Expression of thioredoxin reductase 1 in mammalian cells with regulation by the core promoter and use of alternative splice variants

Abstract: Reactive oxygen species are generated in cells as by-products of aerobic metabolism. To protect from damaging effects the cells possess several antioxidant systems and one of the most important is the thioredoxin system. The thioredoxin system is found in all organisms and consists of thioredoxin (TXN), thioredoxin reductase (TXNRD) and NADPH and it participates in many different types of reactions. Mammalian TXNRD1 is a selenoprotein containing a redox active selenocysteine residue and displays a broad substrate specificity. Three mammalian TXNRD isoenzymes have been identified, one cytosolic (TXNRD1), one mitochondrial (TXNRD2), and one present mainly in testis (TGR). The aim of this thesis project has been to study the regulation of TXNRD1 expression at different levels and in different cells and tissues. The expression of TXNRD1 was analyzed with Northern blots revealing lowest mRNA levels in testis and highest in liver and kidney. To further analyze the expression in kidney in situ hybridization studies were performed. This showed a highly structured expression pattern with the mRNA prominently synthesized in the proximal tubules of the medullary rays. Using bioinformatic tools combined with 5'RACE PCR we found several TXNRD1-specific transcripts with different 5' ends. In total, 20 different splice variants were subsequently identified, arising from at least three separate promoters and encoding 5 different open reading frames, including one novel glutaredoxin motif and one alternative Nterminal domain likely to encompass a signal peptide for mitochondrial targeting. We have cloned and characterized a genomic region upstream of the most common 5'-UTR variant(s) of human TXNRD1. that showed promoter activity as detected with a luciferase reporter vector. Deletion constructs revealed that the promoter activity was maintained within the - 115 to +167 region in all cell lines tested and was considered the core promoter. This core promoter contained a POU motif, which bound the Oct- I transcription factor, and two sites in the promoter were found to bind Sp1 and Sp3. Mutants with only three altered key nucleotides prevented binding of Sp1/Sp3 and resulted in a marked decrease of transcriptional activity. The promoter displayed typical characteristics for a housekeeping gene. However, using RT-PCR analysis we found that oxidized LDL (oxLDL), but not native LDL, dose dependently increased TXNRD1 mRNA in human monocyte-derived macrophages (HMDM). and THP-1 cells or HMDM transiently transfected with TXNRD1 promoter fragments allowed identification of a defined promoter region as specifically responding to the phospholipid component of oxLDL. Gel mobility shift analyses identified a 40-nucleotide stretch of the promoter carrying AP-1 and HoxA5 consensus motifs that responded with an altered shift pattern in THP-1 cells treated with oxLDL, however without evident involvement of the Fos, Jun, Nrf2 or HoxA5 transcription factors. In the literature many reports show that TXNRD1 expression is regulated in response to diverse signaling events. With a housekeeping-type promoter for the most common transcripts, we believe that regulation in response to signaling mainly may be governed on other levels than an altered core promoter activity. TXNRD1 activity in cells hence seems to be modulated by an intricate interplay of mechanisms, involving a housekeeping-type promoter in combination with alternative splice variants and transcriptional start sites, post transcriptional regulation through AU-rich elements, inactivation by electrophilic agents and by the enzyme itself modulating the effects of several key signaling molecules,