A Preliminary Study On Functions Of SSeCKS In Endothelial Cell

[Background and Objective]Src-suppressed C kinase substrate (SSeCKS) is a new scaffolding protein, which belongs to AKAP (A Kinase Anchoring Protein) family. SSeCKS' molecular weight is 280/290 kilodalton, and the amino-terminal of SSeCKS contains membrane location motif and protein kianse C binding site, the carboxyl terminus of SSeCKS contains protein kinase A binding site. SSeCKS is widely expressed in smooth muscle cell, mesangial cell, astrocyte, fibroblasts and endothelial cell. SSeCKS not only functions as a negative regulator of mitogenesis in fibroblasts, but also as a cytoskeletal differentiating factor and as a scaffolder of protein kinases C and A. SSeCKS actually scaffolds PKC and PKA and is involved in the regulation of actin-based cytoskeletal dynamics. The most notable finding was that LPS injection intraperitoneally induced expression of SSeCKS predominantly in vascular endothelial cells of several organs.In order to find out which functions of SSeCKS in endothelial cells plays in physical state and inflammatory state, we firstly work out the relationship between SSeCKS and endothelial cell proliferating,adhesion and migrating, and secondly find out the correlation between SSeCKS and inflammatory, and lastly study the relationship between SSeCKS and inflammation induced cytoskeleton remodeling at endothelial cell. [Methods]1. Cultured ECV304 were stimulated by different concentrations of FN, SSeCKS expression were detected by Western blotting. Cultruled ECV304 were treated with PKC inhibitor (Calphostin C or Ro31-8220). We investigated the ability of ECV304 to adhesion and migration before and after stimulation, and detected the location of SSeCKS,F-actin before and after stimulation with the help of confocal microscopy. Cell cycles of endothelial cell were detected by FACS (fluorescence-activated cell sorting) and the expession of SSeCKS were detected by western blotting at indicated cell cycles.2. The models of acute lung injury were preparated in mice by LPS injection intraperitoneally. The pulmonary tissue content of SSeCKS mRNA and protein were assayed by real-time PCR and western blotting, and the location of SSeCKS mRNA and protein in the pulmonary were detected by methods of in situ hybridization and immumofluorescence.3. Cultured rattus pulmonary microvessel endothelial cells (RPMVEC) and ECV304 were stimulated by LPS, TNF-α,IL-1β. The expression of SSeCKS was detected by in situ hybridization,western blotting and immumofluorescence. Immunofluorescent staining with confocal laser-scanning fluorescence microscope was used to observe the effects of LPS and TNF-αon the morphology of the cytoskeleton of endothelial cells as well as the expression distribution of SSeCKS. The cytoskeleton and phosphorylation of ERK were detected after SSeCKS siRNA plasmid were transfected into endothelial cell.[Results]1. After ECV304 stimulated by FN, the expression of SSeCKS increased at concentrations and time phase dependent manner. After ECV304 pretreated by Ro31-8220 and Calphostin C, the adhesion and migration of EC were restrained, and the reorganization of SSeCKS with F-actin at the edge of ECV304 were retarded. The expression of SSeCKS upregulated after endothelial cells enter S phase of cell cycles.2. After LPS injection intraperitoneally, the expression of SSeCKS of pulmonary increased at concentrations and time phase dependent manner. The expression levels of SSeCKS of 1,5,10 and 15mg/ kg LPS groups were significantly differ with the NS group. The mRNA expression levels of SSeCKS were changing after 5mg/kg LPS administration at different observing time point, and the levels were increasing significantly with peak level at 3h. The protein levels of SSeCKS were identified with the mRNA levels. SSeCKS was located in the capillary endothelial cell in mice induced with LPS administrated.3. LPS,TNF-αand IL-1βcould induce the expressions of SSeCKS at a concentration and time dependent manner. The stimulation of LPS and TNF-αresulted in the redistribution of F-actin and SSeCKS in endothelial cells. PKC inhibitor calphostin c inhibits the effect of LPS,TNF-αon the distribution of f-actin and SSeCKS in endothelial cells. SSeCKS-siRNA could induce F-actin reorganization and partly block ERK phosphorylated induced by LPS,TNF-a and IL-1β.[Conclusions]1. SSeCKS may play important roles on EC adhesion and migration stimulated by fibronectin through interaction with PKC. The expression of SSeCKS was increased after endothelial cell enter S phase of cell cycle, which suggest SSeCKS may play roles in the control of proliferation of endothelial cell.2. After LPS injection intraperitoneally, the expression of SSeCKS of pulmonary were increasing at concentrations and time phase dependent manner, which suggest that SSeCKS, as a PKC substrate, may play roles in PKC signaling during inflammation course.3. LPS,TNF-αand IL-1βcould induce the expression of SSeCKS increased in endothelial cells. PKC inhibitor and SSeCKS siRNA blocked the redistribution of F-actin and SSeCKS in endothelial cells induced by LPS, TNF-αand IL-1β, which suggest that SSeCKS may play roles in cytoskeleton reorganization induced by PKC.