Mast cells secrete various substances that initiate and perpetuate allergic responses. Cross-linking of the high-affinity receptor for IgE (FcɛRI) in RBL-2H3 and bone marrow–derived mast cells activates sphingosine kinase (SphK), which leads to generation and secretion of the potent sphingolipid mediator, sphingosine-1–phosphate (S1P). In turn, S1P activates its receptors S1P1 and S1P2 that are present in mast cells. Moreover, inhibition of SphK blocks FcɛRI-mediated internalization of these receptors and markedly reduces degranulation and chemotaxis. Although transactivation of S1P1 and Gi signaling are important for cytoskeletal rearrangements and migration of mast cells toward antigen, they are dispensable for FcɛRI-triggered degranulation. However, S1P2, whose expression is up-regulated by FcɛRI cross-linking, was required for degranulation and inhibited migration toward antigen. Together, our results suggest that activation of SphKs and consequently S1PRs by FcɛRI triggering plays a crucial role in mast cell functions and might be involved in the movement of mast cells to sites of inflammation.

Airway inflammation is a central feature of asthma and chronic obstructive pulmonary disease. Reactive oxygen species (ROS) contribute to inflammation by damaging DNA, which, in turn, results in the activation of poly(ADP-ribose) polymerase-1 (PARP-1) and depletion of its substrate, nicotinamide adenine dinucleotide. Here we show that prevention of PARP-1 activation protects against both ROS-induced airway epithelial cell injury in vitro and airway inflammation in vivo. H(2)O(2) induced the generation of ROS, PARP-1 activation and concomitant nicotinamide adenine dinucleotide depletion, and release of lactate dehydrogenase in A549 human airway epithelial cells. These effects were blocked by the PARP-1 inhibitor 3-aminobenzamide (3-AB). Furthermore, 3-AB inhibited both activation of the proinflammatory transcription factor nuclear factor-kappaB and expression of the interleukin-8 gene induced by H(2)O(2) in these cells. In a murine model of allergen-induced asthma, 3-AB prevented airway inflammation elicited by ovalbumin. Moreover, PARP-1 knockout mice were resistant to such ovalbumin-induced inflammation. These protective effects were associated with an inhibition of expression of the inducible nitric oxide synthase. These results implicate PARP-1 activation in airway inflammation, and suggest this enzyme as a potential target for the development of new therapeutic strategies in the treatment of asthma as well as other respiratory disorders such as chronic obstructive pulmonary disease.

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, is the ligand for five specific G proteincoupled receptors, named S1P 1 to S1P 5 . In this study, we found that cross-communication between plateletderived growth factor receptor and S1P 2 serves as a negative damper of PDGF functions. Deletion of the S1P 2 receptor dramatically increased migration of mouse embryonic fibroblasts toward S1P, serum, and PDGF but not fibronectin. This enhanced migration was dependent on expression of S1P 1 and sphingosine kinase 1 (SphK1), the enzyme that produces S1P, as revealed by downregulation of their expression with antisense RNA and small interfering RNA, respectively. Although S1P 2 deletion had no significant effect on tyrosine phosphorylation of the PDGF receptors or activation of extracellular signal-regulated kinase 1/2 or Akt induced by PDGF, it reduced sustained PDGF-dependent p38 phosphorylation and markedly enhanced Rac activation. Surprisingly, S1P 2 -null cells not only exhibited enhanced proliferation but also markedly increased SphK1 expression and activity. Conversely, reintroduction of S1P 2 reduced DNA synthesis and expression of SphK1. Thus, S1P 2 serves as a negative regulator of PDGF-induced migration and proliferation as well as SphK1 expression. Our results suggest that a complex interplay between PDGFR and S1P receptors determines their functions.Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite formed by activation of SphK by many stimuli, including platelet-derived growth factor (PDGF) (43, 48). As a specific ligand for a family of five G protein-coupled receptors (GPCRs), S1P 1 to S1P 5 (2, 48), S1P regulates a wide variety of important cellular processes, including cytoskeletal rearrangements and cell movement (17,25,45,49,57), angiogenesis and vascular maturation (14,16,26,32,57), and immunity and lymphocyte trafficking (33,34). Interestingly, all of the S1P receptors (S1PRs) have been shown to play critical roles in cytoskeletal reorganization and cell migration (13,26,57). Activation of S1P 1 or S1P 3 increases directional or chemotactic migration (14, 27, 57), and both mediate activation of Rac via G i (26,38). In contrast, ligation of S1P 2 decreases chemotaxis and membrane ruffling (49), due to suppression of Rac activation, probably by stimulation of a GTPase-activating protein for Rac (38). Interestingly, the repellant receptor S1P 2 and the attractant receptor S1P 3 similarly stimulate RhoA activity, likely via G 12/13 (21). Recent studies suggest that the balance of counteracting signals from the G i -and the G 12/13 -Rho pathways directs either positive or negative regulation of Rac and cell migration (49). Similar to its functions in lower organisms, including yeasts and plants, which do not have S1PRs, S1P may also have intracellular actions important for calcium homeostasis (36), cell growth (40, 56), and stress responses (9,11,37,42). S1P, like various other GPCR agonists, can activate growth factor tyrosine kinase receptors in the absence of added...