The Effect Of Cell Cycle Regulative Protein Hcdc14A On Smooth Muscle Cells Proliferation

Partâ… The expression of protein hcdc14A in HVSMC and other cells with different conditionsOBJECTIVE:To detect the expression of hcdc14A in HVSMC and other cells with different conditions.METHODS:High glucose and insulin were employed as intervention against HVSMCs in different ways,and THP-1 cells were also treated by glucose,LDL and FFA.After the total protein was abstracted from these treated cells, the expression of protein hcdc14A was detected by Western Blot.The proliferation ability of HVSMCs which were treated by glucose was further confirmed by XTT and a special analyzing device.RESULTS:The proliferation ability of HVSMCs was improved after being treated by glucose.The expression of protein hcdc14A was also increased in both HVSMC and THP-1 cells,which were treated by glucose,insulin, LDL and FFA in different ways.CONCLUSIONS:The expression of protein hcdc14A was increased in cells which were treated by glucose,insulin,LDL and FFA in different ways.This result indicated that hcdc14A might influence normal cell division cycle and/or physiologic function by antagonizing activity of Cdk/cyclin complex,influencing intracellular signal transduction pathway,or interfering with cellular mitosis process.Thus hcdc14A may play a role in diabetic vascular disease. Partâ…¡The interaction between hcdc14A and cellular cyclical proteins in diabetic vascular diseaseOBJECTIVE:To detect the interaction between hcdc14A and cellular cyclical proteins in diabetic vascular disease.METHODS:HVSMCs were treated by glucose and insulin.After the total protein was abstracted from these treated cells,the expressions of protein cyclinB and P53 were detected by Western Blot.The full-length cDNA sequence was subcloned into green fluorescence protein vector pEGFP to study the location of hcdc14A in the HEK293 cells and Hela cells by confocal microscope.We also tried to detect the expressions of cyclinB and P53 when hcdc14A was overexpressed.RESULTS:The expressions of protein cyclinB and P53 both were increased in the cells which were treated by glucose and insulin.Hcdc14A was located on cellular centrosome.The expression of P53 increased when hcdc14A was overexpressed,while cyclinB didn't.CONCLUSIONS:Hcdc14A and cyclinB might have synergic interaction to effect cellular mitosis process in cells which were treated by glucose and insulin.Hcdc14A might antagonize the activity of Cdk/cyclin complex by dephosphorylation P53,and also induce the expression of mutational type of P53,so as to decrease the activity of wild type P53.Thus these results suggested that the interaction between hcdc14A and cellular cyclical proteins might play a role in diabetic vascular disease. Partâ…¢The test of hcdc14A candidate interaction partnerOBJECTIVE:To test the interaction between hcdc14A and its candidate partner protein Zipk,which was found by yeast two-hybrid system screening.METHODS: Different expression plasmids were constructed and cotransfected into cells to study their cellular localizations under fluorescence microscopy.Pulldown assay and coimmunoprecipitation assay were used to test the physical interaction between hcdc14A and Zipk.RESULTS:Hcdc14A and Zipk co-localized on centrosome in cells.Their physical interaction was verified by Pulldown assay and coimmunoprecipitation assay.Zipk could interact with the N terminal of hcdc14A. CONCLUSIONS:The proven fact that Zipk could interact with hcdc14A was an important foundation for our further research.The known functions of protein Zipk might relate to hcdc14A,and Zipk also could promote smooth muscle contractility. Thus Zipk might also play a role in diabetic vascular disease,by itself or/and together with hcdc14A. Partâ…£The expression of protein Zipk in HVSMC and its interaction with protein hcdc14AOBJECTIVE:To detect the expression of protein Zipk in HVSMCs,which were treated by glucose and insulin and to confirm its interaction with hcdc14A. METHODS:Different expression plagmids were constructed and cotransfected into cells to study the changes of their cellular localizations so as to investigate their interaction.Total protein was abstracted from HVSMCs which were treated by glucose and insulin and the expressions of protein Zipk was detected by Western Blot. We also tried to observe the expression of hcdc14A when Zipk was overexpressed RESULTS:Zipk altered the cellular location of hcdc14A-N terminal,which was released from nucleus to cytoplasm,when Zipk and hcdc14A-N plasmids were cotransfected into cells.While the mutant plasmid of Zipk without kinase activity could not change the cellular location of hcdc14A-N terminal.The expression of protein Zipk was increased in the cells which were treated by glucose,and decreased when treated by insulin.The expression of hcdc14A was also increased when Zipk was overexpressed in HEK293 cells.CONCLUSIONS:The colocalizations of Zipk and hcdc14A may depend on the kinase activity of Zipk.The release of hcdc14A-N terminal from nucleus to cytoplasm activated by Zipk might promote its phosphatase activity.Both the interaction between Zipk and hcdc14A and Zipk itself,which could promote smooth muscle contractility,might play a role in diabetic vascular disease.