Modulation of thioredoxin reductase-2 expression in EAhy926 cells: implications for endothelial selenoprotein hierarchy.
BACKGROUND
We examined the expression of the mitochondrial selenoenzyme TrxR2 in the endothelial cell line EAhy926 under conditions known to modify its cytoplasmic counterpart TrxR1.


METHODS
Cells 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.


RESULTS
In 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 microM) only modestly increased TrxR2 protein (approximately 1.3-fold), compared with TrxR1 (approximately 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).


CONCLUSIONS
The 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.


CONCLUSIONS
These observations underpin a likely critical antioxidant role for TrxR2 and TrxR1 in the endothelium.
Crane
Michael S
MS
Department of Clinical Biochemistry, Royal Infirmary of Edinburgh, Little France, EH16 4SA Edinburgh, UK. mike.crane@luht.scot.nhs.uk
Howie
Alexander F
AF
Arthur
John R
JR
Nicol
Fergus
F
Crosley
Lynne K
LK
Beckett
Geoffrey J
GJ
D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D004357:Drug Synergism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D017879:Isothiocyanates; D051140:Selenoproteins / Q000378:metabolism*; D018038:Sodium Selenite / Q000494:pharmacology; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology; D054481:Thioredoxin Reductase 1 / Q000096:biosynthesis*; D054482:Thioredoxin Reductase 2 / Q000096:biosynthesis*; D013997:Time Factors
D007476:Ionophores; D017879:Isothiocyanates; D051140:Selenoproteins; D013454:Sulfoxides; D013861:Thiocyanates; D000001:Calcimycin; C516124:TXNRD1 protein, human; C516127:TXNRD2 protein, human; D054481:Thioredoxin Reductase 1; D054482:Thioredoxin Reductase 2; C016766:sulforaphane; D018038:Sodium Selenite