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%; Po0.001). 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.

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 [ 3 H]S1P binding to the cells. In vector-transfected cells, S1P slightly increased cytosolic Ca 2؉ concentration ([Ca 2؉ ] 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 [Ca 2؉ ] i increase was also slightly enhanced in the Edg-1-transfected cells. In all cases, the inositol phosphate and Ca 2؉ 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-Ca 2؉ system, adenylyl cyclase-cAMP system, and cell migration activity, according to the receptor subtype.Sphingosine 1-phosphate (S1P), 1 one of the sphingolipid metabolites, has recently been suggested to affect a variety of cellular processes (1, 2). These cellular responses elicited by S1P have first been ascribed to the intracellular action of the lipid, because S1P accumulated in the cells in response to some kinds of cytokines, and moreover, S1P induced Ca 2ϩ mobilization in a cell-free system (3-5). On the other hand, these S1P-induced responses are also accompanied by the stimulation of several early signaling events that are usually regulated by cell-surface receptors. These signaling events include activation of PLC (6 -9), an increase in [Ca 2ϩ ] i (10 -12), regulation of adenylyl cyclase (6, 9, 10, 13), and Rho activation (14, 15). The presence of the latter mechanism has been supported by the recent identification of several cDNAs encoding G-protein-coupled receptors for S1P, i.e. Edg-1, AGR16/H218, and .The transfection experiments of these S1P receptor subtypes demonstrated that these putative S1P receptors can actually couple to multiple signaling pathways. Thus, the previous transfection experiments suggest the involvement of these putative S1P receptor subtypes in the regulation of multiple signal...

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) ␣ in endothelial cells. HDL inhibited S1P-induced nuclear factor B 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 TNF␣ 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 S1P 1 , 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 S1P 3 receptors severely suppressed the stimulatory actions of S1P. Although G i/o proteins may play roles in either stimulatory or inhibitory S1P actions, as judged from pertussis toxin sensitivity, the coupling of S1P 3 receptors to G 12/13 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 S1P 3 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 TNF␣-induced actions through S1P receptors, especially S1P 1 .The plasma level of HDL 2 has been shown to be inversely correlated with the risk of atherosclerosis and associated cardiovascular disease (1, 2). HDL can remove excess cholesterol from arterial and nonliver cells, transport it to the liver, and excrete it as bile acids. The so-called reverse cholesterol transport is thought to be an important anti-atherogenic action of HDL (1, 2). In recent studies, however, HDL has been shown to exert a variety of actions that are independent of cholesterol metabolism. For example, HDL inhibits LDL oxidation, smooth muscle cell migration, platelet aggregation, and endothelial dysfunction (3, 4). The inhibition of endothelial dysfunction may be achieved by several responses to HDL, including the stimulation of proliferation, cell survival, migration, and NO synthesis, or the inhibition of apoptosis and of the expression of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) (3-5). An increase in the expression of the adhesion molecules stimulates monocyte interaction with endothelial cells and cell penetration into subendothelial space or the intima of arterial walls. Thus, the expr...

Extracellular acidification inhibited LPS-induced TNF-α protein production, which was associated with an inhibition of TNF-α mRNA expression, in mouse peritoneal macrophages. The LPS-induced cytokine production was also inhibited by Gs protein-coupled receptor agonists prostaglandin E1 and isoproterenol. Among OGR1 family proton-sensing GTP-binding regulatory protein-coupled receptors, TDAG8, OGR1, and G2A are expressed in the cells. The inhibitory action by acidic pH on TNF-α production was significantly attenuated in macrophages from TDAG8Tp/Tp mice but not in those from OGR1geo/geo mice. Moreover, small interfering RNA specific to TDAG8, but not to G2A, clearly attenuated the acidification-induced inhibition of TNF-α production. On the other hand, the down-regulation or deficiency of TDAG8 hardly affected prostaglandin E1- or isoproterenol-induced actions. LPS-induced IL-6 production was also inhibited by extracellular acidification in a manner that was sensitive to TDAG8 expression. The acidic pH-induced inhibitory action on the cytokine production was significantly reversed either by a small interfering RNA specific to Gs proteins or by a protein kinase A (PKA)-specific inhibitor H89. Indeed, a PKA-specific cAMP derivative inhibited LPS-induced cytokine production. Moreover, acidification induced cAMP accumulation in a TDAG8-specific way. We conclude that TDAG8, at least partly, mediates the extracellular acidification-induced inhibition of proinflammatory cytokine production through the Gs protein/cAMP/PKA signaling pathway in mouse macrophages.

No abstract