Tamotsu Ishizuka

Comparison of Intrinsic Activities of the Putative Sphingosine 1-Phosphate Receptor Subtypes to Regulate Several Signaling Pathways in Their cDNA-transfected Chinese Hamster Ovary Cells

We examined the actions of sphingosine 1-phosphate (S1P) on signaling pathways in Chinese hamster ovary cells transfected with putative S1P receptor subtypes, i.e.Edg-1, AGR16/H218 (Edg-5), and Edg-3. Among these receptor-transfected cells, there was no significant difference in the expressing numbers of the S1P receptors and their affinities to S1P, which were estimated by [3H]S1P binding to the cells. In vector-transfected cells, S1P slightly increased cytosolic Ca2+ concentration ([Ca2+] i ) in association with inositol phosphate production, reflecting phospholipase C activation; the S1P-induced actions were markedly enhanced in the Edg-3-transfected cells and moderately so in the AGR16-transfected cells. In comparison with vector-transfected cells, the S1P-induced [Ca2+] i increase was also slightly enhanced in the Edg-1-transfected cells. In all cases, the inositol phosphate and Ca2+ responses to S1P were partially inhibited by pertussis toxin (PTX). S1P also significantly increased cAMP content in a PTX-insensitive manner in all the transfected cells; the rank order of their intrinsic activity of S1P receptor subtypes was AGR16 > Edg-3 > Edg-1. In the presence of forskolin, however, S1P significantly inhibited cAMP accumulation at a lower concentration (1–100 nm) of S1P in a manner sensitive to PTX in the Edg-1-transfected cells but not in either the Edg-3 or AGR16-transfected cells. As for cell migration activity evaluated by cell number across the filter of blind Boyden chamber, Edg-1 and Edg-3 were equally potent, but AGR16 was ineffective. Thus, S1P receptors may couple to both PTX-sensitive and -insensitive G-proteins, resulting in the selective regulation of the phospholipase C-Ca2+ system, adenylyl cyclase-cAMP system, and cell migration activity, according to the receptor subtype.

Role of Scavenger Receptor Class B Type I and Sphingosine 1-Phosphate Receptors in High Density Lipoprotein-induced Inhibition of Adhesion Molecule Expression in Endothelial Cells

We characterized the molecular mechanisms by which high density lipoprotein (HDL) inhibits the expression of adhesion molecules, including vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, induced by sphingosine 1-phosphate (S1P) and tumor necrosis factor (TNF) α in endothelial cells. HDL inhibited S1P-induced nuclear factor κB activation and adhesion molecule expression in human umbilical vein endothelial cells. The inhibitory HDL actions were associated with nitric-oxide synthase (NOS) activation and were reversed by inhibitors for phosphatidylinositol 3-kinase and NOS. The HDL-induced inhibitory actions were also attenuated by the down-regulation of scavenger receptor class B type I (SR-BI) and its associated protein PDZK1. When TNFα was used as a stimulant, the HDL-induced NOS activation and the inhibitory action on adhesion molecule expression were, in part, attenuated by the down-regulation of the expression of S1P receptors, especially S1P1, in addition to SR-BI. Reconstituted HDL composed mainly of apolipoprotein A-I and phosphatidylcholine mimicked the SR-BI-sensitive part of HDL-induced actions. Down-regulation of S1P3 receptors severely suppressed the stimulatory actions of S1P. Although Gi/o proteins may play roles in either stimulatory or inhibitory S1P actions, as judged from pertussis toxin sensitivity, the coupling of S1P3 receptors to G12/13 proteins may be critical to distinguish the stimulatory pathways from the inhibitory ones. In conclusion, even though S1P alone stimulates adhesion molecule expression, HDL overcomes S1P3 receptor-mediated stimulatory actions through SR-BI/PDZK1-mediated signaling pathways involving phosphatidylinositol 3-kinase and NOS. In addition, the S1P component of HDL plays a role in the inhibition of TNFα-induced actions through S1P receptors, especially S1P1.

Prognostic significance of L-type amino acid transporter 1 expression in resectable stage I-III nonsmall cell lung cancer

The clinical significance of L-type amino acid transporter 1 (LAT1) expression remains unclear, whereas many experimental studies have demonstrated that LAT1 is associated with the proliferation of cancer cells. The purpose of this study was to evaluate the prognostic value of LAT1 in patients with nonsmall cell lung cancer (NSCLC). A total of 321 consecutive patients with completely resected pathologic stage I–III NSCLC were retrospectively reviewed. Expression of LAT1 and proliferative activity, as determined by the Ki-67 labelling index, was also evaluated immunohistochemically and correlated with the prognosis of patients who underwent complete resection of the tumour. Expression of LAT1 was positive in 163 patients (51%) (29% of adenocaricnoma (58 of 200 patients), 91% of squamous cell carcinoma (91 of 100 patients), and 67% of large cell carcinoma (14 of 21 patients)). The 5-year survival rate of LAT1-positive patients (51.8%) was significantly worse than that of LAT1-negative patients (87.8%; P<0.001). L-type amino acid transporter 1 expression was significantly associated with lymph node metastasis and disease stage. Multivariate analysis confirmed that positive expression of LAT1 was an independent factor for predicting a poor prognosis. There was a significant correlation between LAT1 expression and Ki-67 labelling index. LAT1 expression is a promising pathological factor to predict the prognosis in patients with resectable stage I–III NSCLC.

c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production.

