Induction of dsRNA-activated protein kinase links mitochondrial unfolded protein response to the pathogenesis of intestinal inflammation - PubMed (original) (raw)

Eva Rath et al. Gut. 2012 Sep.

Abstract

Objective: Inflammatory bowel diseases (IBDs) feature multiple cellular stress responses, including endoplasmic reticulum (ER) unfolded protein responses (UPRs). UPRs represent autoregulatory pathways that adjust organelle capacity to cellular demand. A similar mechanism, mitochondrial UPR (mtUPR), has been described for mitochondria. ER UPR in intestinal epithelial cells (IECs) contributes to the development of intestinal inflammation, and since mitochondrial alterations and dysfunction are implicated in the pathogenesis of IBDs, the authors characterised mtUPR in the context of intestinal inflammation.

Methods: Truncated ornithine transcarbamylase was used to selectively induce mtUPR in a murine IEC line. Dextran sodium sulphate (DSS) was administered to PKR (double-stranded-RNA-activated protein kinase) knockout mice to induce IEC stress in vivo and to test for their susceptibility to DSS-induced colitis. Expression levels of the mitochondrial chaperone chaperonin 60 (CPN60) and PKR were quantified in IECs from patients with IBDs and from murine models of colitis using immunohistochemistry and Western blot analysis.

Results: Selective mtUPR induction by truncated ornithine transcarbamylase transfection triggered the phosphorylation of eukaryotic translation initiation factor (eIF) 2α and cJun through the recruitment of PKR. Using pharmacological inhibitors and small inhibitory RNA, the authors identified mtUPR-induced eIF2α phosphorylation and transcription factor activation (cJun/AP1) as being dependent on the activities of the mitochondrial protease ClpP and the cytoplasmic kinase PKR. Pkr(-/-) mice failed to induce CPN60 in IECs upon DSS treatment at early time points and subsequently showed an almost complete resistance to DSS-induced colitis. Under inflammatory conditions, primary IECs from patients with IBDs and two murine models of colitis exhibited a strong induction of the mtUPR marker protein CPN60 associated with enhanced expression of PKR.

Conclusion: PKR integrates mtUPR into the disease-relevant ER UPR via eIF2α phosphorylation and AP1 activation. Induction of mtUPR and PKR was observed in IECs from murine models and patients with IBDs. The authors' results indicate that PKR might link mitochondrial stress to intestinal inflammation.

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Figures

Figure 1

Figure 1

Mitochondrial UPR (mtUPR) induces PKR via AP1. Ornithine transcarbamylase (OTCΔ) induces mtUPR in Mode-K cells. Mode-K cells were transfected with OTCΔ cDNA for 36 h. (A) OTCΔ expression and translocation to the mitochondria and CPN60 recruitment to the mitochondria analysed in mitochondrial and whole-cell protein lysates by Western blot analysis. Cytochrome c oxidase (COX) IV serves as mitochondrial loading control. Bar charts: relative (B) Grp78 gene or (C) Pkr gene expression 36 h after OTCΔ transfection. Data are shown as mean±SD (**p<0.01, t test). (B) Phosphorylation of eukaryotic translation initiation factor (eIF) 2α and cJun 30 h after OTCΔ transfection, and of glucose-regulated protein 78 (GRP78) and CHOP expression 36 h after OTCΔ transfection, as determined by Western blot analysis. (C) PERK phosphorylation 30 h after OTCΔ transfection and PKR expression 36 h after OTCΔ transfection, as determined by Western blot analysis. (D) Cell lysates were prepared 30 h after OTCΔ transfection, followed by immunoprecipitation with anti-eIF2α or anti-PKR antibody and Western blot analysis for PKR or eIF2α. (E) AP1 recruitment to the Pkr promoter and (F) CHOP recruitment to the Cpn60 promoter following OTCΔ transfection analysed by chromatin immunoprecipitation (ChIP) using anti-P-cJun or anti-CHOP antibodies and subsequent PCR analysis.

Figure 2

Figure 2

Mitochondrial unfolded protein response (mtUPR) signalling is dependent on PKR and ClpP in Mode-K cells. (A) Mode-K cells were transfected with small inhibitory RNA (siRNA) specific for Pkr or control siRNA (10 nmol/l) for 12 h, (B) or pretreated with PKR inhibitor (1 μmol/l) (C) or the ClpP inhibitor Z-LY-CMK (1 μmol/l) for 10 h. Subsequently, cells were transfected with ornithine transcarbamylase (OTCΔ) cDNA for 30 h or 36 h to determine protein phosphorylation (eukaryotic translation initiation factor (eIF) 2 and cJun) and expression (PKR and CHOP), respectively.

