The Mechanism Of HCASK-Id1 Pathway On The Regulation Of P53 Expression In Vascular Endothelial Cells

BackgroundMultiple organ dysfunction syndrome (MODS) was one of the most important causes of burn mortality. The hypoperfusion of tissues and ischemia-hypoxia injury were the pathophysiological bases of postburn organ dysfunction. Many factors could activate and harm vascular endothelial cells (VECs), which could elevate vascular permeability and lead to organ edema and aggravate organ dysfunction. The activated vascular endothelial cells could release cytokines and inflammatory mediators contributing to inflammation, influencing vasomotion, causing hypercoagulable state and promoting neutrophil-vascular endothelial cell adhesion. The adhesive neutrophils release large amount of inflammatory mediators and in turn deteriorate vascular injury, worsen microcirculation dysfunction,decrease blood supply of important organs and lead to MODS. Thus, the activation and injury of vascular endothelial cells plays a key role in post-burn MODS.In early stage, vascular endothelial cells were activated and produced cytokines and mediators which functioned in inflammation. While injury persisted, the VECs would shed off from vascular wall and even proceed to cell death; the exposure of collagens under endothelium could trigger DIC and worsen microcirculation disturbance. Reconstruction of vascular function relys on damage repair of vascular cells, which was very important in the rehabilitation of MODS.There are many cellular factors and pathways involved in this process. The interfering of vascular repair could only be achieved after the thorough understanding of the complicated networks of cellular proliferation signal pathways.hCASK (human calcium/calmodulin-dependent serine protein kinase)is a member of the membrane associated guanylate kinase (MAGUK) family. As a scaffolding protein, it participates in many physiological and pathological processes including cytoskeleton assembly, cell junction formation and adhesion, protein sub-cellular localization, signal transduction and gene expression by binding associated protein with its multi-protein domain.Previous studies, using mammalian two-hybrid protein-protien interaction assays, immunoprecipitation and laser con-focal microscopy techniques, have shown that hCASK could interact with Id1 in vivo, compelling expression of hCASK could restrain cell proliferation and induce the expression of p21WAF1/CIP1 and p16INK4a.The regulation of p21 expression by hCASK attributes to the E-box in p21 gene promoter, which could bind the E protein family members like E12,E47and HEB.Thus a putative signal pathway involving proliferation effects of hCASK on ECV-304 cell might be: the binding of hCASK with Id1 repress the dominant negative effect of Id1 on bHLH protein,which promotes the binding of bHLH transcription factors (E12,E47 and HEB) with the E-box on p21 gene promoter. The activated p21 expression could lead to G1/S arrest and inhibit cell proliferation.It was also found that compelling expression of hCASK could increase the expression of p53, which may also inhibit cell proliferation and involve in cell cycle and differentiation control. It was speculated that p53 might also be one of the downstream signals of the hCASK-Id1 pathway.AimThe study was focused on cell proliferation regulation function of hCASK-Id1 pathway and whether it could promote cell proliferation via regulating p53 expression and try to find out the mechanism of p53 regulation by hCASK-Id1 pathway.Methods1. Recombinant siRNA expression plasmids, pGenesil-1-hCASK, were transfected into ECV-304 cells. Stable hCASK knockdown cell strain of ECV-304 cells was obtained under sustained pressure of G418.The influences on cell proliferation and p53 expression were examined in the screened cell strain using MTT, flow cytomytry and Western Blot techniques.2. Recombinant Id1 compelling expression plasmids were constructed using pIRES2-EGFP vector and were transfected into ECV-304 cells. Stable Id1 compelling expression cell strain of ECV-304 cells were obtained under sustained pressure of G418.The influences on cell proliferation and p53 expression were examined in screened cell strain using MTT,flow cytomytry and Western Blot techniques.3. p53 gene promoter was subcloned into the luciferase reporter vector, PGL3-basic, as wild type PGL3-p53p-luc vector.To create truncated PGL3-p53p-luc constructs, a series of 5'truncated p53 gene promoter sequences were obtain by PCR using PGL3-p53p-luc as template. The reporter gene constructs were transiently transfected into ECV-304 cells, hCASK knock down and Id1 compelling expression cell lines. Luciferase activity of cell lysate was detected using a dual-luciferase reporter assay kit. The ratio of firefly luciferase activity to renilla luciferase activity was taken as the relative activity of the PGL3 plasmids.Results1. The inhibition rate of hCASK interfering was associated with sequences selected in siRNA design, the pGenesil-1-hCASK based on the No.2 target sequence exhibited 70% inhibition rate compared with that of No.1, which had nearly no effect. The inhibition of CASK expression could promote proliferation of ECV-304 cells, which was revealed by the left shift of growth curve and the increase of G2M+S cells in hCASK-knockdown ECV-304 cells.2. Id1 compelling expression plasmid (pIRES2-EGFP-Id1)was successfully constructed and was transfected into ECV-304 cells. The stable cell strain of Id1 compelling expression , with EGFP positive rate over 90%, was achieved by G418 screening. Id1 compelling expression could lead to a left shift of cell growth curve and increase of G2M+S ratio and promote the proliferation of ECV-304 cells.3. hCASK inhibition and Id1 compelling expression could decrease the expression of p53 protein.4. The luciferase reporter gene vector containing p53 gene promoter was constructed successfully. The activation of p53 gene promoter was decreased under CASK inhibition and Id1 compelling expression, which implied that hCASK and Id1 transactivates the expression of p53 by stimulating the promoter activity of p53 and regulate its expression at transcription level.5. Deletion analysis of the p53 gene promoter revealed that a 150bp sequence in the 3'end of p53 gene promoter, containing potent transcription factor binding sequences of E-box, Ets, AP-2, HIF-1, HES1 and Sum1, was required for the activation of p53 gene expression by hCASK and Id1. 6. The expression of Id1 could influence p53 gene promoter activity in hCASK knock down cells. The inhibited activation of p53 gene promoter by hCASK down-regulation was elevated at Id1 inhibition and even lower at Id1 compelling expression.Conclusion1. Id1 compelling expression and hCASK inhibition could promote cell proliferation and inhibit p53 expression.2. hCASK and Id1 regulated p53 expression at transcriptional level, and a 150bp sequence in the 3'end of the promoter , containing binding sequences of E-box, Ets, AP-2, HIF-1, HES1 and Sum1, was required for its trans-activation by hCASK and Id1 stimulation.3. Influence of Id1 expression on p53 gene promoter activity in hCASK-knockdown cells proved a direct effect of Id1 in the p53 regulation by hCASK.4. p53 plays very important rolls in cell proliferation, cell cycle regulation and repair, as a downstream molecule of hCASK-Id1 pathway, it might endow this pathway with more physiological and pathological significances and shine a new light on the hCASK gene function .