Tsutomu Ogura

Expression of the inducible isoform of nitric oxide synthase in the central nervous system of mice correlates with the severity of actively induced experimental allergic encephalomyelitis.

A cytokine-mediated excessive increase in nitric oxide (NO) by macrophages or glial cells via an inducible isoform of NO synthase (iNOS) has been proposed to play an important role in demyelinating diseases. To further investigate the role of iNOS in demyelination, experimental allergic encephalomyelitis (EAE), a known animal model of multiple sclerosis (MS) in mice, was chosen in this study. A semiquantitative reverse transcriptase-polymerase chain reaction (RT/PCR) analysis revealed an increase in the mRNA levels of iNOS and cytokines known to induce iNOS or inflammatory cytokines (interleukin (IL)-1 alpha, IL-1 beta, IL-2, IL-6, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and TNF-beta) in the spinal cord corresponding to the severity of the disease without significant change in the mRNA levels of immunoregulatory cytokines (IL-4, IL-10 and transforming growth factor (TGF)-beta) during the course of EAE. An immunohistochemical examination of the spinal cord using an iNOS-specific antibody showed iNOS-positive cells to be mainly inflammatory cells with a higher frequency of iNOS-positive cells at the peak of EAE than in the early phase. These iNOS-positive cells at the peak appeared to be composed of infiltrating macrophages and most of them were located in the necrotic area. These results suggested that cytokine-induced excessive NO via iNOS by macrophages caused tissue damage in the central nervous system in EAE.

Induction of vascular endothelial growth factor by nitric oxide in human glioblastoma and hepatocellular carcinoma cells

We evaluated the effect of nitric oxide (NO) on vascular endothelial growth factor (VEGF) gene expression in human A-172 glioblastoma cells and human HepG2 hepatocellular carcinoma cells. The mRNA level of VEGF increased in response to S-Nitroso-N-acetly-D,L-penicillamine (SNAP) in both cell lines, and increased in mRNA level well coincided with VEGF protein production in A-172 cells. SNAP at 0.5 mM induced maximal stimulation of 4.4 and 3.7 kb VEGF mRNA expression after 6 h about 11 and 8 fold increase, respectively above control level. Similar VEGF mRNA accumulation was observed also with NOR3, another chemical NO generator. To evaluate the effect of SNAP on VEGF mRNA stability, half-lives of VEGF mRNA were measured in A-172 cells cultured with or without 0.5 mM SNAP and treated with actinomycin D (25 μg/ml). Half-life for VEGF mRNA was found to be prolonged about 2.4 fold by SNAP. VEGF expression induced by SNAP was inhibited by guanylate cyclase inhibitors, methylene blue (10 μM) and LY-83583 (1 μM), and by the protein synthesis inhibitor, cycloheximide (25 μg/ml). These results suggest that induction of VEGF gene expression by NO is mediated through guanylate cyclase activity and requires on-going protein synthesis.

ARK5 is a tumor invasion-associated factor downstream of Akt signaling

ABSTRACT AMP-activated protein kinases (AMPKs) are a class of serine/threonine protein kinases that are activated by an increase in intracellular AMP concentration. They are a sensitive indicator of cellular energy status and have been found to promote tumor cell survival during nutrient starvation. We recently identified a novel AMPK catalytic subunit family member, ARK5, whose activation is directly regulated by Akt, which, in turn, has been reported to be a key player in tumor malignancy. In this study, we attempted to determine whether ARK5 is involved in tumor malignancy under regulation by Akt. Matrigel invasion assays demonstrated that both overexpressed and endogenous ARK5 showed strong activity dependent on Akt. In addition, ARK5 expression induced activation of matrix metalloproteinase 2 (MMP-2) and MMP-9 following new expression of membrane type 1 MMP (MT1-MMP), and the MT1-MMP expression induced by ARK5 was initiated by rapamycin-sensitive signaling. In nude mice, ARK5 expression was associated with a significant increase in tumor growth and significant suppression of necrosis in tumor tissue. Interestingly, only the ARK5-overexpressing PANC-1 cell line (P/ARK) tumor showed invasion and metastasis in nude mice, although Akt was activated in tumors derived from both P/ARK and its parental cell line. We report that a novel AMPK catalytic subunit family member, ARK5, plays a key role in tumor malignancy downstream of Akt.

ARK5 suppresses the cell death induced by nutrient starvation and death receptors via inhibition of caspase 8 activation, but not by chemotherapeutic agents or UV irradiation

AMPK is a serine/threonine protein kinase family and we recently identified a novel member, ARK5. The activation of ARK5 is triggered by Akt, and ARK5 induces tumor cell survival during nutrient starvation. In the current study, we investigated the mechanisms of induction of cell survival by ARK5. Human hepatoma HepG2 cells undergo necrotic cell death within 24 h after the start of glucose starvation, and the cell death signaling has been found to be mediated by death-receptor-independent activation of caspase 8. When HepG2 cells were transfected with ARK5 expression vector and subjected to several cell death stimuli, ARK5 was found to suppress cell death by glucose starvation, TRAIL, and TNF-α, but not by ultraviolet irradiation, camptothecin, or doxorubicin. Western blotting analysis revealed that both TRAIL and glucose starvation induced Bid cleavage and FLIP degradation following caspase 8 activation in a time-dependent manner, and ARK5 overexpression clearly delayed Bid cleavage, FLIP degradation, and caspase 8 activation. On the basis of the results of this study, we report that cell survival induced by ARK5 is, at least in part, due to inhibition of caspase 8 activation.

