Abstract
The selenoenzyme thioredoxin reductase regulates redox-sensitive proteins involved in inflammation and carcinogenesis, including ribonucleotide reductase, p53, NFkappaB, and others. Little is known about endogenous cellular factors that modulate thioredoxin reductase activity. Here we report that several metabolites of 15-lipoxygenase-1 inhibit purified thioredoxin reductase in vitro. 15(S)-Hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic acid, a metastable hydroperoxide generated by 15-lipoxygenase-1, and 4-hydroxy-2-nonenal, its non-enzymatic rearrangement product inhibit thioredoxin reductase with IC(50) = 13 +/- 1.5 microm and 1 +/- 0.2 microm, respectively. Endogenously generated metabolites of 15-lipoxygenase-1 also inhibit thioredoxin reductase in HEK-293 cells that harbor a 15-LOX-1 gene under the control of an inducible promoter complex. Conditional, highly selective induction of 15-lipoxygenase-1 caused an inhibition of ribonucleotide reductase activity, cell cycle arrest in G(1), impairment of anchorage-independent growth, and accumulation of the pro-apoptotic protein BAX. All of these responses are consistent with inhibition of thioredoxin reductase via 15-lipoxygenase-1 overexpression. In contrast, metabolites of 5-lipoxygenase were poor inhibitors of isolated thioredoxin reductase, and the overexpression of 5-lipoxygenase did not inhibit thioredoxin reductase or cause a G cell cycle arrest. The influences of 15-lipoxygenase-1 on (1)inflammation, cell growth, and survival may be attributable, in part, to inhibition of thioredoxin reductase and several redox-sensitive processes subordinate to thioredoxin reductase.
Highlights
The selenoenzyme thioredoxin reductase regulates redox-sensitive proteins involved in inflammation and carcinogenesis, including ribonucleotide reductase, p53, NFB, and others
To determine if LOX enzymes modulate Thioredoxin reductase (TrxR), we developed HEK-293 cells that stably express either 15-LOX-1 or 5-LOX under the control of a ponasterone-responsive, heterologous transcription complex [13]
Induction of 15-LOX-1, and the resulting inhibition of TrxR caused a corresponding inhibition of ribonucleotide reductase activity, cell cycle arrest in G1, impairment of anchorage-independent growth, and accumulation of the proapoptotic protein BAX
Summary
TrxR, thioredoxin reductase; AA, arachidonic acid; COX, cyclooxygenase; DMEM, Dulbecco’s modified essential medium; DTNB, 5,5Ј-dithiobis-(2-nitrobenzoic acid); ETE, eicosatetraenoic acid; FACL-4, fatty acid CoA ligase-4; HETE, hydroxyeicosatetraenoic acid; HIF, hypoxia-inducible factor; HODE, hydroxyoctadecadienoic acid; 4-HNE, 4-hydroxy-2-nonenal; HpETE, hydroperoxy-eicosatetraenoic acid; HpODE, hydroperoxy-octadecadienoic acid; HRP, horseradish peroxidase; LA, linoleic acid; LOX, lipoxygenase; NSAID, non-steroidal anti-inflammatory drug; PBS, phosphatebuffered saline; PPAR, peroxisome proliferator activator receptor; Trx, thioredoxin; FACS, fluorescent-activated cell sorter. LOX enzymes [12] might modulate TrxR, and the regulatory control it exerts over other proteins and processes, such as ribonucleotide reductase and the cell cycle. To determine if LOX enzymes modulate TrxR, we developed HEK-293 cells that stably express either 15-LOX-1 or 5-LOX under the control of a ponasterone-responsive, heterologous transcription complex [13]. Induction of 15-LOX-1, and the resulting inhibition of TrxR caused a corresponding inhibition of ribonucleotide reductase activity, cell cycle arrest in G1, impairment of anchorage-independent growth, and accumulation of the proapoptotic protein BAX. These effects are consistent with known regulatory roles for the TrxR-Trx system. These findings support the concept that molecular interactions between 15-LOX-1 and TrxR, a vital selenoenzyme, might be relevant to carcinogenesis [17, 18] and inflammation [19, 20]
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