sphingosine-kinase and Pulmonary-Edema

Two mammalian sphingosine kinase (SphK) isoforms, SphK1 and SphK2, possess identical kinase domains but have distinct kinetic properties and subcellular localizations, suggesting each has one or more specific roles in sphingosine-1-phosphate (S1P) generation. Although both kinases use sphingosine as a substrate to generate S1P, the mechanisms controlling SphK activation and subsequent S1P generation during lung injury are not fully understood. In this study, we established a murine lung injury model to investigate LPS-induced lung injury in SphK1 knockout (SphK1(-/-)) and wild-type (WT) mice. We found that SphK1(-/-) mice were much more susceptible to LPS-induced lung injury compared with their WT counterparts, quantified by multiple parameters including cytokine induction. Intriguingly, overexpression of WT SphK1 delivered by adenoviral vector to the lungs protected SphK1(-/-) mice from lung injury and attenuated the severity of the response to LPS. However, adenoviral overexpression of a SphK1 kinase-dead mutant (SphKKD) in SphK1(-/-) mouse lungs further exacerbated the response to LPS as well as the extent of lung injury. WT SphK2 adenoviral overexpression also failed to provide protection and, in fact, augmented the degree of LPS-induced lung injury. This suggested that, in vascular injury, S1P generated by SphK2 activation plays a distinctly separate role compared with SphK1-dependent S1P generation and survival signaling. Microarray and real-time RT-PCR analysis of SphK1 and SphK2 expression levels during lung injury revealed that, in WT mice, LPS treatment caused significantly enhanced SphK1 expression ( approximately 5x) levels within 6 h, which declined back to baseline levels by 24 h posttreatment. In contrast, expression of SphK2 was gradually induced following LPS treatment and was elevated within 24 h. Collectively, our results for the first time demonstrate distinct functional roles of the two SphK isoforms in the regulation of LPS-induced lung injury.

Topics: Adenoviridae; Animals; Gene Deletion; Gene Expression Regulation, Enzymologic; Gene Transfer Techniques; Lipopolysaccharides; Lung; Lung Injury; Lysophospholipids; Membrane Proteins; Mice; Mice, Inbred C57BL; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Pulmonary Edema; Sphingosine; Time Factors; Tumor Necrosis Factor-alpha

Little is known about the contribution of bone marrow-derived progenitor cells (BMPCs) in the regulation endothelial barrier function as defined by microvascular permeability alterations at the level of adherens junctions (AJs).. We investigated the role of BMPCs in annealing AJs and thereby in preventing lung edema formation induced by endotoxin (LPS).. We observed that BMPCs enhanced basal endothelial barrier function and prevented the increase in pulmonary microvascular permeability and edema formation in mice after LPS challenge. Coculture of BMPCs with endothelial cells induced Rac1 and Cdc42 activation and AJ assembly in endothelial cells. However, transplantation of BMPCs isolated from sphingosine kinase-1-null mice (SPHK1(-/-)), having impaired S1P production, failed to activate Rac1 and Cdc42 or protect the endothelial barrier.. These results demonstrate that BMPCs have the ability to reanneal endothelial AJs by paracrine S1P release in the inflammatory milieu and the consequent activation of Rac-1 and Cdc42 in endothelial cells.

Topics: Adherens Junctions; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Capillary Permeability; cdc42 GTP-Binding Protein; Cell Movement; Cell Separation; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Flow Cytometry; Humans; Lipopolysaccharides; Lung; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuropeptides; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Edema; rac GTP-Binding Proteins; rac1 GTP-Binding Protein; Signal Transduction; Sphingosine; Stem Cells; Time Factors

Topics: Adherens Junctions; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Capillary Permeability; cdc42 GTP-Binding Protein; Cell Movement; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Humans; Lung; Lysophospholipids; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Edema; rac GTP-Binding Proteins; Signal Transduction; Sphingosine; Stem Cells; Time Factors

The lipid mediator sphingosine-1-phosphate (S1P), the product of sphingosine kinase (SPHK)-induced phosphorylation of sphingosine, is known to stabilize interendothelial junctions and prevent microvessel leakiness. Here, we investigated the role of SPHK1 activation in regulating the increase in pulmonary microvessel permeability induced by challenge of mice with lipopolysaccharide or thrombin ligation of protease-activating receptor (PAR)-1. Both lipopolysaccharide and thrombin increased mouse lung microvascular permeability and resulted in a delayed activation of SPHK1 that was coupled to the onset of restoration of permeability. In contrast to wild-type mice, Sphk1(-/-) mice showed markedly enhanced pulmonary edema formation in response to lipopolysaccharide and PAR-1 activation. Using endothelial cells challenged with thrombin concentration (50 nmol/L) that elicited a transient but reversible increase in endothelial permeability, we observed that increased SPHK1 activity and decreased intracellular S1P concentration preceded the onset of barrier recovery. Thus, we tested the hypothesis that released S1P in a paracrine manner activates its receptor S1P1 to restore the endothelial barrier. Knockdown of SPHK1 decreased basal S1P production and Rac1 activity but increased basal endothelial permeability. In SPHK1-depleted cells, PAR-1 activation failed to induce Rac1 activation but augmented RhoA activation and endothelial hyperpermeability response. Knockdown of S1P1 receptor in endothelial cells also enhanced the increase in endothelial permeability following PAR-1 activation. S1P treatment of Sphk1(-/-) lungs or SPHK1-deficient endothelial cells restored endothelial barrier function. Our results suggest the crucial role of activation of the SPHK1-->S1P-->S1P1 signaling pathway in response to inflammatory mediators in endothelial cells in regulating endothelial barrier homeostasis.

Topics: Animals; Capillary Permeability; Cells, Cultured; Enzyme Activation; Hemostatics; Humans; Inflammation Mediators; Intercellular Junctions; Lipopolysaccharides; Lung; Lysophospholipids; Mice; Mice, Knockout; Neuropeptides; Paracrine Communication; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Edema; rac GTP-Binding Proteins; rac1 GTP-Binding Protein; Receptor, PAR-1; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Thrombin