Lysophosphatidic acid (LPA), a bioactive lipid produced by several cell types including postmitotic neurons and activated platelets, is thought to be involved in various biological processes, including brain development. Three cognate G protein-coupled receptors encoded by lpa 1 /lp A1 /Edg-2/Gpcr26, lpa 2 /lp A2 /Edg-4, and lpa 3 /lp A3 / Edg-7 mediate the cellular effects of LPA. We have previously shown that deletion of lpa 1 in mice results in craniofacial dysmorphism, semilethality due to defective suckling behavior, and generation of a small fraction of pups with frontal hematoma. To further investigate the role of these receptors and LPA signaling in the organism, we deleted lpa 2 in mice. Homozygous knockout (lpa 2 (؊/؊) ) mice were born at the expected frequency and displayed no obvious phenotypic abnormalities. fibroblasts. Thus, although LPA 2 is not essential for normal mouse development, it does act redundantly with LPA 1 to mediate most LPA responses in fibroblasts.

These results indicate a nonessential role for LP B3 in normal development of mouse but show nonredundant cellular signaling mediated by a single type of S1P receptor. Sphingosine 1-phosphate (S1P)1 is a potent lysophospholipid mediator that exerts diverse biological effects on many types of cells and tissues. S1P is produced from activated platelets and many other cell types and affects fundamental cellular processes including proliferation, differentiation, survival, adhesion, migration, morphogenesis, and cytoskeletal rearrangement (reviewed in Refs. 1-6). S1P has been proposed to act both as an extracellular mediator and an intracellular second messenger. Recent progress in the identification of specific G protein-coupled receptors that can account for the extracellular effects induced by S1P has improved our understanding of the mechanisms of action of this lipid (reviewed in Refs. 1-6).To date, five cognate G protein-coupled receptors have been identified as mammalian high affinity S1P receptors: LP B1 / EDG-1, LP B2 /H218/AGR16/EDG-5, LP B3 /EDG-3, LP B4 /NRG-1/ EDG-8, and LP C1 /EDG-6 (reviewed in Refs. 6 -8). All S1P receptors belong to a larger lysophospholipid (LP) receptor subfamily, which also includes receptors for lysophosphatidic acid (LPA), a bioactive lipid that is structurally and biologically related to S1P. Each of the LP receptors couples to multiple subsets of heterotrimeric G proteins (including G q , G i/o , and G 12/13 ) and thus drives different signal transduction pathways. Also, the receptor genes are expressed in spatially and temporally different patterns in mice (9, 10), 2 suggesting specific roles for each receptor in vivo. It was reported recently that LP B1 -null mice are embryonic lethal because of incomplete vascular maturation (11), showing the essential role of LP B1 in mouse development. In zebrafish, a single point mutation in an lp B ortholog, mil, led to abnormal heart development (12). To determine in vivo functions and roles of the LP B3 receptor in mammals, we have disrupted lp B3 in mice. Unexpectedly, LP B3 -null mice were viable and fertile and developed normally with no gross phenotypic abnormality, although selective loss of S1P signal transduction pathways was observed in mouse embryonic fibroblast (MEF) cells. These results continue to clarify physiological functions and roles of the LP B3 receptor in vivo. EXPERIMENTAL PROCEDURES Materials-[␣-32 P]Deoxy-CTP and myo-[2-3 H]inositol were purchased from PerkinElmer Life Sciences. S1P and LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate) were purchased from Avanti Polar Lipids (Alabaster, AL). GTP␥S, platelet-derived growth factor (BB homodimer), and pertussis toxin (PTX) were purchased from Calbiochem. Anti-EDG-3 (LP B3 ) carboxyl and amino terminus monoclonal antibodies were purchased from Exalpha Biological Inc. (Boston, MA). Rhotekin Rho binding domain agarose and PAK-1 p21-binding domain agarose were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The pFlox targeting vector (13) and R1 embryonic ...

