Abstract
Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras - ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H2O2, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases.
Highlights
Thioredoxin-1 (TRX-1), a 12 kDa protein with conserved cysteines at its redox active site, plays major roles in cellular redox balance and signaling by maintaining a reducing intracellular microenvironment [1]
The aim of the present study is to investigate the role of Thioredoxin Interacting Protein (TXNIP) on TRX-1 nuclear migration induced by nitrosative/ oxidative stress conditions; the participation of the ERK1/2 MAP kinases as mediators of this process is investigated
Since the evidence associated with the oxidative-stressmediated negative regulation of TXNIP expression is circumstantial, we investigated the effects of 0.5 mM H2O2 and 0.5 mM SNAP on TXNIP promoter activity. 2056 base pairs corresponding to the TXNIP promoter were cloned, as well as three other fragments of 1293, 765, and 385 base pairs, before the ATG initial codon
Summary
Thioredoxin-1 (TRX-1), a 12 kDa protein with conserved cysteines at its redox active site, plays major roles in cellular redox balance and signaling by maintaining a reducing intracellular microenvironment [1]. In addition to the redox regulation of transcription factors, TRX-1 nuclear localization was associated with cell survival [3]. Nerve growth factor a major survival factor of sympathetic neurons induced TRX-1 nuclear translocation in rat pheochromocytoma PC12 cells. PD98059, an inhibitor of MEK, which phosphorylates and activates the ERK1/2 MAP kinases, suppressed nuclear translocation of TRX-1 and neuron survival [4]. Our previous work demonstrated that in HeLa cells exposed to increasing concentrations of the low molecular weight nitrosothiol S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 nuclear migration was stimulated. The SNAP-induced TRX-1 nuclear migration was directly associated with the activation of the p21Ras – ERK1/2 MAP kinases survival signaling pathway. Inhibition of p21Ras or MEK in HeLa cells prevented TRX-1 nuclear migration and increased the rate of cell death [3]
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