Modulation of thioredoxin reductase-2 expression in EAhy926 cells: Implications for endothelial selenoprotein hierarchy

BackgroundWe examined the expression of the mitochondrial selenoenzyme TrxR2 in the endothelial cell line EAhy926 under conditions known to modify its cytoplasmic counterpart TrxR1.MethodsCells were cultured with varying concentrations of selenite, sulforaphane or the Ca2+ ionophore A23187 for 72-h, prior to assay of TrxR concentration and activity. Further cultures underwent prolonged (7-day) Se-depletion before selenoprotein measurement.ResultsIn Se-deficient cultures, neither Se, A23187 or sulforaphane affected TrxR2 concentration, while these treatments induced TrxR1 concentration (p < 0.05). When co-incubated, optimal concentrations of Se (40 nM) and sulforaphane (4 μM) only modestly increased TrxR2 protein (∼ 1.3-fold), compared with TrxR1 (∼ 4-fold). In Se-deficient cells, TrxR activity was unaffected by sulforaphane or A23187. Prolonged Se-depletion caused a comparatively small reduction in TrxR2 (66% TrxR2 retained) against TrxR1 and glutathione peroxidase-1 activity (38% and 17% retained, respectively).ConclusionsThe relative resistance of TrxR2 to Se-deprivation and induction by sulforaphane and A23187 suggests TrxR2 lies near the top of the selenoprotein hierarchy in EAhy926 cells and exhibits near maximum expression under a range of culture conditions. In Se deficiency an inactive (possibly truncated) TrxR1 is produced in response to stimulus by sulforaphane and A23187.General significanceThese observations underpin a likely critical antioxidant role for TrxR2 and TrxR1 in the endothelium.