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. 2012 Jan 4;31(1):83-94.
doi: 10.1038/emboj.2011.360. Epub 2011 Sep 30.

Nuclear retention of importin α coordinates cell fate through changes in gene expression

Yoshinari Yasuda  1 , Yoichi Miyamoto, Tomoko Yamashiro, Munehiro Asally, Ayumi Masui, Chin Wong, Kate L Loveland, Yoshihiro Yoneda
Affiliations

Nuclear retention of importin α coordinates cell fate through changes in gene expression

Yoshinari Yasuda et al. EMBO J. .

Abstract

Various cellular stresses including oxidative stress induce a collapse of the Ran gradient, which causes accumulation of importin α in the nucleus and a subsequent block of nuclear protein import. However, it is unknown whether accumulated importin α performs roles in the nucleus after its migration in response to stress. In this study, we found that nuclear-retained importin α2 binds with DNase I-sensitive nuclear component(s) and exhibits selective upregulation of mRNA encoding Serine/threonine kinase 35 (STK35) by microarray analysis. Chromatin immunoprecipitation and promoter analysis demonstrated that importin α2 can access to the promoter region of STK35 and accelerate its transcription in response to hydrogen peroxide exposure. Furthermore, constitutive overexpression of STK35 proteins enhances caspase-independent cell death under oxidative stress conditions. These results collectively reveal that nuclear-localized importin α2 influences gene expression and contributes directly to cell fate outcomes including non-apoptotic cell death.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Importin α2 accumulation in the nucleus in response to the stress is efficiently released by DNase I treatment. (A) HeLa cells were either exposed to UV (254 nm, 0.3 J/cm2) or incubated with 200 μM H2O2 for 1 h or at 42°C for 1 h, as indicated. The cells were permeabilized with 0.5% Triton X-100 for 5 min and then incubated for 1 h at 30°C with DNase I, RNase or NaCl. After incubation, the cells were fixed with 3.7% formalin and indirect immunofluorescence was performed to detect endogenous importin α2 using a specific antibody. (B) HeLa cells were permeabilized with 0.5% Triton X-100 for 5 min and then incubated for 1 h at 30°C with DNase I, RNase or NaCl. After incubation, the cells were fixed with 3.7% formalin and indirect immunofluorescence was performed to detect either endogenous RCC1 or CRM1 using specific antibodies. DNA was detected with DAPI.
Figure 2
Figure 2
Transfection of importin α2 into HeLa cells. (A) Schematic illustration of enhanced green fluorescent protein (EGFP)-fused importin α2 proteins used in this transfection analysis. The C-mutant was mutated between 469 and 474 a.a., as indicated. IBB: importin β1-binding, ARM repeats: armadillo repeats. (B) Cellular localization of these encoded proteins in transiently transfected HeLa cells. HeLa cells expressing the EGFP-importin α2 full-length proteins are shown with different exposure times for the same image.
Figure 3
Figure 3
Validation of mRNA expression levels of STK35 and histone genes by quantitative PCR. HeLa cells were transfected with EGFP as a control, EGFP–importin α2 full-length or C-mutant constructs, and mRNA expression levels of STK35, each histone genes and β-actin (ACTB) as a control were analysed by qPCR. The results were from three independent experiments and were presented in comparison with values in the EGFP-expressing cells as the mean±s.e.m. (n=3 each). **P<0.01; Student's t-test. Primers are shown in Supplementary Table S-III.
Figure 4
Figure 4
