Differential Roles Of Mapks In Thermal Injury Induced Endothelial Cell Dysfunction

Objectives:Thermal injury induced blood vessel hyper-permeability and leukocytes adhesion are the major inflammatory phenomena in burned patients.The mechanisms behind are still obscure.We previously found that inhibition of a G-protein-coupled receptor kinase,namely Rho kinase(ROCK),and p38 mitogen-activated protein kinase (MAPK) significantly attenuated burn-induced endothelium stress fiber formation, and decreased blood vessel hyper-permeability.Here we further investigated the role of their downstream signal transduction in the activation of endothelial cells in thermal injury.Methods:1.Human dermal micro-vascular endothelial cells(HMVEC-1) were pretreated with RO31-7549,Y27632,SB203580,SP600125 and PD98059 respectively,which are inhibitors of PKC,Rho kinase,p38,ERK and JNK MAPKs accordingly,and then exposed to plasma extracted from burned-rat.2.Morphology of endothelial cells was visualized by immuno-staining cytoskeleton F-actin,tight junction protein ZO-1 and adhesion molecule ICAM-1.3.Expression of ICAM-1 and activation of MAPKs on endothelial cells were determined by western blot. 4.The substrates of p38-MK2 and PRAK were excited or inhibited by recombinant adenovirus.Then the morphology of endothelial cell was observed by staining F-actin and neucleus.5.Phosphorylation level of HSP27 was detected by western blot on adenovirus treated endothelial cells.Results:1.Burned-plasma indeed induced re-arrangement of F-actin,and ensued formation of stress fiber,damage of inter-endothelial tight junction,and expression of adhesion molecule ICAM-1.2.Inhibition of PKC or Rho kinase restrained the formation of stress fiber in burned-plasma treated endothelial cells,while only inhibition of Rho kinase could attenuated the phosphorylation of p38 MAPK.2.Stress fiber formation was blocked with inhibition of p38 or ERK pathways, while only inhibition of p38 MPAK could attenuate the damage of intercellular tight junction.3.Inhibiting the activities of MK2 and PRAK by transfecting the cells with their dominant negative forms alleviated the formation of stress fiber induced by burned-plasma;constitutively activation of MK2 induced rearrangement of F-actin obviously.4.Burned-serum stimulation increased the phosphorylation of HSP27,while both MK2 and PRAK activation with transfection of their constitutively active forms could also phosphorylate HSP27.The inhibition of MK2 and PRAK by transfecting the cells with their dominant negative forms before burned-plasma administration diminished the phosphorylation of HSP27. 5.Burned-plasma evoked endothelial expression of ICAM-1 was significantly reduced by pretreating of the cells with JNK MAPK inhibitor-SP600125,but other MAPKs inhibitors had no such effect.Conclusions:1.Burned-plasma induced rearrangement of endothelial cytoskeleton F-actin is regulated through p38/MK2/PRAK/HSP27 pathway,p38 even participate in damage of tight junction protein ZO-1 triggered by thermal injury.2.Rho and PKC also take part in formation of stress fiber in endothelial cells stimulated by burn serum.The former one may exert its influence through p38 signal pathway.3.ICAM-1 expression on burn endothelial cell is mediated by JNK MAPK,but not ERK or p38.Thus,members of MAPKs have differential functions in thermal injury and the results in this study may have implications for future design of pharmacologic agents for thermal injury therapy.