Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that induces a variety of biological responses in diverse cell types. Many, if not all, of these responses are mediated by members of the EDG (endothelial differentiation gene) family G protein-coupled receptors EDG1, EDG3, and EDG5 (AGR16). Among prominent activities of S1P is the regulation of cell motility; S1P stimulates or inhibits cell motility depending on cell types. In the present study, we provide evidence for EDG subtype-specific, contrasting regulation of cell motility and cellular Rac activity. In CHO cells expressing EDG1 or EDG3 (EDG1 cells or EDG3 cells, respectively) S1P as well as insulin-like growth factor I (IGF I) induced chemotaxis and membrane ruffling in phosphoinositide (PI) 3-kinase-and Rac-dependent manners. Both S1P and IGF I induced a biphasic increase in the amount of the GTP-bound active form of Rac. In CHO cells expressing EDG5 (EDG5 cells), IGF I similarly stimulated cell migration; however, in contrast to what was found for EDG1 and EDG3 cells, S1P did not stimulate migration but totally abolished IGF I-directed chemotaxis and membrane ruffling, in a manner dependent on a concentration gradient of S1P. In EDG5 cells, S1P stimulated PI 3-kinase activity as it did in EDG1 cells but inhibited the basal Rac activity and totally abolished IGF I-induced Rac activation, which involved stimulation of Rac-GTPase-activating protein activity rather than inhibition of Rac-guanine nucleotide exchange activity. S1P induced comparable increases in the amounts of GTP-RhoA in EDG3 and EDG5 cells. Neither S1P nor IGF I increased the amount of GTP-bound Cdc42. However, expression of N 17 -Cdc42, but not N 19 -RhoA, suppressed S1P-and IGF I-directed chemotaxis, suggesting a requirement for basal Cdc42 activity for chemotaxis. Taken together, the present results demonstrate that EDG5 is the first example of a hitherto-unrecognized type of receptors that negatively regulate Rac activity, thereby inhibiting cell migration and membrane ruffling.

Abstract-Ca2ϩ sensitization of vascular smooth muscle (VSM) contraction involves Rho-dependent and Rho-kinasedependent suppression of myosin phosphatase activity. We previously demonstrated that excitatory agonists in fact induce activation of RhoA in VSM. In this study, we demonstrate a novel Ca 2ϩ -dependent mechanism for activating RhoA in rabbit aortic VSM. High KCl-induced membrane depolarization as well as noradrenalin stimulation induced similar extents of sustained contraction in rabbit VSM. Both stimuli also induced similar extents of time-dependent, sustained increases in the amount of an active GTP-bound form of RhoA. Consistent with this, the Rho kinase inhibitors HA1077 and Y27632 inhibited both contraction and the 20-kDa myosin light chain phosphorylation induced by KCl as well as noradrenalin, with similar dose-response relations.

Class II α-isoform of phosphatidylinositol 3-kinases (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles, however, its functional role is little understood. Global or endothelial cell (EC)-specific targeted disruption of PI3K-C2α resulted in embryonic lethality due to defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in ECs induced decreased phospatidylinositol 3-phosphate-enriched endosomes, impaired endosomal trafficking, and defective delivery of VE-cadherin to EC junctions and its assembly. PI3K-C2α knockdown also impeded cell signaling including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. These together led to defective EC migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deletion suppressed post-ischemic and tumor angiogenesis, and diminished vascular barrier function, with greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α plays a crucial role in vascular formation and barrier integrity, and represents a new therapeutic target for vascular diseases. 3Formation of the vascular network by vasculogenesis and angiogenesis is essential for embryonic development, repair and remodeling of tissues in adults, as well as tumor growth. The angiogenic response to vascular endothelial growth factor (VEGF) and other factors begins with vascular leakage and dissolution of the subendothelial basement membrane, followed by proliferation and migration of vascular EC 1,2 . Then, formation of the intercellular junctions results in initial sprouts from existing vessels. The newly formed endothelial tubes are associated with mural cells, i.e. smooth muscle cells (SMC) and pericytes, thus becoming mature and stabilized 3 . Tightness of the intercellular junctions, particularly adherens junctions composed of VE-cadherin, controls vascular permeability 4,5 . Quiescent, stabilized vasculature with intact barrier integrity dominates in the healthy condition. In contrast, in pathological conditions, such as tumors, the vasculature is generally inmaturate and leaky. In the case of vascular insult such as excessive angiotensin II (Ang II) activity, increased vascular permeability is asssociated with leukocyte infiltration in the vascular wall and vascular disruption 6,7 . Therefore, stabilization of the vasculature and maintenance of vascular integrity is essential for vascular and tissue homeostasis 8,9 .PI3Ks are an enzyme family that phosphorylates membrane inositol lipids at the 3' position of the inositol ring. The lipid products of PI3Ks serve as important intracellular messengers by interacting with effector proteins, which include protein kinases, guanine nucleotide exchangers for G proteins, and actin cytoskeleton-regulating proteins. Through these actions, PI3Ks regulate a diverse array of cellular processes 10-12 .PI3Ks comprise three classes. Class I PI...