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.

A major role for the Rho‐associated coiled coil forming protein kinase in G‐protein‐mediated Ca2+ sensitization through inhibition of myosin phosphatase in rabbit trachea

G protein‐mediated Ca2+ sensitization of airway smooth muscle contraction was investigated with respect to the relative importance of Rho‐associated coiled coil forming protein kinase (ROCK) and protein kinase C (PKC). We examined the effects of Y‐27632, a ROCK inhibitor, and GF 109203X, a PKC inhibitor, on guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS)‐induced contraction in α‐toxin‐ or β‐escin‐permeabilized rabbit trachea. Although pre‐treatment with Y‐27632 dose‐dependently inhibited GTPγS (10 μM)‐induced Ca2+ sensitization of α‐toxin‐permeabilized trachea, a Y‐27632‐insensitive component (approximately 16% of the maximum contraction) was retained during the early phase of the GTPγS response in the presence of Y‐27632 (100 μM). GF 109203X (5 μM) abolished 1 μM 4β‐phorbol 12, 13‐dibutyrate (PDBu)‐induced, but only partially inhibited the GTPγS‐induced Ca2+ sensitization. A combination of Y‐27632 (100 μM) and GF 109203X (5 μM) totally abolished the GTPγS response. GTPγS caused only a small contraction in the absence of Ca2+. Wortmannin (30 μM), a myosin light chain kinase (MLCK) inhibitor, completely inhibited Ca2+‐induced contraction. ATP‐triggered contraction of the strip which had been treated with calyculin A (1 μM), a phosphatase inhibitor, in rigor solutions was markedly slowed by worthmannin (30 μM), but not by Y‐27632 (100 μM), in the presence of GTPγS and Ca2+. GTPγS, but not PDBu, contracted the β‐escin‐permeabilized trachea in the absence of Ca2+, but the presence of Ca2+‐independent MLCK. We conclude that ROCK plays a primary role in G‐protein‐mediated Ca2+ sensitization, which requires MLCK activity, with minor contribution of PKC to the early phase of contraction, and PDBu utilizes conventional PKC(s) in airway smooth muscle.

MEKK2 gene disruption causes loss of cytokine production in response to IgE and c‐Kit ligand stimulation of ES cell‐derived mast cells

Ligation of the high‐affinity IgE receptor (FcϵRI) or of c‐Kit stimulates cytokine production in mast cells. We show that MEK kinase 2 (MEKK2), a MAPK kinase kinase (MAP3K) that regulates the JNK and ERK5 pathways, is required for cytokine production in embryonic stem (ES) cell‐derived mast cells (ESMC). Targeted disruption of the MEKK2 or MEKK1 gene was used to abolish expression of the respective kinases in ESMC. Transcription of specific cytokines in response to IgE or c‐Kit ligand was markedly reduced in MEKK2−/− ESMC relative to wild‐type ESMC. Cytokine production in MEKK1−/− ESMC was similar to that of wild‐type ESMC, demonstrating the specificity of MEKK2 in signaling cytokine gene regulation. MEKK2−/− ESMC also lost receptor‐mediated stimulation of JNK. In contrast, JNK activation in response to UV irradiation was normal, showing that MEKK2 is required for receptor signaling but not for cellular stress responses. MEKK2 is the first MAP3K shown to be required for mast cell tyrosine kinase receptor signaling controlling cytokine gene expression.

l-type amino acid transporter 1 and CD98 expression in primary and metastatic sites of human neoplasms.

The significance of l‐type amino acid transporter (LAT) 1 expression remains unclear in the metastatic process of human neoplasms, whereas experimental studies have demonstrated that LAT1 is associated with the metastatic process of cancer cells. We compared the immunohistochemical expression of LAT1 and CD98 between the primary site and a concordant pulmonary metastatic site in 93 cancer patients, all of whom had undergone thoracotomy. LAT1, CD98, Ki‐67 labeling index, vascular endothelial growth factor (VEGF), CD31, and CD34 were analyzed by immunohistochemical staining in the resected tumors of 93 cancer patients: 45 colon cancers; nine breast cancers; eight head and neck cancers; 11 genital cancers; 14 soft‐tissue sarcomas; and six other cancers. The expression of these markers was significantly higher in the metastatic sites than in the primary sites. In total, the positive rates of LAT1, CD98, Ki‐67, VEGF, CD31, and CD34 were 40, 24, 56, 41, 45, and 39%, respectively, in the primary sites and 65, 45, 84, 67, 73, and 61%, respectively, in the metastatic sites. LAT1 expression was closely correlated with CD98 expression, angiogenesis, and cell proliferation. The association between LAT1 and CD98 expression was strongest in the primary and metastatic sites. The present study suggests that overexpression of LAT1 and CD98 has an important role to play in the metastatic process of variable human neoplasms. Moreover, LAT1 expression was significantly correlated with cell proliferation and angiogenesis. (Cancer Sci 2008; 99: 2380–2386)

