Intracellular shuttling and mitochondrial function of thioredoxin-interacting protein

Abstract

The thioredoxin-interacting protein TXNIP is a ubiquitously expressed redox protein that promotes apoptosis. Recently, we found that TXNIP deficiency protects against type 1 and 2 diabetes by inhibiting beta cell apoptosis and maintaining pancreatic beta cell mass, indicating that TXNIP plays a key role in beta cell biology. However, very little is known about the intracellular localization and function of TXNIP, and although TXNIP has been thought to be a cytoplasmic protein, our immunohistochemistry studies in beta cells surprisingly revealed a nuclear TXNIP localization, suggesting that TXNIP may shuttle within the cell. Using immunohistochemistry/confocal imaging and cell fractionation/co-immunoprecipitation, we found that, under physiological conditions, TXNIP is localized primarily in the nucleus of pancreatic beta cells, whereas oxidative stress leads to TXNIP shuttling into the mitochondria. In mitochondria, TXNIP binds to and oxidizes Trx2, thereby reducing Trx2 binding to ASK1 and allowing for ASK1 phosphorylation/activation, resulting in induction of the mitochondrial pathway of apoptosis with cytochrome c release and caspase-3 cleavage. TXNIP overexpression and Trx2 (but not cytosolic Trx1) silencing mimic these effects. Thus, we discovered that TXNIP shuttles between subcellular compartments in response to oxidative stress and identified a novel redox-sensitive mitochondrial TXNIP-Trx2-ASK1 signaling cascade.

FIGURE 1.

Nuclear localization of TXNIP in pancreatic beta cells. A, immunofluorescent detection of endogenous TXNIP in wild-type C57BL/6 mouse pancreas sections by confocal microscopy. TXNIP was detected with the JY2 antibody and fluorescein isothiocyanate-labeled secondary antibody (green); beta cells were visualized by anti-insulin antibody and Cy3-conjugated secondary antibody (red). The pancreases of three mice were analyzed by staining of two separate 10-μm sections each; a representative pancreatic islet is shown. B, effects of importin-α₁ siRNA knockdown on subcellular localization of TXNIP in INS-1 cells as assessed by cell fractionation and immunoblotting. N, nuclear fractions; C, cytoplasmic fractions. β-Actin was used as a loading control. One representative of three independent experiments is shown. C, confocal imaging of changes in subcellular localization of TXNIP in response to importin-α₁ siRNA knockdown in INS-1 cells. Merged images were taken after 48 h: TXNIP (green) and MitoTracker Red (red)).

FIGURE 2.

Shuttling of TXNIP into the mitochondria in response to oxidative stress. Shown is the immunofluorescent double staining of TXNIP (Alexa Fluor 488, green) and mitochondria (MitoTracker Red, red) in INS-1 beta cells treated without (A) or with (B) 15 μm H₂O₂ for 4 h. Merged confocal images (×200) are shown and are representative of four independent experiments. C, effects of H₂O₂ on the percentage of TXNIP protein localized in nuclear (nuc), cytoplasmic (cyto), and mitochondrial (mito) INS-1 cell fractions as analyzed by immunoblotting. Error bars represent means ± S.E. of five independent experiments.

FIGURE 3.

TXNIP interaction with mitochondrial Trx2. A, effects of oxidative stress on TXNIP-Trx2 co-immunoprecipitation. INS-1 cells were treated without (control (C)) or with H₂O₂ (15 μm for 4 h) prior to isolation of their mitochondrial fractions and immunoprecipitation (IP) with anti-TXNIP antibody JY2 and immunoblotting for Trx2. B, quantification of TXNIP-Trx2 binding in mitochondrial fractions of INS-1 cells treated with or without H₂O₂. Error bars represent means ± S.E. of three independent experiments. C, subcellular specificity of the TXNIP-Trx2 interaction. Co-immunoprecipitation experiments were performed using nuclear (nuc), mitochondrial (mito), and cytoplasmic (cyto) fractions of H₂O₂-treated INS-1 cells, and anti-TXNIP and anti-Trx2 antibodies were used for pulldown assays. One representative of three independent immunoblots is shown. D and E, representative confocal images of immunohistochemical localization of TXNIP (green) and Trx2 (red) in control and H₂O₂-treated INS-1 cells, respectively.

