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Naotoshi Sugimoto

Publications and source records attributed to Naotoshi Sugimoto.

28 records · Page 2Linked to original sources
PubMed2003

Ligand-dependent inhibition of B16 melanoma cell migration and invasion via endogenous S1P2 G protein-coupled receptor. Requirement of inhibition of cellular RAC activity.

We investigated mechanisms for inhibition of B16 melanoma cell migration and invasion by sphingosine-1-phosphate (S1P), which is the ligand for the Edg family G protein-coupled receptors and also implicated as an intracellular second messenger. S1P, dihydro-S1P, and sphingosylphosphorylcholine inhibited B16 cell migration and invasion with the relative potencies expected as S1P2 receptor agonists. The S1P2-selective antagonist JTE013 completely abolished the responses to these agonists. In addition, JTE013 abrogated the inhibition by sphingosine, which is the S1P precursor but not an agonist for S1P receptors, indicating that the sphingosine effects were mediated via S1P2 stimulation, most likely by S1P that was converted from sphingosine. S1P induced inhibition and activation, respectively, of Rac and RhoA in B16 cells, which were abrogated by JTE013. Adenovirus-mediated expression of N17Rac mimicked S1P inhibition of migration, whereas C3 toxin pretreatment, but not Rho kinase inhibitors, reversed the S1P inhibition. Overexpression of S1P2 sensitized, and that of either S1P1 or S1P3 desensitized, B16 cells to S1P inhibition of Rac and migration. In JTE013-pretreated, S1P3-overexpressing B16 cells, S1P stimulated cellular RhoA but failed to inhibit either Rac or migration, indicating that RhoA stimulation itself is not sufficient for inhibition of migration. These results provide compelling evidence that endogenously expressed S1P2 negatively regulates cell motility and invasion through ligand-dependent reciprocal regulation of cellular Rac and RhoA activities. In the presence of JTE013, S1P instead stimulated Rac and migration in B16 cells that overexpress either S1P1 or S1P3, unveiling counteractions between S1P2 and S1P1 or S1P3 chemotactic receptor.

Adenoviridae↗
PubMed2003

The organum vasculosum laminae terminalis in immune-to-brain febrigenic signaling: a reappraisal of lesion experiments.

The organum vasculosum laminae terminalis (OVLT) has been proposed to serve as the interface for blood-to-brain febrigenic signaling, because ablation of this structure affects the febrile response. However, lesioning the OVLT causes many "side effects" not fully accounted for in the fever literature. By placing OVLT-lesioned rats on intensive rehydration therapy, we attempted to prevent these side effects and to evaluate the febrile response in their absence. After the OVLT of Sprague-Dawley rats was lesioned electrolytically, the rats were given access to 5% sucrose for 1 wk to stimulate drinking. Sucrose consumption and body mass were monitored. The animals were examined twice a day for signs of dehydration and treated with isotonic saline (50 ml/kg sc) when indicated. This protocol eliminated mortality but not several acute and chronic side effects stemming from the lesion. The acute effects included adipsia and gross (14% of body weight) emaciation; chronic effects included hypernatremia, hyperosmolality, a suppressed drinking response to hypertonic saline, and previously unrecognized marked (by approximately 2 degrees C) and long-lasting (>3 wk) hyperthermia. Because the hyperthermia was not accompanied by tail skin vasoconstriction, it likely reflected increased thermogenesis. After the rats recovered from the acute (but not chronic) side effects, their febrile response to IL-1beta (500 ng/kg iv) was tested. The sham-operated rats developed typical monophasic fevers ( approximately 0.5 degrees C), the lesioned rats did not. However, the absence of the febrile response in the OVLT-lesioned rats likely resulted from the untreatable side effects. For example, hyperthermia at the time of pyrogen injection was high enough (39-40 degrees C) to solely prevent fever from developing. Hence, the changed febrile responsiveness of OVLT-lesioned animals is given an alternative interpretation, unrelated to febrigenic signaling to the brain.

Animals↗
PubMed2003

Integral role of RhoA activation in monocyte adhesion-triggered tissue factor expression in endothelial cells.

