Kenji Akita

Involvement of Caspase-1 and Caspase-3 in the Production and Processing of Mature Human Interleukin 18 in Monocytic THP.1 Cells

Recently, human interleukin 18 (hIL-18) cDNA was cloned, and the recombinant protein with a tentatively assigned NH2-terminal amino acid sequence was generated. However, natural hIL-18 has not yet been isolated, and its cellular processing is therefore still unclear. To clarify this, we purified natural hIL-18 from the cytosolic extract of monocytic THP.1 cells. Natural hIL-18 exhibited a molecular mass of 18.2 kDa, and the NH2-terminal amino acid was Tyr37. Biological activities of the purified protein were identical to those of recombinant hIL-18 with respect to the enhancement of natural killer cell cytotoxicity and interferon-γ production by human peripheral blood mononuclear cells. We also found two precursor hIL-18 (prohIL-18)-processing activities in the cytosol of THP.1 cells. These activities were blocked separately by the caspase inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO. Further analyses of the partially purified enzymes revealed that one is caspase-1, which cleaves prohIL-18 at the Asp36-Tyr37 site to generate the mature hIL-18, and the other is caspase-3, which cleaves both precursor and mature hIL-18 at Asp71-Ser72 and Asp76-Asn77 to generate biologically inactive products. These results suggest that the production and processing of natural hIL-18 are regulated by two processing enzymes, caspase-1 and caspase-3, in THP.1 cells.

Cloning and expression of interleukin-18 binding protein

Interleukin‐18 binding protein is a novel glycoprotein that we successfully cloned and expressed. First, murine interleukin‐18 binding protein was purified from the sera of mice with endotoxin shock using ligand affinity chromatography. The murine interleukin‐18 binding protein cDNA was cloned after RT‐PCR using mixed primer pair sequences based on partial murine interleukin‐18 binding protein amino acid sequence analysis. Subsequently, human interleukin‐18 binding protein cDNA was cloned from cDNA libraries of normal human liver using murine interleukin‐18 binding protein cDNA as a probe. Next, we transiently expressed recombinant human and murine interleukin‐18 binding proteins in COS‐1 cells and purified them from culture supernatants. Both recombinant interleukin‐18 binding proteins did not exhibit species specificity and prevented interleukin‐18 binding to its receptor. In addition, they inhibited interleukine‐18 dependent IFN‐γ production from KG‐1 cells effectively. These results suggest that the interleukin‐18 binding protein may possess interleukine‐18 antagonist activity.

INTRACELLULAR PRODUCTION OF INTERLEUKIN-18 IN HUMAN EPITHELIAL-LIKE CELL LINES IS ENHANCED BY HYPEROSMOTIC STRESS IN VITRO

Abstract Interleukin-18 is a novel multifunctional cytokine, which enhances natural killer cell activity and promotes the induction of cytokine production, including that of interferon-γ by T cells and antitumor effects. Interleukin-18 is produced by cells of several different tissues (e.g., macrophages, keratinocytes, osteoblasts, and intestinal epithelium); however, it is unclear what physiological conditions or stimuli induce interleukin-18 production. To determine physiological conditions for the production of interleukin-18, we have examined the effect of mannitol-induced hyperosmotic conditions on normal human umbilical vein endothelial cells (HUVEC) and eight established human epithelial-like cell lines (Intestine 407, Caco-2, A253, HeLa, SCC25, HT1197, ACHN, A549). Hyperosmotic conditions induced interleukin-18 immunoreactivity in all the human cell lines tested, as detected by immunocytochemistry. The enhanced interleukin-18 production was also observed when mannitol was replaced with NaCl as the inducer of hyperosmotic stress. Enzyme-linked immunosorbent assays revealed that interleukin-18 concentrations in cell extracts were significantly increased by hyperosmotic conditions. Reporter gene assays also revealed that hyperosmotic conditions stimulated transcriptional activity of the interleukin-18 promoter. These results show for the first time that hyperosmotic stress is a stimulator of interleukin-18 production in epithelial-like cells.