Regulation of cell migration is critical in such diverse biological processes as organogenesis, neuronal axon pathfinding, wound healing, inflammatory responses, vascular remodeling, and tumor cell dissemination (21). Extracellular cues called attractants and repellants, which are either soluble or membrane bound, instruct cells to advance and to retreat, respectively (36,40). A number of chemokines, growth factors, cytokines, and other inflammatory mediators have been shown to stimulate directed cell migration, whereas a much more limited number of biological mediators have been shown to inhibit cell motility in a manner dependent on their concentration gradients. The latter include metastin (28), Slit, semaphorins, ephrins (44), and a lipid mediator, sphingosine 1-phosphate (S1P) (42). S1P is a bioactive lysophospholipid that exerts a wide variety of biological activities, most of which are mediated via Edg family G protein-coupled receptors (GPCRs), including S1P 1 /Edg1, S1P 2 /Edg5/AGR16/H218, and S1P 3 /Edg3 (7,16,39,43). S1P has been demonstrated to be quite unique as an extracellular regulator of motility in that it exerts either stimulatory or inhibitory actions on cell motility (42). These bimodal actions are apparently cell type specific; thus, S1P stimulates chemotaxis in vascular endothelial cells (22) and embryonic fibroblasts (24), whereas it inhibits cell migration in vascular smooth muscle cells (3, 33) and melanoma cells (34). We recently showed that this bimodal regulation by S1P is based upon a diversity of S1P receptor isotypes, which mediate either stimulatory or inhibitory regulation for cell migration (31, 42). Thus, we found that S1P 2 acts as a repellant receptor to mediate inhibition of chemotaxis toward attractants, whereas S1P 1 and S1P 3 act as attractant receptors to mediate migration directed toward S1P. Elimination of the S1P receptor gene in mice (24) and development of a drug to target S1P receptors (4, 25) have revealed that S1P is involved in regulation of cell migration in vivo, thus contributing to morphogenesis and regulation of lymphocyte homing.Small GTPases of the Rho family, primarily Rac, Cdc42, and Rho, are well-known regulators of actin organization and myosin motor function and thereby of cell motility (10,14,47). These Rho GTPases show distinct activities on actin cytoskeletons: Rho mediates stress fiber formation and focal adhesion, while Rac and Cdc42 direct peripheral actin assembly that results in formation of lamellipodia and filopodia, respectively. Despite limitation of our understanding of intracellular signaling from the membrane to the cytoskeleton, a model has emerged from the observations in a variety of cell types that attractive extracellular cues activate Rac or Cdc42, while repulsive cues inhibit Rac or Cdc42 and stimulate Rho (9,38,42,48). In fact, the repellant receptor S1P 2 negatively regulates cellular Rac activity through mechanisms involving stimulation of a GTPase-activating protein (GAP) for Rac (31). In contrast, the attractant receptors ...

