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.

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 ...

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.

To clarify mechanisms of hypothermia in lipopolysaccharide (LPS) shock, four experiments were conducted in 72 chronically instrumented Wistar rats. They were intended to accomplish the following: experiment 1, determine the dose of intravenous Escherichia coli LPS that induces a body temperature (Tb) fall at a minimal mortality [the dose chosen (0.5 mg/kg) was then used in experiments 2-4]; experiment 2, identify the time course of the arterial blood pressure (BP) fall (shock) during the response to LPS; experiment 3, measure threshold Tb values for skin vasodilation and activation of metabolic heat production (M) during the LPS shock; and experiment 4, ascertain behavioral thermoregulation in LPS shock. For experiments 1-3, rats were kept in restrainers; ambient temperature (Ta) was 26 degrees C. In experiment 4, rats freely moved in a thermogradient (18-33 degrees C). Variables monitored were colonic (Tc) and tail skin (Tsk) temperatures (experiment 1); BP (experiment 2); hypothalamic temperature (Thy), M (from oxygen consumption), and Tsk (experiment 3); and preferred Ta (Tpr) and abdominal temperature (experiment 4). In experiment 1, LPS induced no Tc changes at 0 mg/kg, a biphasic fever (no mortality) at 0.05 mg/kg, a biphasic hypothermia (42% mortality) at 0.5 mg/kg, and a rapid fall of Tc (100% mortality) at 5 mg/kg. LPS-induced (0.5 mg/kg) hypotension (experiment 2) occurred simultaneously with the first hypothermic phase; both Tc and BP reached their nadirs (-0.8 +/- 0.1 degrees C and -34 +/- 12 mmHg) at approximately 1.5 h post-LPS. The major autonomic mechanism of the shock hypothermia was a shift in the threshold Thy for M from 37.9 +/- 0.3 to 36.0 +/- 0.3 degrees C (experiment 3; P < 0.05). In experiment 4, rats selected Tpr below 25 degrees C (vs. 28-30 degrees C in control; P < 0.05) throughout the duration of the shock; their Tb dropped to 36.2 +/- 0.3 degrees C (P < 0.05). In sum, the LPS shock-associated hypothermia involves a decrease in the threshold Tb for M, the resultant widening of the interthreshold zone, and cold-seeking behavior.