Induction of matrix metalloproteinase gene transcription by nitric oxide and mechanisms of MMP-1 gene induction in human melanoma cell lines.

Expression of 12 matrix metalloproteinases (MMPs) after exposure of human melanoma cell lines C32TG and Mewo to nitric oxide (NO) was investigated by the reverse transcription‐polymerase chain reaction. Expression of the mRNA of MMP‐1, ‐3, ‐10 and ‐13 in C32TG cells was transcriptionally enhanced in a dose‐dependent manner by exposure to an NO donor, S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) and mRNA expression of MMP‐1 and ‐10 was similarly enhanced in Mewo cells. Exposure of C32TG cells to NO increased the MMP‐1 protein concentration in the culture medium. Testing with the luciferase gene fused to the 1.5 Kbp 5′‐flanking region of the human MMP‐1 gene showed that exposure to NO upregulated MMP‐1 promoter activity in C32TG cells. Endogenous NO production after introduction of inducible NO synthase cDNA also enhanced MMP‐1 promoter activity in C32TG cells. Deletion and mutational analysis identified a critical AP‐1 binding site required for NO regulation of MMP‐1. A neighboring Ets motif from the AP‐1 site in the promoter region acted as an accessory to enhance MMP‐1 expression. Electromobility shift analysis using the AP‐1 binding site showed that NO enhanced the AP‐1 binding ability of nuclear factors in C32TG cells. PD98059, a selective MEK inhibitor and SB202190, a p38 MAPK inhibitor, attenuated the MMP‐1 mRNA expression enhanced by NO. Thus, MMP‐1 was transcriptionally enhanced by NO via MAPK (ERK and p38) pathways. The results of our study suggest that the increased expression of MMPs in response to NO may be associated with tumor progression under inflammation.

Loss of Heterozygosity on Chromosomes 9q and 16p in Atypical Adenomatous Hyperplasia Concomitant with Adenocarcinoma of the Lung

Atypical adenomatous hyperplasia (AAH) has recently been implicated as a precursor to lung adenocarcinoma. We previously reported loss of heterozygosity (LOH) in tuberous sclerosis (TSC) gene-associated regions to frequently be observed in lung adenocarcinoma with multiple AAHs. In this study, we analyzed LOH in four microsatellite loci on 9q, including the TSC1 gene-associated region, and four loci on 16p, including the TSC2 gene-associated region, in both 18 AAHs and 17 concomitant lung adenocarcinomas from 11 patients. Seven of 18 (39%) AAHs and 9 of 17 (53%) adenocarcinomas displayed LOH on 9q. Five (28%) AAHs and seven (41%) adenocarcinomas harbored LOH at loci adjacent to the TSC1 gene. Four of 18 (22%) AAHs and 6 of 17 (35%) adenocarcinomas displayed LOH on 16p. One (6%) AAH and five (29%) adenocarcinomas harbored LOH at loci adjacent to the TSC2 gene. These findings may indicate a causal relationship of LOH on 9q and 16p in a fraction of AAH lesions and adenocarcinomas of the lung. Especially, the frequencies of LOH on 9q and at the TSC1 gene-associated region were high. The TSC1 gene or another neighboring tumor suppressor gene on 9q might be involved in an early stage of the pathogenesis of lung adenocarcinoma.

Expression of two types of nitric oxide synthase mRNA in human neuroblastoma cell lines.

Abstract: Expression of nitric oxide synthase (NOS) was studied in nine human neuroblastoma and two human glioblastoma cell lines. Neuronal NOS (n‐NOS) mRNA of ∼10 kb was detected in four of the nine neuroblastoma cell lines by northern blot analysis using human n‐NOS cDNA as a probe. Expression of the n‐NOS mRNA was also detected in another neuroblastoma cell line in a subsequent reverse transcriptase polymerase chain reaction (RT‐PCR) study, but no n‐NOS mRNA expression was observed in the other four neuroblastoma cell lines or in the glioblastoma cell lines. The level of NOS activity correlated well with that of n‐NOS mRNA expression in neuroblastoma cell lines expressing n‐NOS mRNA. Western blot analysis showed that the n‐NOS expressed in neuroblastoma cells was a 160‐kDa protein reacted with anti‐n‐NOS antibody. By using the RT‐PCR method, a short n‐NOS (n‐NOS‐2) mRNA with a 315‐bp inframe deletion from the entire n‐NOS (n‐NOS‐1) mRNA was detected in the human neuroblastoma cells. The structural diversity of human n‐NOS mRNA was demonstrated for the first time.

Regulation of caspase-6 and FLIP by the AMPK family member ARK5.

