Study On The Mechanism Of Peptidase Inhibitor PI16 Inhibiting Warfarin-induced Aortic Valve Calcification

Aims:Warfarin induced cardiac aorticvalvular disease(CAVD)is an newly indispensable side effect needing more concem.The exact mechanism of warfarin causing calcification remains unknown.Peptidase inhibitor 16(PI 16),also called PSP94-binding protein(PSPBP),was firstly identified as a binding protein of prostate secretory protein of 94 amino acids(PSP94)in prostate cancer patients and associated with prognosis.Our study was designed to investigate the role of PI 16 in aortic valve calcification induced by warfarin.Methods:Expression of PI 16 was determined by quantitative reverse transcription polymerase chain reactions(qPCRs)and immunostaining in normal and calcific aortic valvestissue samples.Porcine aortic valve interstitial cells(AVICs)were isolated and cultured in complete medium,and then infected with recombinant adenovirous Ad-MCMV-PI16-HA-P2A-GFP according to manufacturer's instruction.After 48h,cells were cultured in promineralizing medium containing DMEM supplemented with 1%FBS,1%penicillin-streptomycin,10 ?M warfarin(Sigma),inorganic phosphate(Pi;final concentration 1.6 mM)to induce calcification.Cells harvested to used for qPCR,western blot,prolifieration assays,migration assays,and Alizarin red S staining,Calcium Quantification.And we constructed a series of PI 16 fragment expression vectors to transfected 293T cells with p53 overexpression vector to further confirm the interaction between PI16 and p53 by using reciprocalCo-immunoprecipitation(Co-IP).Then we explored the epigenetic mechanism of P53 downregulation by PI16 through Chromatin Immunoprecipitation(Ch-IP).In vivo,we test the levels of calcium deposition and ostegenic markers in wildtype and PI 16 transgenic mice to verify anti-calcific effect of PI16.Results:1.Immunostaining and quantitative PCR showed PI 16 was specificly expressed by AVICs and downregulated in calcific aortic valve tissues.2Intracellular signaling analysis revealed PI 16 inhibited warfarin induced P53 and Akt signaling.3.Co-immunoprecipitation and immunostaining showed PI 16 directly bind to P53 via 41-50 amino acids,then reduced P53 nuclear import and HDAC1 nuclear export.Chromatin immunoprecipitation(ChIP)assays revealed PI 16 epigenetically regulated P53 transcription status via reducing the recruitment of acetylated histone H3,H4 and P300/CBP,but increasing HDAC1 to P53 and P21 promoters.4.P53 overexpression or HDAC inhibition reversed the anti-calcific effects and histone deacetylation by PI16 suggesting a P53 and histone acetylation dependent mechanism.Conclusions:Our data suggest a P53 involved mechanism in warfarin induced aortic valve calcification.PI 16 is a novel P53 binding protein which attenuates AVIC and aortic valve calcification via epigenetically modulating P53 and associated osteogenic genes expression.