Role Of Src-suppressed C Kinase Substrate In Changes Of Pulmonary Microvascular Endothelial Permeability Triggered By Inflammatory Cytokines

BackgroundPulmonary microvascular endothelial cells (PMVECs) lined at the critical interface between the blood and microvessel are primary targets of inflammatory cytokines during lung inflammation. A pathophysiologic response of PMVECs to inflammatory cytokines is permeability increase that contributes to high-protein pulmonary edema, which is a characteristic of acute lung injury and acute respiratory distress syndrome (ALI/ARDS). It is well known that the protein kinase C (PKC) signaling pathway significantly contributes to the breakdown of PMVECs barrier though phosphorylation of proteins which can promote cytoskeletal reorganization or dissolution of the adherens junctions. Given the complex interplay between PKC signaling pathway and the possible contribution of other signaling pathways, further studies should be required to delineate downstream targets of PKC and the mechanisms that regulate endothelial barrier function in PMVECs. In addition, it is very difficult in vivo to study changes of PMVECs permeability, and considerable achievements in this research area come from in vitro experiments of cultured cells, therefore, the integrality of study can be determined by the confluent state of PMVECs.Src-suppressed C kinase substrate (SSeCKS) is a PKC substrate that is identified to be phosphorylated by PKC. Of particular interest, evidences that SSeCKS is involved in a dramatic rearrangement of the actin cytoskeleton and the modulation of the blood-brain barrier permeability have been presented. Moreover, in rat PMVECs (RPMVECs), increased levels of SSeCKS have been found after challenge of lipopolysaccharide (LPS). These implicate that SSeCKS is possibly one of the critical proteins in the pathogenesis of endothelial hyperpermeability after exposure of PMVECs to various inflammatory factors, such as interleukin (IL)-1βand tumor necrosin factor (TNF)-α. Nevertheless, the exact role of SSeCKS in the regulation of PMVECs permeability is not well understood. Additionally, besides LPS, other potential inducers of SSeCKS in RPMVECs remain ill-defined.Interleukin-17F is a novel proinflammatory cytokine. Accumulating studies have showed that IL-17F triggers a variety of inflammatory responses such as inducing inflammatory factors, including IL-6, IL-8, and intracellular adhesion molecule-1, in airway epithelial cells and vein endothelial cells. Of note, in lung, increased expression of IL-17F has been found in animal models after Klebsiella pneumoniae, Mycoplasma pneumoniae and Candida albicans infection, and to induce pulmonary neutrophilia and an additive effect on antigen-induced allergic inflammatory responses. These findings have suggested that IL-17F is one of the key cytokines regulating lung inflammation. However, up to date, studies about mechanisms of IL-17F in lung focus on bronchial epithelium, and signaling pathways activated by IL-17F in cells have not been elucidated. It is well known that PMVECs are involved in lung inflammatory disease, yet, little information exists in the role of IL-17F and its signaling pathway in PMVECs.ObjectiveThe aim of this study is firstly to observe some biological characteristics of PMVECs monolayers before and after confluence, and then, we sought to investigate the effects of IL-17F on confluent PMVECs monolayers and assess the role of PKC in this process. The role of SSeCKS in the modulation of PMVECs permeability elicited by IL-1β, TNF-αand IL-17F is also investigated. These may be helpful for elucidating the pathogenesis of ALI/ARDS.MethodsAfter primary RPMVECs were successfully cultured in vitro, the cells were seeded on Transwell polyester membranes or nitric-acid/acetic-acid membranes to construct in vitro models of PMVECs monolayers. The biological characteristics of cells monolayers before and after confluence were observed by the inverted microscope, staining with hematoxylin-eosin, transendothelial electrical resistance (TER) and changes of permeability as measured by means of fluorescein isothiocyanate (FITC)-dextran (Pd) and Hank's solution (LP) across monolayers. Additional monolayers were stained using FITC-phalloin for observing the filamentous actin (F-actin) cytoskeletal changes. The gene expression of SSeCKS was analyzed by the reverse transcription-polymerase chains. Immunoblotting was used to determine the PKC activity, the levels of SSeCKS protein expression and the biological alterations of SSeCKS protein in PMVECs. In addition, SSeCKS-specific small interfering RNA (siRNA) was transfected into PMVECs.Results1) In group one, after a cell suspension (1×105 cells /cm2) was cultured on Transwell, the normalized TER increased steadily in a time-dependent manner and reached the summit on the fourth day post-seeding, then was sustained. Subsequently, on the tenth day post-seeding, the increased TER began to decrease. However, the monolayer under inversed microscope reached the state of confluence on the third day post-seeding. In group two, a cell suspension (2×105 cells /cm2) was seeded, changes of the normalized TER were similar to that of the group one, reaching the summit on the second day post-seeding and beginning to decrease on the ninth day post-seeding. Of note, no significant difference in the maximum TER was found between both groups. The TER, Pd and LP of confluent PMVECs monolayers under quiescent state were 48.07±2.84 ?