Neurotrophic Effects of a Cyanine Dye via the PI3K-Akt Pathway: Attenuation of Motor Discoordination and Neurodegeneration in an Ataxic Animal Model

Background Neurotrophic factors may be future therapeutic agents for neurodegenerative disease. In the screening of biologically active molecules for neurotrophic potency, we found that a photosensitizing cyanine dye, NK-4, had remarkable neurotrophic activities and was a potent radical scavenger. Methodology/Principal Findings In this study, we evaluated the effect of NK-4 on the protection of neurons against oxidative damage and investigated the associated intracellular signaling pathways. Subsequently, we evaluated the effect of NK-4 in an animal model of neurodegeneration. In vitro, NK-4 showed dose-dependent protection of PC12 cells from toxicity induced by oxidative stress caused by hydrogen peroxide (H2O2) or 6-hydroxydopamine (6-OHDA). Comparison of extracellular signal-regulated kinase signaling pathways between treatment with NK-4 and nerve growth factor (NGF) using K252a, an inhibitor of the NGF receptor TrkA, revealed that NK-4 activity occurs independently of NGF receptors. LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocked the protective effect of NK-4, and NK-4 caused activation of Akt/protein kinase B, a downstream effector of PI3K. These results suggest that the neuroprotective effects of NK-4 are mediated by the PI3K-Akt signaling pathway. NK-4 treatment also attenuated stress-induced activation of SAPK/JNK, which suggests that NK-4 activates a survival signaling pathway and inhibits stress-activated apoptotic pathways independently of the TrkA receptor in neuronal cells. In vivo, administration of NK-4 improved motor coordination in genetic ataxic hamsters, as assessed by rota-rod testing. Histological analysis showed that cerebellar atrophy was significantly attenuated by NK-4 treatment. Notably, the Purkinje cell count in the treated group was threefold higher than that in the vehicle group. Conclusions/Significance These results suggest that NK-4 is a potential agent for therapy for neurodegenerative disorders based on the activation of survival signaling pathways.

Effects of NK-4 in a Transgenic Mouse Model of Alzheimer's Disease

Beta-amyloid (Aβ) peptides are considered to play a major role in the pathogenesis of Alzheimers disease (AD) and molecules that can prevent pathways of Aβ toxicity may be potential therapeutic agents for treatment of AD. We have previously reported that NK-4, a cyanine photosensitizing dye, displays neurotrophic and antioxidant activities. In this study, we report the effects of NK-4 on the toxicity of Aβ and on cognitive function and Aβ concentration in a transgenic mouse model of AD (Tg2576). In vitro, NK-4 effectively protected neuronal cells from toxicity induced by Aβ. In addition, it displayed profound inhibitory activities on Aβ fibril formation. In vivo, Tg2576 mice received an intraperitoneal injection at 100 or 500 µg/kg of NK-4 once a day, five times a week for 9 months. Administration of NK-4 to the mice attenuated impairment of recognition memory, associative memory, and learning ability, as assessed by a novel object recognition test, a passive avoidance test, and a water maze test, respectively. NK-4 decreased the brain Aβ concentration while increasing the plasma amyloid level in a dose-dependent manner. NK-4 also improved memory impairments of ICR mice induced by direct intracerebroventricular administration of Aβ. These lines of evidence suggest that NK-4 may affect multiple pathways of amyloid pathogenesis and could be useful for treatment of AD.

SUPPRESSED NNA1 GENE EXPRESSION IN THE BRAIN OF ATAXIC SYRIAN HAMSTERS

Ataxic Syrian hamsters with an autosomal recessive trait were analyzed. Homozygotes showed moderate ataxia beginning at seven to eight weeks of age. They were fertile and lived more than two years. The affected hamsters exhibited an adult-onset degeneration of cerebellar Purkinje neurons, followed by a slow, mild reduction in the density of granule cells. Northern hybridization demonstrated that expression of Nna1, the gene responsible for the Purkinje cell degeneration (pcd) phenotype, was almost negligible in the brain of homozygous hamsters. These results strongly suggest that pcd-type mutation is involved in the ataxic phenotype of mutant hamsters.