Recent studies (1-3) provide increasing evidence of roles for lysosphingolipids as mediators to elicit a variety of physiological and pathophysiological responses. Thus, the lysosphingolipids SP 1 and SPC have been shown to evoke diverse cellular responses in various cell types, including mitogenesis (1, 2), inhibition of migration (4, 5), cell shape change (6), and microfilament reorganization (6, 7). Stimulation of cells with the lysosphingolipids triggers the activation of multiple intracellular signaling molecules, including phospholipase C (2, 5, 8, 9), phospholipase D (8), PKC (10), MAPK (5, 11), and K ϩ channel (muscarinic K ϩ current) (12). Many of the lysosphingolipidinduced responses are demonstrated to be inhibited by PTX pretreatment (5, 8 -13). In addition, either an increase or a decrease in cellular cAMP content in response to SP has been reported, depending on cell types used (5, 13). These observations suggest the existence of multiple G protein-coupled cell surface receptors for SP and SPC.Recently, the orphan G protein-coupled receptor EDG2 was identified as a functional receptor for LPA (14). Moreover, EDG4 was very recently identified to be the second LPA receptor (15). EDG2 and EDG4 are members of the EDG family of receptors comprising EDG1 (16), EDG3 (17), and AGR16 (18)/ H218 (19), which have 36 -58% homology in amino acid sequences with each other. SP is related in its structure to LPA, and in some cell types, LPA and SP have been suggested to share a cell surface receptor (20, 21). These observations prompted us to examine the possibility that members of the EDG family receptors could function as a receptor for the lysosphingolipids. Many of cell lines usually used for expression of exogenous genes, including COS, NIH3T3 and HEK293 cells, respond to SP (13), which hampered expression cloning of SP receptor gene and functional analysis of cloned SP receptor gene. In the present study, by using carefully selected mammalian cell expression systems, we found that EDG1 is a functional receptor with a high specificity and affinity for SP. We demonstrate that EDG1 is coupled via a G i/o protein to multiple effector pathways, including phospholipase C, adenylate cyclase, and Ras/MAPK. MATERIALS AND METHODS Cells-CHO-K1(CHO) and HEL cells, obtained from RIKEN CellBank and the Japanese Cancer Research Resources Bank (Tokyo, Japan), respectively, were grown in Ham's F-12 (CHO) and RPMI (HEL) media supplemented with 10% fetal calf serum (Equitech-Bio, Ingram, TX), 100 units/ml penicillin, and 100 g/ml streptomycin (Wako Pure Chemicals, Osaka, Japan). Before each experiment, cells were switched to the respective medium supplemented with 1% fetal calf serum.

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-β 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.

It is well documented that Ras functions as a molecular switch for reentry into the cell cycle at the border between G 0 and G 1 by transducing extracellular growth stimuli into early G 1 mitogenic signals. In the present study, we investigated the role of Ras during the late stage of the G 1 phase by using NIH 3T3 (M17) fibroblasts in which the expression of a dominant negative Ras mutant, p21Ha-Ras(Asn17) , is induced in response to dexamethasone treatment. We found that delaying the expression of Ras(Asn17) until late in the G 1 phase by introducing dexamethasone 3 h after the addition of epidermal growth factor (EGF) abolished the downregulation of the p27 kip1 cyclin-dependent kinase (CDK) inhibitor which normally occurred during this period, with resultant suppression of cyclin Ds/CDK4 and cyclin E/CDK2 and G 1 arrest. The immunodepletion of p27 When serum-deprived quiescent cells are exposed to mitogenic stimuli, including growth factors, G protein-coupled receptor agonists, and hemopoietic cytokines, cellular Ras activates within a few minutes (12,44,45,54). Active Ras then interacts with and activates a number of downstream effectors, including Raf family kinases and p110 catalytic subunits of phosphatidylinositol-3-kinase (43,55,60). The serine and threonine protein kinase cascade consisting of Raf, MEK, and mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase [ERK]) is one of the best-characterized Ras effector systems (9). Accumulated evidence indicates that ERKs participate in the transcriptional activation of certain immediate early genes by directly phosphorylating p62 TCF / Elk-1 (13, 21), an Ets family transcription factor involved in ternary complex formation at the serum response element (19). It has also been reported recently (57) that ERKs mediate gene expression through the phosphorylation and activation of p90 RSK2 , which phosphorylates CREB to activate its transactivation potential. These findings provide compelling evidence that Ras acts as a molecular switch for reentry into the cell cycle at the border between G 0 and G 1 by transducing extracellular stimuli into a number of early G 1 mitogenic signals.By contrast, the role of Ras in later phases of the cell cycle is poorly understood. The ratio of GTP-GDP bound to Ras promptly rises after the addition of growth factors and then declines over a period of hours to a steady-state level that is slightly higher than or very close to the basal unstimulated value (31). Stacey and coworkers (11, 30) previously demonstrated that the microinjection of a neutralizing anti-Ras antibody into NIH 3T3 fibroblasts potently inhibited the initiation of DNA synthesis whether the microinjection was performed before serum stimulation or 6 h afterward. These researchers also showed that the anti-Ras antibody introduced into cells after entry into the S phase was much less inhibitory for the ongoing DNA synthesis (30). These results support the notion that the function of Ras is required for passage through the restriction (R) po...