OBJECTIVE: The role of Rho activation in the regulation of tissue factor (TF) is not clear. This study was undertaken to investigate this in endothelial cells induced by monocyte adhesion. METHODS AND RESULTS: Isolated human peripheral blood monocytes were added to cultured human coronary endothelial cells. Monocyte adhesion to endothelial cells increased the levels of TF antigen in the endothelial cells. The results of transient transfection of the human TF promoter/luciferase gene into endothelial cells indicated that the increase in endothelial expression of the TF gene caused by monocyte adhesion occurred at the transcriptional level. The upregulation of TF was inhibited by statins, and the suppressive effect of statins was reversed by geranylgeranylpyrophosphate. Monocyte adhesion rapidly upregulated the membrane translocation and GTP/GDP exchange of RhoA, but not of Cdc42 or Rac, in endothelial cells. Rho inhibition by C3 exoenzyme or adenovirus-mediated expression of N19RhoA prevented the endothelial upregulation of TF caused by monocyte adhesion, and this was mimicked by Rho-kinase inhibitors. Moreover, monocyte adhesion increased the phosphorylation of nuclear factor-kappaB p65 in endothelial cells, and this was prevented by statins and Rho inhibition. CONCLUSIONS: Our study shows that RhoA activation plays an integral role in TF expression in endothelial cells.

Cell Adhesion↗
PubMed2003

Differential requirements for JAK2 and TYK2 in T cell proliferation and IFN-gamma production induced by IL-12 alone or together with IL-18.

IL-12 activates TYK2 and Janus kinase (JAK)-2 to induce the phosphorylation of various signal transducers and activators of transcription (STAT) proteins. However, little is known regarding how these JAK exhibit the distinct biological effects of IL-12. Using two JAK inhibitors, tyrphostin A1 (A1) for TYK2 and tyrphostin B42 (B42) for JAK2, we investigated the involvement of JAK2 and TYK2 in IL-12-induced T cell proliferation and IFN-gamma production. B42, but not A1, inhibited T cell proliferation along with down-regulation of IL-12-induced c-Myc expression and STAT5 phosphorylation. In contrast, A1 but not B42 inhibited STAT4/STAT3 phosphorylation and IFN-gamma production. IL-18, but not IL-12, induced activator protein-1 (AP-1) responsible for high levels of IFN-gamma promoter activation. However, this IL-18 effect depended on the interaction of AP-1 with STAT4. A1 prevented AP-1 binding by inhibiting STAT4 involvement and down-regulated synergistic IFN-gamma promoter activation. These results indicate that JAK2 activation is required for IL-12-mediated T cell growth, whereas the TYK2-STAT4 signaling pathway is critical for IFN-gamma expression that is mediated by IL-12 alone and enhanced synergistically by combination with IL-18.

Animals↗
PubMed2003

Inhibitory and stimulatory regulation of Rac and cell motility by the G12/13-Rho and Gi pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform.

The G protein-coupled receptors S1P2/Edg5 and S1P3/Edg3 both mediate sphingosine-1-phosphate (S1P) stimulation of Rho, yet S1P2 but not S1P3 mediates downregulation of Rac activation, membrane ruffling, and cell migration in response to chemoattractants. Specific inhibition of endogenous Galpha12 and Galpha13, but not of Galphaq, by expression of respective C-terminal peptides abolished S1P2-mediated inhibition of Rac, membrane ruffling, and migration, as well as stimulation of Rho and stress fiber formation. Fusion receptors comprising S1P2 and either Galpha12 or Galpha13, but not Galphaq, mediated S1P stimulation of Rho and also inhibition of Rac and migration. Overexpression of Galphai, by contrast, specifically antagonized S1P2-mediated inhibition of Rac and migration. The S1P2 actions were mimicked by expression of V14Rho and were abolished by C3 toxin and N19Rho, but not Rho kinase inhibitors. In contrast to S1P2, S1P3 mediated S1P-directed, pertussis toxin-sensitive chemotaxis and Rac activation despite concurrent stimulation of Rho via G12/13. Upon inactivation of Gi by pertussis toxin, S1P3 mediated inhibition of Rac and migration just like S1P2. These results indicate that integration of counteracting signals from the Gi- and the G12/13-Rho pathways directs either positive or negative regulation of Rac, and thus cell migration, upon activation of a single S1P receptor isoform.

3T3 Cells↗
PubMed2003

The unique target specificity of a nonpeptide chemokine receptor antagonist: selective blockade of two Th1 chemokine receptors CCR5 and CXCR3.

