G Protein-Coupled Receptor Kinase4-Induced Cellular Senescence And Its Senescence-A Ssociated Gene Expression Profiling

Objective: To explore the effect of GRK4 on cellular senescence and its mechanism(s).Methods: HEK293 cells were seeded onto?10cm plates and transiently transfected with pEGFP-GRK4 by using lipofectamine 2000 reagent at 70%-80% cell confluence according to manufacturer's instructions.Twenty-four hours after the transfection,later,isolated GRK4-GFP expressing positive(GRK4(+))and negative(GRK4(-))cell populations were isolated by fluorescence-activated cell sorting(FACS).Cell immunofluorescence staining and Western Blot testing for GRK4 protein were conducted.Cell proliferative ability was determined by Cell Counting Kit(CCK-8).Cell cycle distribution was analyzed by a flow cytometer.Senescence-associated ?-galactosidase(SA-?-Gal)activity staining was used for determining the cellular senescence phonotype.Expression profiling of 78 senescence-related genes was analyzed by qRT-PCR,and further validated partially by Western blotting.SPSS 18.0 software was used for the data analysis.Results: GRK4-positive and-negative cells were successfully sorted out by fluorescence-activated cell sorting(FACS).Overexpression of GRK4 inhibits cell proliferation and arrests cell cycle progression in G0/G1 phase,accompanied with significant increase of senescence-associated-?-galactosidase(SA-?-Gal)activity.Expression profiling analysis of 78 senescence-related genes by qRT-PCR showed a total of 17 genes significantly changed in GRK4(+)cells(? 2 fold,p<0.05).Among these,9 genes — AKT1,p16INK4,p27KIP1,p19INK4,IGFBP3,MAPK14,PLAU,THBS1,TP73 — were up-regulated,while 8 genes,Cyclin A2,Cyclin D1,CDK2,CDK6,ETS1,NBN,RB1,SIRT1,were down-regulated.The increase in cyclin-dependent kinase inhibitors(p16,p27)and p38 MAPK proteins(MAPK14)was validated by Western blotting.Neither p53 nor p21Waf1/Cip1 protein was detectable,suggesting no p53 activation in the HEK293 cells.Conclusion: These results unveil a novel function of GRK4 on triggering a p53-independent cellular senescence,which involves an intricate signaling network.