title: A requirement for thioredoxin in redox-sensitive modulation of T-cadherin expression in endothelial cells.
abstract: T-cad (T-cadherin), a glycosylphosphatidylinositol-anchored cadherin superfamily member, is expressed widely in the brain and cardiovascular system, and absent, decreased, or even increased, in cancers. Mechanisms controlling T-cad expression are poorly understood. The present study investigated transcriptional regulation of T-cad in ECs (endothelial cells). Conditions of oxidative stress (serum-deprivation or presence of H(2)O(2)) elevate T-cad mRNA and protein levels in ECs. Reporter gene analysis, using serially deleted T-cad promoter stretches ranging from -99 to -2304 bp, located the minimal promoter region of T-cad within -285 bp from the translation start site. Reporter activity in ECs transfected with the -285 bp construct increased under conditions of oxidative stress, and this was normalized by antioxidant N-acetylcysteine. An electrophoretic-mobility-shift assay revealed a specific nucleoprotein complex unique to -156 to -203 bp, which increased when nuclear extracts from oxidatively stressed ECs were used, suggesting the presence of redox-sensitive binding element(s). MS analysis of the nucleoprotein complex unique to -156 to -203 bp after streptavidin-agarose pull-down detected the presence of the redox-active protein thioredoxin. The presence of thioredoxin-1 in a nuclear extract from oxidatively stressed ECs was demonstrated after immunoprecipitation and immunoblotting. Transfection of ECs with thioredoxin-1 small interfering RNA abrogated oxidative-stress-induced up-regulation of T-cad transcripts and protein. We conclude that thioredoxin-1 is an important determinant of redox-sensitive transcriptional up-regulation of T-cad in ECs.
authors.affiliation: Department of Research, Cardiovascular Laboratories, Basel University Hospital, Hebelstrasse 20, CH4031 Basel, Switzerland.
authors.forename: Manjunath B
authors.initials: MB
authors.lastname: Joshi
authors.forename: Danila
authors.initials: D
authors.lastname: Ivanov
authors.forename: Maria
authors.initials: M
authors.lastname: Philippova
authors.forename: Emmanouil
authors.initials: E
authors.lastname: Kyriakakis
authors.forename: Paul
authors.initials: P
authors.lastname: Erne
authors.forename: Thérèse J
authors.initials: TJ
authors.lastname: Resink
mesh_terms: D001483:Base Sequence; D015820:Cadherins / Q000235:genetics*; D002470:Cell Survival; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013879:Thioredoxins / Q000378:metabolism*; D014162:Transfection; D014471:Umbilical Veins
chemical_list: D015820:Cadherins; D017931:DNA Primers; C099972:H-cadherin; D034741:RNA, Small Interfering; D013879:Thioredoxins