·cm2, 6.19±0.43×10-6 cm/s and 6.80±0.62×10-7 cm/s/cm H2O, respectively.2) After confluent PMVECs monolayers were stimulated with 10 mg/L LPS for both 0.5 h and 2 h, there were significant decreases in TER and increases in Pd and LP when respectively compared with the untreated group.3) Interleukin-1β(5 ng/ml) and TNF-α(10 ng/ml) activated PKC signaling pathway in PMVECs, and up-regulated the gene and protein expression of SSeCKS. Meanwhile, we found that the expression of SSeCKS was the greatest after stimulation with the cytokine combination of 5 ng/ml IL-1βand 10 ng/ml TNF-α. PKC inhibitor, Bisindolylmaleimide I (BIM, 1μmol/L) significantly decreased both the increased PKC activity and SSeCKS expression induced by the cytokines in PMVECs. However, both the selective protein kinase A (PKA) inhibitor H89 (10μmol/L) and the broad-spectrum protein tyrosine kinase (PTK) inhibitor genistein (50μg/ml) showed no significant inhibitory effects on cytokine-induced SSeCKS gene expression and protein production in PMVECs.4) Transfection with special SSeCKS-siRNA resulted in a decrease in the expression of SSeCKS gene and protein in a dose-dependent manner (10, 20, 50 nmol/L) and in a time-dependent manner. When compared with the untreated group, significantly inhibitory effects of SSeCKS-siRNA (50 nmol/L) in the expression of SSeCKS gene and protein began at 12 h and 24 h, reached a peak at 24 h and 48 h and were sustained to 96 h and 120 h after PMVECs were transfected, respectively. At 48 h post-transfection, the expression of SSeCKS in PMVECs was inhibited by 50 %. Furthermore, SSeCKS depletion, the mock trasfection and the non-silencing control siRNA transfection induced a slight, but not significant (P > 0.05), decrease in quiescent TER and increase in quiescent Pd across confluent PMVECs monolayers.5) When compared with the untreated group, application of the cytokine combination (IL-1βand TNF-α) to PMVECs monolayers resulted in a significant increase of Pd and a significant decrease of TER, and then, prior transfection with 50 nmol/L SSeCKS-siRNA significantly attenuated the cytokine-induced effects. Notably, it was found that the effect of BIM in blocking cytokine-induced endothelial barrier dysfunction is more significant than that of SSeCKS depletion.6) Interleukin-17F induced, in a dose-dependent manner (0.1, 1, 10, 100ng/ml), significant increases of Pd and decreases of TER after PMVECs monolayers were exposed to stimulations for 3 h. The effects of IL-17F were also time dependent. Pd increased ~15 % as compared with quiescent levels after monolayers were treated by 100ng/ml IL-17F for 0.5 h, peaked at 3h and prolonged lag time of 24 h. Similarly, the progressive decrease in TER produced by IL-17F was time-dependent when compared with untreated monolayer. After PMVECs monolayers were pretreated with BIM (1 or 10μmol/L, respectively), the increased Pd and decreased TER induced by IL-17F can be significantly blocked, but did not return to normal levels.7) In PMVECs, IL-17F stimulation for 3 h produced numerous intercellular gaps and a marked reorganization of F-actin from a web-like peripheral distribution to centrally located parallel stress fibers. The latter became thicker. Meanwhile, after IL-17F stimulation, the flow cytometric profile was shifted to the right and the mean fluorescence intensity of F-actin was conspicuously increased when compared with untreated monolayers. IL-17F-induced cytoskeletal changes indicated above can be significantly amended after PMVECs monolayers were pretreated by 1 and 10μmol/L BIM.8) After PMVECs were treated with increasing concentrations of IL-17F (0.1, 1, 10, 100 ng/ml) and 100 ng/ml IL-17F for various times, the expression of SSeCKS gene and protein were remarkably up-regulated in a dose-dependent manner, occurred maximally at 3 h and 6 h post-stimulation, respectively. The serine phosphorylation of SSeCKS was weakly detected at 5-minutes point, remarkably occurred at 30-minutes point, reached maximal level at 3 ~ 6 h, and stayed at a slightly high level until 24 h after PMVECs were exposed to100 ng/ml IL-17F. BIM (1μmol/L) can significantly abrogate this phosphorylation induced by IL-17F. In addition, our results demonstrated that treatment with IL-17F significantly increased the amount of SSeCKS in the insoluble fraction in a time-dependent manner, beginning at 30 min and peaking at 3 ~ 6 h post-stimulation. Finally, under quiescent conditions, SSeCKS existed for the most part in the cytosolic fraction of PMVECs, after 30 min treatment, IL-17F resulted in significant translocation of SSeCKS from the cytosol to the membrane. At 90-minutes point, SSeCKS predominantly existed in the membrane fraction.Conclusions1) The combination of TER and an inverted microscope is better than the solo application of an inverted microscope to assess the confluent state of PMVECs monolayers.2) Three methods, TER, Pd and LP, can be availably used to determine PMVECs permeability. Combination of Pd and TER maybe greatly reflect the information of changes in permeability across the in vitro PMVECs monolayer model.3) The increased expression of SSeCKS is involved in the regulation of PMVECs hyperpermeability that maybe play an important role in the process of ALI/ARDS. The expression of SSeCKS gene and protein can be potently induced by IL-1β, TNF-αand IL-17F in PMVECs. It is PKC signaling pathway, neither PKA signaling pathway nor PTK signaling pathway, which is involved in the regulation of SSeCKS expression.4) IL-17F promotes the lung inflammatory response though the activated PKC-SSeCKS signaling pathway and the PKC-dependent reorganization of actin cytoskeleton that all contribute to PMVECs hyperpermeabilty induced by IL-17F.