Fluorine-18-α-Methyltyrosine Positron Emission Tomography for Diagnosis and Staging of Lung Cancer: A Clinicopathologic Study

Purpose:l-[3-18F]-α-Methyltyrosine ([18F]FMT) is an amino acid tracer for positron emission tomography (PET). We evaluated the diagnostic usefulness of [18F]FMT PET in non–small-cell lung cancer (NSCLC) patients. Tumor uptake of [18F]FMT was compared with that of 2-[18F]-fluoro-2-deoxy-d-glucose ([18F]FDG) and correlated with L-type amino acid transporter 1 (LAT1) expression. Experimental Design: Fifty NSCLC patients were enrolled in this study, and a pair of PET study with [18F]FMT and [18F]FDG was done. LAT1 expression and Ki-67 labeling index of the resected tumors were analyzed by immunohistochemical staining. Results: For the primary tumor detection, [18F]FMT PET exhibited a sensitivity of 90% whereas the sensitivity for [18F]FDG PET was 94%. For lymph node staging, the sensitivity and specificity of [18F]FMT PET were 57.8% and 100%, and those of [18F]FDG PET were 65.7% and 91%, respectively. The expression of LAT1 in squamous cell carcinoma and large cell carcinoma was significantly higher than that in adenocarcinoma. [18F]FMT uptake was also higher in squamous cell carcinoma and large cell carcinoma than in adenocarcinoma. Uptake of [18F]FMT in the tumor is closely correlated with LAT1 expression (ρ = 0.890). Conclusion: [18F]FMT PET had no false-positives in the detection of primary tumor and lymph node metastasis and could improve the diagnostic performance in NSCLC. Uptake of [18F]FMT correlated with the expression of LAT1 that showed a significant association with cellular proliferation.

Aggregation of the FcϵRI on Mast Cells Stimulates c-Jun Amino-terminal Kinase Activity A RESPONSE INHIBITED BY WORTMANNIN

Aggregation of the high-affinity Fc receptors for immunoglobulin E (IgE) (FcεRI) on the surface of mast cells initiates intracellular signal transduction pathways including the tyrosine phosphorylation of cellular proteins, phosphoinositide hydrolysis, an increase in intracellular calcium, and protein kinase C activation. These signals are believed to be involved in the exocytic release of inflammatory mediators such as vasoactive amines, cytokines, and lipid metabolites. However, the downstream consequences of these early activation events are not well defined. One exception is the activation of the extracellular signal-regulated kinases/mitogen-activated protein kinases. One member of the mitogen-activated protein kinase superfamily, designated c-Jun amino-terminal kinase (JNK), has been recently identified. JNK is activated following dual phosphorylation at a Thr-Pro-Tyr motif in response to diverse stimuli including tumor necrosis factor-α, heat shock, or ultraviolet irradiation. We found that JNK was strongly activated by antigen cross-linking in a mouse mast cell line passively sensitized with ovalbumin-specific IgE. Anti-mouse IgE antibody also activated JNK. MEK kinase 1 (MEKK1) which activates the JNK activator, JNK kinase (JNKK), was similarly activated by antigen stimulation. JNK but not p42erk2 activation induced by antigen was significantly inhibited in the presence of wortmannin, a known inhibitor of phosphatidylinositol 3-kinase. These results indicate that in response to the aggregation of FcεRI on mast cells, phosphatidylinositol 3-kinase activation is involved in the stimulation of the MEKK1, JNKK, JNK pathway.

Glutathione redox regulates lipopolysaccharide-induced IL-12 production through p38 mitogen-activated protein kinase activation in human monocytes: role of glutathione redox in IFN-γ priming of IL-12 production

We examined whether changes in intracellular reduced (GSH) or oxidized (GSSG) glutathione of human monocytes regulate lipopolysaccharide (LPS)‐induced IL‐12 production and defined the molecular mechanism that underlies glutathione redox regulation. Monocytes exposed to glutathione reduced form ethyl ester (GSH‐OEt) or maleic acid diethyl ester (DEM) increased or decreased the intracellular GSH/GSSG ratio, respectively. LPS‐induced IL‐12 production and p38 mitogen‐activated protein (MAP) kinase activation were enhanced by GSH‐OEt but suppressed by DEM. Selective p38 inhibitors showed that p38 promoted GSH‐OEt‐enhanced IL‐12 production. Furthermore, IFN‐γ priming increased the GSH/GSSG ratio and enhanced IL‐12 production through p38, and DEM negated the priming effect of IFN‐γ on p38 activation and IL‐12 production as well as on the GSH/GSSG ratio. These findings reveal that glutathione redox regulates LPS‐induced IL‐12 production from monocytes through p38 MAP kinase activation and that the priming effect of IFN‐γ on IL‐12 production is partly a result of the glutathione redox balance.