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.

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.

In smooth muscle, a Rho-regulated system of myosin phosphatase exists; however, it has yet to be established whether Rho kinase, one of the downstream effectors of Rho, mediates the regulation of myosin phosphatase activity in vivo. In the present study, we demonstrate in permeabilized vascular smooth muscle cells (SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dose dependently inhibits Rho-mediated enhancement of Ca(2+)-induced 20-kDa myosin light chain (MLC(20)) phosphorylation due to abrogating Rho-mediated inhibition of MLC(20) dephosphorylation. By an immune complex phosphatase assay, we found that guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) stimulation of permeabilized SMCs caused a decrease in myosin phosphatase activity with an increase in the extent of phosphorylation of the 130-kDa myosin-binding regulatory subunit (MBS) of myosin phosphatase in a Rho-dependent manner. HA-1077 abolished both of the Rho-mediated events. Moreover, we observed that the pleckstrin homology/cystein-rich domain protein of Rho kinase, a dominant negative inhibitor of Rho kinase, inhibited GTPgammaS-induced phosphorylation of MBS. These results provide direct in vivo evidence that Rho kinase mediates inhibition of myosin phosphatase activity with resultant enhancement of MLC(20) phosphorylation in smooth muscle and reveal the usefulness of HA-1077 as a Rho kinase inhibitor.