Colorectal cancer cells are unique in that they escape Fas-mediated cell death in the presence of Fas ligand, and we recently reported that AMP-activated protein kinase-related kinase 5 (ARK5) suppresses cell death signaling mediated by cell death receptor in Akt-dependent manner. In the current study, therefore, we examined whether ARK5 is involved in the escape from Fas-mediated cell death of colorectal cancer cells. Among 10 cell lines, ARK5 mRNA expression was observed in LoVo, SW480, and SW1116 cell lines. Interestingly, SW480 and SW1116 cell lines, but not LoVo cell line, showed expressions of both Fas ligand (FasL) and Fas mRNAs. SW620 cell line also showed FasL mRNA; however, Fas and ARK5 mRNAs were not detected. Furthermore, well-coincided expression among ARK5, FasL, and Fas mRNAs was observed in tumor tissues from patients with colorectal cancer, suggesting the suppression of FasL/Fas system-induced cell death by ARK5 in colorectal cancer cell lines. Intensive cell death, which was dependent on the FasL/Fas system was encountered when ARK5 antisense RNA (ARK5/AS) was introduced into SW480 cells. FLIP was expressed in only ARK5 mRNA-expressing cell lines, and ARK5/AS induced FLIP cleavage in a caspase-6-dependent manner. Amino-acid sequence analysis of caspase-6 revealed two putative sites of phosphorylation by ARK5 at Ser80 and Ser257. Although active caspase-6 overexpression induced cell death in SW480 and DLD-1 cell lines, SW480 cells, but not DLD-1 cells, exhibited strong resistance to procaspase-6 overexpression. Moreover, mutant caspase-6, in which the Ser257 was substituted by Ala (caspase-6/SA), induced cell death and FLIP degradation, even in SW480 cells. Active ARK5 was found to phosphorylate wild-type caspase-6 in vitro, but not caspase-6/SA, and the prevented activation of caspase-6 was promoted due to its phosphorylation by active ARK5 in vitro. On the basis of the results of this study, we propose that ARK5 negatively regulates procaspase-6 by phosphorylation at Ser257, leading to resistance to the FasL/Fas system.

Inducible Nitric Oxide Synthase in a Human Glioblastoma Cell Line

Abstract: Nitric oxide synthase (NOS) activity was induced in the cytosol of A‐172 cells by treatment with lipopolysaccharide, tumor necrosis factor‐α, and interferon‐γ. A 130‐kDa human inducible NOS (iNOS) protein was detected with anti‐rat iNOS antibody by western blot analysis. Northern blot analysis showed that the iNOS mRNA was ∼4.5 kb, using a cDNA fragment for human iNOS, isolated from stimulated A‐172 cells by reverse transcriptase‐PCR, as a probe. The mRNA was induced by interferon‐γ at a trace level, and its expression was synergistically enhanced by simultaneous addition of lipopolysaccharide, tumor necrosis factor‐α, and, to a larger extent, interleukin‐1β. The mRNA expression was blocked by coincubation with actinomycin D or cycloheximide. Furthermore, by transfecting the mouse iNOS gene promoter into A‐172 cells, transcriptional activation of the iNOS gene was detected in these cells upon stimulation with lipopolysaccharide and cytokines. The pattern of promoter activation correlated well with that of iNOS mRNA expression upon stimulation. These data indicate that expression of iNOS is transcriptionally regulated in A‐172 cells. This process requires de novo protein synthesis with a mechanism similar to that in place in mouse macrophages.

Prostaglandin D2 Inhibits Inducible Nitric Oxide Synthase Expression in Rat Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMCs) as well as macrophages have been shown to generate a substantial amount of NO in inflammatory vascular lesions. Prostaglandin (PG) D2 (PGD2) is produced by inflammatory cells, including mast cells and macrophages. We investigated whether PGD2 modulates NO metabolism in rat VSMCs. PGD2 at a concentration of 10(-7) mol/L or greater dose-dependently inhibited nitrite accumulation in the medium of cultured VSMCs stimulated with interleukin 1beta (IL-1beta). In a dose-response analysis of IL-1beta and nitrite accumulation, PGD2 was seen to decrease the maximal ability of VSMCs to generate NO, arguing against competition by PGD2 at cytokine receptors. Northern analysis showed that PGD2 suppresses induction of inducible NO synthase (iNOS) mRNA in IL-1beta-stimulated VSMCs, with consequent inhibition of iNOS protein expression in Western analysis. A thromboxane A2 (TXA2) analogue, U46619 (10(-5) mol/L), produced less inhibition of NO generation than did PGD2. Neither the PGI2 analog carbaprostacyclin nor PGE1 showed any inhibition. PGD2 dose-dependently inhibited NO generation despite the addition of the TXA2 antagonist SQ29548. PGJ2, delta12-PGJ2, and 15-deoxy-delta12,14-PGJ2, all metabolites of PGD2, were as potent as or slightly stronger than PGD2 in the inhibition of NO generation. These data suggest that PGD2 suppresses NO generation in VSMCs by inhibiting iNOS mRNA expression, most likely through the cascade of the PGJ2 series rather than through the TX receptor or cAMP upregulation. Such action makes it likely that PGD2 regulates NO metabolism in vascular lesions.