CC chemokine receptor (CCR) 5 and CXC chemokine receptor (CXCR)3 are expressed on T helper cell type 1 cells and have been implicated in their migration to sites of inflammation. Our preceding study demonstrated that a nonpeptide synthetic CCR5 antagonist, TAK-779 (N, N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6, 7-dihydro-5H-benzocyclohepten-8-yl]carbon-yl]amino]benzyl]-tetrahydro-2H-pyran4-aminium chloride, inhibits the development of experimentally induced arthritis by modulating the migration of CCR5(+)/CXCR3(+) T cells to joints. The present study investigated the functional properties of TAK-779, including the effect of this antagonist on CXCR3 function. For this purpose, transfectants expressing mouse CCR5 (mCCR5) or mCXCR3 and expressing mCCR4 or mCXCR4 as controls were established by introducing each relevant gene into 2B4 T cells and were subjected to the following assays. First, the ligand binding to chemokine receptors was assayed by incubating transfectants with [(125)I]-labeled relevant ligand or with the unlabeled relevant ligand followed by staining with anti-ligand antibody. Second, chemokine-induced lymphocyte function-associated antigen-1 (LFA-1) activation was assayed by measuring the adhesion of cells to microculture plates coated with purified intercellular adhesion molecule-1. Third, chemokine-stimulated chemotaxis was assayed by observing the cell migration through transwells. In these assays, TAK-779 blocked the ligand binding as well as LFA-1 up-regulating and chemotactic function of mCXCR3 and mCCR5 but did not elicit a biologically significant inhibition of those functions of mCCR4 and mCXCR4. These observations indicate the unique target specificity of TAK-779 and explain why this antagonist efficiently blocks the migration of T cells expressing CCR5 and CXCR3 to sites of inflammation.

Amides↗
PubMed2002

Sphingosine-1-phosphate, a platelet-derived lysophospholipid mediator, negatively regulates cellular Rac activity and cell migration in vascular smooth muscle cells.

Previous studies demonstrated that sphingosine-1-phosphate (S1P) induced migration of human umbilical vein endothelial cells (HUVECs) whereas it inhibited that of vascular smooth muscle cells (SMCs). This study explored the molecular mechanisms underlying the contrasting S1P actions on vascular cell motility. In rat and human aortic SMCs, the chemoattractant platelet-derived growth factor B-chain (PDGF) induced rapid 5- to 6-fold increases in the cellular amount of GTP-bound, active form of Rac. S1P did not affect PDGF-stimulated tyrosine phosphorylation of PDGF-beta receptor, but strongly inhibited PDGF-induced Rac activation, with a dose-response relationship similar to that for inhibition of PDGF-elicited chemotaxis. Dihydrosphingosine-1-phosphate, which is a weaker agonist for the S1P receptors, but not an inactive ligand sphingosine, also inhibited PDGF-stimulated chemotaxis and Rac activation although to lesser extents compared with S1P, suggesting that negative regulation by S1P of both chemotaxis and Rac was a receptor-mediated process. In contrast, S1P by itself stimulated Rac activity in HUVECs. Among the five S1P receptor isoforms, SMCs prominently expressed Edg-5 mRNA, whereas HUVECs expressed abundant Edg-1 mRNA but lacked detectable expression of Edg-5 mRNA. Adenovirus-mediated expression of a dominant-negative form of either Rac or Cdc42, but not RhoA, markedly attenuated chemotaxis of SMCs and HUVECs toward PDGF and S1P, respectively. Overexpression of Edg-1 in SMCs and Edg-5 in HUVECs reduced S1P-induced inhibition and stimulation, respectively, of Rac activity and migration. These results together indicate that Edg isoform-specific, negative or positive regulation of cellular Rac activity is critically involved in S1P-mediated bimodal regulation of cell motility in SMCs and HUVECs.

Animals↗
PubMed2002

The Edg family G protein-coupled receptors for lysophospholipids: their signaling properties and biological activities.

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are blood-borne lysophospholipids with a wide spectrum of biological activities, which include stimulation of cell growth, prevention of apoptosis, regulation of actin cytoskeleton, and modulation of cell shape, cell migration, and invasion. Activated platelets appear to be a major source of both S1P and LPA in blood. Despite the diversity of their biosynthetic origins, they are considered to share substantial structural similarity. Indeed, recent investigation has revealed that S1P and LPA act via a single family of G protein-coupled receptors designated as Edg. Thus, the Edg isoforms, Edg1 (also called S1P(1)), Edg5 (S1P(2)), Edg3 (S1P(3)), Edg6 (S1P(4)), and Edg8 (S1P(5)), are specific receptors for S1P (and SPC with a lower affinity), whereas Edg2 (LPA(1)), Edg4 (LPA(2)), and Edg7 (LPA(3)) serve as receptors specific for LPA. Each receptor isoform displays a unique tissue expression pattern and coupling to a distinct set of heterotrimeric G proteins, leading to the activation of an isoform-specific panel of multiple intracellular signaling pathways. Recent studies on knockout mice have unveiled non-redundant Edg receptor functions that are essential for normal development and vascular maturation. In addition, the Edg lysophospholipid signaling system may play a role in modulating cell motility under such pathological conditions as inflammation, tumor cell dissemination and vascular remodeling.

Animals↗