Five cognate G protein-coupled receptors (S1P 1-5 ) have been shown to mediate various cellular effects of sphingosine 1-phosphate (S1P). Here we report the generation of mice null for S1P 2 and for both S1P 2 and S1P 3 . S1P 2 -null mice were viable and fertile and developed normally. The litter sizes from S1P 2 S1P 3 double-null crosses were remarkably reduced compared with controls, and double-null pups often did not survive through infancy, although double-null survivors lacked any obvious phenotype. Mouse embryonic fibroblasts (MEFs) were examined for the effects of receptor deletions on S1P signaling pathways. Wild-type MEFs were responsive to S1P in activation of Rho and phospholipase C (PLC), intracellular calcium mobilization, and inhibition of forskolin-activated adenylyl cyclase. S1P 2 -null MEFs showed a significant decrease in Rho activation, but no effect on PLC activation, calcium mobilization, or adenylyl cyclase inhibition. Double-null MEFs displayed a complete loss of Rho activation and a significant decrease in PLC activation and calcium mobilization, with no effect on adenylyl cyclase inhibition. These data extend our previous findings on S1P 3 -null mice and indicate preferential coupling of the S1P 2 and S1P 3 receptors to Rho and PLC/Ca 2؉ pathways, respectively. Although either receptor subtype supports embryonic development, deletion of both produces marked perinatal lethality, demonstrating an essential role for combined S1P signaling by these receptors.Sphingosine 1-phosphate (S1P) 1 is a bioactive lysophospholipid that elicits diverse physiological effects on most types of cells and tissues. Several lines of evidence from a wealth of in vitro studies revealed that these effects are induced by S1P activation of any of five cognate G protein-coupled receptors: S1P 1 (LP B1 /EDG-1), S1P 2 (LP B2 /H218/AGR16/EDG-5), S1P 3 (LP B3 /EDG-3), S1P 4 (LP C1 /EDG-6), and S1P 5 (LP B4 /NRG-1/ EDG-8) (reviewed in Refs. 1-7). In part reflecting a lack of receptor subtype-specific agonists/antagonists and the universal expression of multiple S1P receptor genes in many single cell types, the in vivo roles of each receptor were unclear until the recent use of genetic approaches in mice (reviewed in Ref. 44). Liu et al. (8) reported that S1P 1 -null mice are lethal. S1P 1 -null mice exhibit embryonic hemorrhage and incomplete vascular maturation, which lead to intrauterine death. S1P-induced cell migration and activation of the small GTPase Rac are severely defective in S1P 1 -null mouse embryonic fibroblasts (MEFs), suggesting that the loss of S1P cellular signaling is relevant to those phenotypes found in S1P 1 -null mice (8). We observed that S1P 3 -null mice are without marked phenotypic differences compared with controls (9). However, S1P-induced phospholipase C (PLC) activation is severely defective in S1P 3 -null MEFs (9). In this study, we generated S1P 2 -null mice. S1P 2 -null mice were viable and apparently normal, as were S1P 3 -null mice. However, S1P-induced Rho activation was impaired. B...

The Rho family small GTPases play a crucial role in mediating cellular responses to stretch. However, it remains unclear how force is transduced to Rho signaling pathways. We investigated the effect of stretch on the activation and caveolar localization of RhoA and Rac1 in neonatal rat cardiomyocytes. In unstretched cardiomyocytes, RhoA and Rac1 were detected in both caveolar and non-caveolar fractions as assessed using detergent-free floatation analysis. Stretching myocytes for 4 min activated RhoA and Rac1. By 15 min of stretch, RhoA and Rac1 had dissociated from caveolae, and there was decreased coprecipitation of RhoA and Rac1 with caveolin-3. To determine whether compartmentation of RhoA and Rac1 within caveolae was necessary for stretch signaling, we disrupted caveolae with methyl ␤-cyclodextrin (M␤CD). Treatment with 5 mM M␤CD for 1 h dissociated both RhoA and Rac1 from caveolae. Under this condition, stretch failed to activate RhoA or Rac1. Stretch-induced actin cytoskeletal organization was concomitantly impaired. Interestingly the ability of stretch to activate extracellular signal-regulated kinase (ERK) was unaffected by M␤CD treatment, but ERK translocation to the nucleus was impaired. Stretch-induced hypertrophy was also inhibited. Actin cytoskeletal disruption with cytochalasin-D also prevented stretch from increasing nuclear ERK, whereas actin polymerization with jasplakinolide restored nuclear translocation of activated ERK in the presence of M␤CD. We suggest that activation of RhoA or Rac1, localized in a caveolar compartment, is essential for sensing externally applied force and transducing this signal to the actin cytoskeleton and ERK translocation.

Myocardial ischemia/reperfusion activates a calcium-dependent protease, calpain, in the ischemic myocytes. It is not known whether calpain is involved in the mechanism of ischemia/reperfusion injury in hearts. Thus the purpose of this study was to clarify the effect of a selective calpain inhibitor (CAI) on infarct size and the extent of DNA damage in ischemic/reperfused rat hearts. Rats were divided in four groups (n = 7 each). In saline group, 0.3 ml of saline was administered (i.v.) 10 min before 30-min coronary occlusion followed by 6-h reperfusion. In vehicle group, 0.3 ml of 10% dimethyl sulfoxide (DMSO) was administered 10 min before the 30-min ischemia. CAI (0.5 mg/kg) was administered 10 min before the 30-min ischemia (CAI-A group) and 10 min before the 6-h reperfusion period (CAI-B group). Infarct size was detected with triphenyl tetrazolium chloride, and DNA fragmentation was detected by agarose gel electrophoresis and by in situ nick end labeling (ISEL). Infarct size was significantly smaller in the CAI-A group compared with the vehicle group (13+/-9% vs. 48+/-12%; p < 0.01), and the incidence of ISEL-positive myocyte nuclei in the subendocardial region was significantly reduced in the CAI-A group compared with the vehicle group (26+/-3% vs. 59+/-6%; p < 0.01). However, the effects of CAI in CAI-B group were not significant. Activation of calpain is involved in the mechanism of ischemia/reperfusion injury, and the preischemic administration of CAI was effective in reducing myocardial infarct size and the DNA damage of the myocytes in ischemic/reperfused rat heart.