second response in a PTX‐sensitive manner. A protein kinase A inhibitor mimicked the effect of EP3 agonist. These results demonstrate that the activation of EP3 restores the response to BK by attenuating the desensitization of BK B2 receptor activity via Gi protein.

The POU-domain protein Oct-1 is widely expressed in adult rat organs

The cDNA encoding a rat Oct-1 POU-domain was cloned by the reverse transcription-polymerase chain reaction method and subsequently Oct-1 mRNA expression was investigated. Our results show that the POU-domain of Oct-1 has been highly conserved during vertebrate evolution and that Oct-1 mRNA is widely expressed in various organs of adult rat.

Interleukin 8 and Biocompatibility of Dialysis Membranes

To determine whether interleukin 8 (IL-8) can be used as an index of biocompatibility of dialysis membranes, the effects of hemodialysis (HD) on plasma IL-8 levels and the expression of IL-8 mRNA in peripheral blood mononuclear cell (PBMC) were compared among regenerated cellulose (RC), polyacrylonitrile (PAN) and polymethylmethacrylate (PMMA) dialyzers. HD using RC dialysers significantly increased plasma IL-8 levels and induced abundant expression of IL-8 mRNA in PBMC. HD using PMMA dialyzers also increased plasma IL-8 levels and induced slight expression of IL-8 mRNA. In contrast, HD using PAN dialyzers neither increased plasma IL-8 levels nor induced the expression of IL-8 mRNA. In vitro studies demonstrated that IL-8 was considerably adsorbed by PAN membranes and not at all by RC and PMMA. These studies indicate that plasma IL-8 level and expression of PBMC IL-8 mRNA can be used as indices of biocompatibility of dialysis membranes.