Effects of NK-4, a Cyanine Dye with Antioxidant Activities: Attenuation of Neuronal Deficits in Animal Models of Oxidative Stress-Mediated Brain Ischemia and Neurodegenerative Diseases

Cyanine photosensitizing dyes have been studied for over 150 years and continue to be of interest in their biology and medicine. They have been shown to possess various biological activities, including antimicrobial, antioxidant, macrophage activating, and oxidative phosphorylation uncoupling activities (Hayami, 1984; Ushio et al., 2009; Ishihara & Fujisawa, 2007; Kunikata et al., 2002; Takeguchi et al., 1985). Some dyes have been used as immunomodulators to treat allergy and rheumatoid arthritis, cancer, and to promote wound healing (Trauner et al., 1998; Motoyoshi et al., 1991). Since cumulative evidence suggests an involvement of oxidative stress and neuroinflammation as the common feature in the pathogenesis of neurodegenerative disorders (Amor et al., 2010; Wolozin & Behl, 2000a, 2000b), it would be reasonable to expect that cyanine dyes with anti-oxidative and antiinflammatory properties could protect the central nervous system from neuroinflammatoryrelated brain insults.

Anti-neurodegenerative activity of photosensitizing dyes (3): The effect on a hereditary cerebellar ataxia

Dehydroevodiamine (DHED), derived from Evodia rutaecarpa, was synthesized and it was previously reported that DHED has anti-dementic activities through various tests. In the present study, it was investigated whether DHED could attenuate cognitive deficits and A proudction in Tg2576 mice. We used memory tests, A 40 and A 42 ELISA kit, and western blotting to confirm total A proteins. The cognitive impairments in Tg2576 mice were significantly improved by DHED treatment. Interestingly, DHED significantly lowered the soluble A 40 and A 42 levels and total A proteins in the cerebral cortex of Tg2576 mice. These results show that DHED might attenuate cognitive dysfunction through blocking production of A . Thus DHED might be one of the potential therapeutic candidates for Alzheimer’s disease.

Anti-neurodegenerative activity of photosensitizing dyes (2): The effect on transient focal cerebral ischemia in rats

Dehydroevodiamine (DHED), derived from Evodia rutaecarpa, was synthesized and it was previously reported that DHED has anti-dementic activities through various tests. In the present study, it was investigated whether DHED could attenuate cognitive deficits and A proudction in Tg2576 mice. We used memory tests, A 40 and A 42 ELISA kit, and western blotting to confirm total A proteins. The cognitive impairments in Tg2576 mice were significantly improved by DHED treatment. Interestingly, DHED significantly lowered the soluble A 40 and A 42 levels and total A proteins in the cerebral cortex of Tg2576 mice. These results show that DHED might attenuate cognitive dysfunction through blocking production of A . Thus DHED might be one of the potential therapeutic candidates for Alzheimer’s disease.

Anti-neurodegenerative activity of photosensitizing dyes (1): The neurotrophic effects of photosensitizing dye and potential mechanisms of action

Dehydroevodiamine (DHED), derived from Evodia rutaecarpa, was synthesized and it was previously reported that DHED has anti-dementic activities through various tests. In the present study, it was investigated whether DHED could attenuate cognitive deficits and A proudction in Tg2576 mice. We used memory tests, A 40 and A 42 ELISA kit, and western blotting to confirm total A proteins. The cognitive impairments in Tg2576 mice were significantly improved by DHED treatment. Interestingly, DHED significantly lowered the soluble A 40 and A 42 levels and total A proteins in the cerebral cortex of Tg2576 mice. These results show that DHED might attenuate cognitive dysfunction through blocking production of A . Thus DHED might be one of the potential therapeutic candidates for Alzheimer’s disease.