FIGURE 4.

Effects of Trx2 siRNA knockdown in INS-1 beta cells. INS-1 cells were transfected with specific siRNAs targeting rat Trx2 (siTrx2) or Trx1 (siTrx1) or with control scrambled (scram) siRNA 72 h prior to fractionation into cytoplasmic (C, cyto) and mitochondrial (M, mito) fractions and analysis of phosphorylated ASK1 (p-ASK1; A), cytochrome c (cyt C; B), and cytoplasmic cleaved caspase-3 (C) by immunoblotting. Three independent experiments were performed, and error bars represent means ± S.E. corrected for β-actin. Cyclooxygenase IV (COX-IV) was run as a mitochondrial marker.

FIGURE 5.

Overexpression of mitochondrial TXNIP and Trx2 oxidation. A and B, whole cell and mitochondrial TXNIP expression, respectively, in control INS-LacZ and TXNIP-overexpressing INS-TXNIP cells as assessed and quantified by immunoblotting. Error bars represent means ± S.E. of at least three independent experiments corrected for β-actin. Insets, representative immunoblots. C, redox immunoblot and quantification of Trx2 in mitochondrial fractions of control and H₂O₂-treated INS-1 cells and control INS-LacZ and INS-TXNIP cells. Cell extracts were alkylated with AMS as described under “Experimental Procedures.” Upper bands represent reduced (red) Trx2, and lower bands represent oxidized (ox) Trx2. β-Actin was used as a loading control. One representative of three independent experiments is shown. Error bars represent means ± S.E.

FIGURE 6.

Effects of increased TXNIP expression on Trx2-ASK1 interaction and ASK1 phosphorylation/activation. Shown are the effects of increased mitochondrial TXNIP induced by H₂O₂ treatment of INS-1 cells (A) or by TXNIP overexpression in INS-TXNIP cells (B) on the interaction between Trx2 and ASK1 as determined by co-immunoprecipitation (IP). Protein complexes were precipitated with anti-ASK1 antibody and analyzed for Trx2 by Western blotting, and the percent binding was quantified. C, control. Shown are the TXNIP effects on ASK1 phosphorylation/activation (C). Phosphorylated ASK1 (p-ASK1) expression in mitochondrial fractions of control INS-LacZ and INS-TXNIP cells was assessed by Western blotting with phosphorylated ASK1 (Ser⁹⁶⁷)-specific antibody and normalized for total ASK1 (t-ASK1). Error bars represent means ± S.E. of three independent experiments.

FIGURE 7.

Effects of TXNIP knockdown on Trx2-ASK1 interaction and ASK1 phosphorylation/activation. A, demonstration of specific TXNIP knockdown by siRNA in INS-1 cells. Cells were transfected with TXNIP siRNA (siTXNIP) or scrambled (scram) oligonucleotides as described under “Experimental Procedures” and analyzed 48 h later by immunoblotting for TXNIP as well as for Trx1 and Trx2 protein expression. B and C, Trx2-ASK1 interaction and ASK1 phosphorylation/activation, respectively, in response to TXNIP knockdown as assessed by co-immunoprecipitation (IP). Error bars represent means ± S.E. of at least three independent experiments. p-ASK1, phosphorylated ASK1; t-ASK1, total ASK1.

FIGURE 8.

Schematic representation of the effects of oxidative stress-induced subcellular TXNIP shuttling. A, in the cytosol, TXNIP has been shown to bind to Trx1, leading to its inactivation and dissociation from ASK1. However, our data now indicate that, under basal conditions, importin-α ensures that TXNIP is primarily localized in the nucleus. This also allows mitochondrial Trx2 to remain bound to ASK1, preventing its activation and promoting cell survival. B, in contrast, in response to oxidative stress, TXNIP translocates from the nucleus into the mitochondria (Mito), where it competes for Trx2 binding, leading to ASK1 phosphorylation/activation and initiation of the apoptotic cascade. p-ASK1, phosphorylated ASK1.