Determinants of human plasma glutathione peroxidase (GPx-3) expression.
Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing protein with antioxidant properties. GPx-3 deficiency has been associated with cardiovascular disease and stroke. The regulation of GPx-3 expression remains largely uncharacterized, however, and we studied its transcriptional and translational determinants in a cultured cell system. In transient transfections of a renal cell line (Caki-2), the published sequence cloned upstream of a luciferase reporter gene produced minimal activity (relative luminescence (RL) = 0.6 +/- 0.4). Rapid amplification of cDNA ends was used to identify a novel transcription start site that is located 233 bp downstream (3') of the published site and that produced a >25-fold increase in transcriptional activity (RL = 16.8 +/- 1.9; p < 0.0001). Analysis of the novel GPx-3 promoter identified Sp-1- and hypoxia-inducible factor-1-binding sites, as well as the redox-sensitive metal response element and antioxidant response element. Hypoxia was identified as a strong transcriptional regulator of GPx-3 expression, in part through the presence of the hypoxia-inducible factor-1-binding site, leading to an almost 3-fold increase in expression levels after 24 h compared with normoxic conditions (normalized RL = 3.5 +/- 0.3 versus 1.2 +/- 0.1; p < 0.001). We also investigated the role of the translational cofactors tRNA(Sec), SECIS-binding protein-2, and SelD (selenophosphate synthetase D) in GPx-3 protein expression. tRNA(Sec) and SelD significantly enhanced GPx-3 expression, whereas SECIS-binding protein-2 showed a trend toward increased expression. These results demonstrate the presence of a novel functional transcription start site for the human GPx-3 gene with a promoter regulated by hypoxia, and identify unique translational determinants of GPx-3 expression.
Bierl
Charlene
C
Whitaker Cardiovascular Institute and the Evans Department of Medicine, Boston, MA 02118, USA.
Voetsch
Barbara
B
Jin
Richard C
RC
Handy
Diane E
DE
Loscalzo
Joseph
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015687:Cell Hypoxia / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005979:Glutathione Peroxidase / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D017279:Selenocysteine / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D024363:Transcription Initiation Site; D014162:Transfection; D014982:Xenopus laevis / Q000235:genetics
D018076:DNA, Complementary; D016601:RNA-Binding Proteins; C121530:SECISBP2 protein, human; D013438:Sulfhydryl Compounds; D017279:Selenocysteine; D012343:RNA, Transfer; C486484:GPX3 protein, human; D005979:Glutathione Peroxidase; D008156:Luciferases; D010770:Phosphotransferases