Importin α2 can access to a promoter region of STK35. (A) Genomic DNA sequences of Stk35 described were constructed into the pGL3-Basic luciferase vector and were transfected into GC2 cells with an internal control plasmid pRL-TK. After 24 h incubation, the relative luciferase activity was determined by the ratio of the activities of firefly luciferase and Renilla luciferase. Values are means±s.e.m. (n=3 each). *P<0.05 and ***P<0.001; Student's t-test. (B) pGL3-Basic/−2 kbp plasmid was co-transfected with EGFP as a control (Cont.) or each EGFP–importin α construct, as indicated and with pRL-TK into GC2 cells and incubated for 48 h. Values are means±s.e.m. (n=3 each). *P<0.05 and ***P<0.001. (C) pGL3-Basic/−2 kbp plasmid was co-transfected with EGFP as a control (Cont.), EGFP–importin α2 C-mutant, EGFP–importin α2 ΔIBB-mutant, EGFP–importin α4 ΔIBB-mutant or EGFP–importin α2 ED-mutant constructs and with pRL-TK into GC2 cells and incubated for 24 h. Values are means±s.e.m. (n=3 each). **P<0.01 and ***P<0.001. Because the transfection condition for the ED mutant was different from that for the other constructs as described in Materials and methods, the data are separated by a vertical line. (D) pGL3-Basic/−0.25 kbp plasmid was co-transfected with EGFP as a control (Cont.), EGFP–importin α2 or EGFP–importin α4 and with pRL-TK into GC2 cells and incubated for 48 h. Values are means±s.e.m. (n=3 each). *P<0.05 and **P<0.01. (E) HeLa cells were exposed to 200 μM H2O2 for 30 min and subjected to immunoprecipitation using an anti-importin α2 antibody or anti-importin α3 antibody. After DNA purification from the precipitated the chromatin sample, PCR was performed to amplify sequences in the STK35 promoter region. GAPDH was used as a negative control.
Figure 5
Figure 5
Nuclear accumulation of importin α is a feature in caspase-independent cell death mode. (A) HeLa cells were exposed either to 5 mM H2O2 or to 0.5 μM staurosporine (STS) at the indicated times. After incubation, the intracellular ATP levels were measured using CellTiter-Glo assay kit. The results were risen from three independent experiments and presented in comparison with the values in 0 h cells as the mean±s.e.m. (n=3 each). (B) HeLa cells were exposed either to 5 mM H2O2 or to 0.5 μM STS for 4 h. After incubation, the cells were fixed with 3.7% formalin and indirect immunofluorescence was performed to detect endogenous importin α2 using a specific antibody. DNA was visualized by Hoechst 33342. (C) HeLa cells were pretreated in the presence or absence of 50 μM zVAD-fmk for 30 min and treated with 5 mM H2O2 or 0.5 μM STS for 4 h. Equal amounts of cellular proteins contained in total cell extracts were subjected to SDS–PAGE and analysed by western blotting for PARP and GAPDH. (D) HeLa cells were exposed to 5 mM H2O2 for 4 h and were stained with PI and then sorted by FACS. Untreated cells were showed as a control (Cont.). Values are means±s.e.m. (n=3 each) of PI-positive cells. **P<0.01; Student's t-test.
Figure 6
Figure 6
Ectopic expression of STK35 enhances non-apoptotic cell death during oxidative stress. (A) HeLa cells were treated with 1 mM H2O2 for 1 h and then placed in fresh medium. The STK35 mRNA was measured by qPCR at the indicated times. Quantification of GAPDH was used as a control for data normalization. Values are means±s.e.m. (n=3 each). **P<0.01 and ***P<0.001; Student's t-test. (B) HeLa cells were exposed to 0.5 μM STS at the indicated times and the mRNA expression levels were analysed by qPCR. Quantification of GAPDH was used as a control for data normalization. Values are means±s.e.m. (n=3 each); ***P<0.001. (C) 293F stably expressing wtag as a control (Cont.), wtag-STK35S or wtag-STK35L1 was pretreated for 30 min in the absence or presence of 50 μM zVAD-fmk and exposed to 2 mM H2O2 for 2 h. Cells were stained with PI and then sorted by FACS. Values are means±s.e.m. (n=4 each) of PI-positive cells; *P<0.05. (D) HeLa cells transfected with indicated siRNA oligonucleotides were exposed to 5 mM H2O2 for 4 h. Cells were stained with PI and then sorted by FACS. Values are means±s.e.m. (n=3 each) of PI-positive cells. **P<0.01; Student's t-test.
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