Phosphatidylinositol (PI) 3-kinase is required for G 1 to S phase cell cycle progression stimulated by a variety of growth factors and is implicated in the activation of several downstream effectors, including p70 S6K . However, the molecular mechanisms by which PI 3-kinase is engaged in activation of the cell cycle machinery are not well understood. Here we report that the expression of a dominant negative (DN) form of either the p110␣ catalytic or the p85 regulatory subunit of heterodimeric PI 3-kinase strongly inhibited epidermal growth factor (EGF)-induced upregulation of cyclin D1 protein in NIH 3T3(M17) fibroblasts. The PI 3-kinase inhibitors LY294002 and wortmannin completely abrogated increases in both mRNA and protein levels of cyclin D1 and phosphorylation of pRb, inducing G 1 arrest in EGF-stimulated cells. By contrast, rapamycin, which potently suppressed p70 S6K activity throughout the G 1 phase, had little inhibitory effect, if any, on either of these events. PI 3-kinase, but not rapamycin-sensitive pathways, was also indispensable for upregulation of cyclin D1 mRNA and protein by other mitogens in NIH 3T3 (M17) cells and in wild-type NIH 3T3 cells as well. We also found that an enforced expression of wild-type p110 was sufficient to induce cyclin D1 protein expression in growth factor-deprived NIH 3T3(M17) cells. The p110 induction of cyclin D1 in quiescent cells was strongly inhibited by coexpression of either of the PI 3-kinase DN forms, and by LY294002, but was independent of the Ras-MEK-ERK pathway. Unlike mitogen stimulation, the p110 induction of cyclin D1 was sensitive to rapamycin. These results indicate that the catalytic activity of PI 3-kinase is necessary, and could also be sufficient, for upregulation of cyclin D1, with mTOR signaling being differentially required depending upon cellular conditions. Phosphatidylinositol (PI) 3-kinase is implicated in the receptor-mediated regulation of diverse mammalian cell functions, including insulin-stimulated glucose uptake and glycogen synthesis, exocytosis, neurite outgrowth, prevention of apoptosis, and mitogenesis (for reviews, see references 21,25,70,74). Growth factor stimulation of receptor-protein tyrosine kinases rapidly activates heterodimeric isoforms of PI 3-kinase, which consist of p110 catalytic and p85 regulatory subunits (74). p85 possesses adaptor modules in its structure, among which are two SH2 regions that mediate binding to specific phosphotyrosine residues presented on either cytoplasmic region of the activated growth factor receptors or their associated substrate proteins such as insulin receptor substrate 1 (IRS-1), thereby recruiting p110 to the plasma membrane where the lipid substrates are localized. Binding of p110 via its N-terminal region to p85 in the inter-SH2 region is indispensable for its enzymatic activity (references 30, 31, and 39 and references therein), which generates the lipid second messengers 3-polyphosphoinositides (29,70,74,82). In addition, p110 could directly interact with the GTP-bound active form ...