title: Ca2+ influx via TRPC channels induces NF-kappaB-dependent A20 expression to prevent thrombin-induced apoptosis in endothelial cells.
abstract: NF-kappaB signaling is known to induce the expression of antiapoptotic and proinflammatory genes in endothelial cells (ECs). We have shown recently that Ca(2+) influx through canonical transient receptor potential (TRPC) channels activates NF-kappaB in ECs. Here we show that Ca(2+) influx signal prevents thrombin-induced apoptosis by inducing NF-kappaB-dependent A20 expression in ECs. Knockdown of TRPC1 expressed in human umbilical vein ECs with small interfering RNA (siRNA) suppressed thrombin-induced Ca(2+) influx and NF-kappaB activation in ECs. Interestingly, we observed that thrombin induced >25% of cell death (apoptosis) in TRPC1-knockdown ECs whereas thrombin had no effect on control or control siRNA-transfected ECs. To understand the basis of EC survival, we performed gene microarray analysis using ECs. Thrombin stimulation increased only a set of NF-kappaB-regulated genes 3- to 14-fold over basal levels in ECs. Expression of the antiapoptotic gene A20 was the highest among these upregulated genes. Like TRPC1 knockdown, thrombin induced apoptosis in A20-knockdown ECs. To address the importance of Ca(2+) influx signal, we measured thrombin-induced A20 expression in control and TRPC1-knockdown ECs. Thrombin-induced p65/RelA binding to A20 promoter-specific NF-kappaB sequence and A20 protein expression were suppressed in TRPC1-knockdown ECs compared with control ECs. Furthermore, in TRPC1-knockdown ECs, thrombin induced the expression of proapoptotic proteins caspase-3 and BAX. Importantly, thrombin-induced apoptosis in TRPC1-knockdown ECs was prevented by adenovirus-mediated expression of A20. These results suggest that Ca(2+) influx via TRPC channels plays a critical role in the mechanism of cell survival signaling through A20 expression in ECs.
authors.affiliation: Dept. of Pharmacology, College of Medicine, Univ. of Illinois, 835 South Wolcott Ave., Chicago, IL 60612, USA.
authors.forename: Prabhakar B
authors.initials: PB
authors.lastname: Thippegowda
authors.forename: Vandana
authors.initials: V
authors.lastname: Singh
authors.forename: Premanand C
authors.initials: PC
authors.lastname: Sundivakkam
authors.forename: Jiaping
authors.initials: J
authors.lastname: Xue
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
authors.forename: Chinnaswamy
authors.initials: C
authors.lastname: Tiruppathi
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000235:genetics; D001665:Binding Sites; D001896:Boron Compounds / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D020869:Gene Expression Profiling / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: C494826:BAX protein, human; D001896:Boron Compounds; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C498143:RELA protein, human; D050052:TRPC Cation Channels; D051996:Transcription Factor RelA; D051028:bcl-2-Associated X Protein; C096351:transient receptor potential cation channel, subfamily C, member 1; C109986:2-aminoethoxydiphenyl borate; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D013917:Thrombin; C505407:CASP3 protein, human; D053148:Caspase 3