Figure 3

Figure 3

Pkr−/− mice fail to induce chaperonin 60 (CPN60) in intestinal epithelial cells (IECs) in response to stress induced by dextran sodium sulphate (DSS). Pkr−/− and CTRL mice received 1% DSS for 3 days, and colonic IECs were isolated (n 5). (A) IECs were analysed for expression of CPN60 and PKR by Western blot analysis. (B) Cpn60 and Pkr gene expression in IECs analysed by quantitative RT PCR. Bars represent fold induction±SD compared to Pkr−/− or CTRL mice receiving water. (a, b) Different from CTRL mice receiving water (two-way ANOVA followed by Tukey test; p=0.005 and p=0.019, respectively).

Figure 4

Figure 4

Chaperonin 60 (CPN60) and PKR are induced in primary intestinal epithelial cells (IECs) in experimental colitis. Rag2−/− and Rag2−/− ×IL-10−/− recipients were adoptively transferred with CD4 T cells from Wt or IL-10−/− mice (n=5). Mice were killed 1 week and 4 weeks later. (A) Bar charts: mean histopathologic score±SD: (b) different from (a), (f) different from (e), (h) different from (g) and (i) (ANOVA on Ranks followed by Holm–Sidak test, p<0.01); (d) different from (c) (ANOVA on Ranks followed by Dunn's test, p<0.05). Isolated large IECs for expression of CPN60 and PKR were determined by Western blot analysis. (B) Immunohistochemical staining of CPN60 and PKR in colonic tissue sections 4 weeks after T cell transfer (CPN60; PKR (green), DAPI (blue), 1800×). (C) Bar charts show the mean intensity/μm2±SD of the fluorescence signal of each group (n=5 per group; five IEC regions per mouse): (b) different from (a) and (c), (e) different from (d) and (f) (one-way ANOVA followed by Holme–Sidak test, p<0.001). Immunohistochemical double staining of CPN60 with PKR or E-cadherin, respectively, in colonic tissue sections of Rag2−/− (D) and Rag2−/− ×IL-10 −/− (E) recipients reconstituted with CD4 T cells from IL-10−/− mice for 4 weeks (PKR, E-cadherin (red), CPN60 (green), DAPI (blue), 1800×). (F) Germ-free Wt and IL-10−/− mice were mono-associated or dual-associated with En faecalis and/or E coli. Mice were killed 6 weeks later. Bar charts: mean histopathologic score±SD: (b) different from (c) and (d), (d) different from (a) and (c) (ANOVA on Ranks followed by Holme–Sidak test, p<0.01). Isolated large IECs for expression of CPN60 and PKR were determined by Western blot analysis.

Figure 5

Figure 5

Chaperonin 60 (CPN60) and PKR are induced in primary intestinal epithelial cells (IECs) from patients with inflammatory bowel diseases. (A) Primary IECs were isolated from the surgical specimens of patients with colorectal cancer (CC; non-inflammatory control), active Crohn's disease (CD) and ulcerative colitis (UC). UC patient 6: IECs of non-inflamed (N) and inflamed (I) tissue regions. Expression of CPN60 and PKR was determined by Western blot analysis. Patients 1–5 and 7–9 were analysed on the same Western blot. (B) Immunohistochemical staining of CPN60 and PKR in the surgical specimens of patients with colorectal cancer (control), active CD or UC (not the same patients as in (A)). (C) Bar charts show the mean intensity/μm2±SD of the fluorescence signal of each group (n=5 for CTRL and CD, n=3 for UC; 10 IEC regions per patient). *Different from CTRL (ANOVA on Ranks followed by Dunn's test, p<0.05). The fluorescence intensity measurements of individual patients can be found in supplemental table 2. (D) Immunohistochemical double staining of CPN60 with PKR or E-cadherin, respectively, in the surgical specimens of patients with CD and UC (PKR and E-cadherin (red), CPN60 (green), DAPI (blue), 1800×).

Figure 6

Figure 6

Schematic illustration of the integration of mitochondrial unfolded protein response (mtUPR) and endoplasmic reticulum unfolded protein response (ER UPR). PKR is activated by ClpP-dependent mtUPR signalling and in turn induces its own transcription via MEK, JNK2 and AP1. Enhanced PKR signalling then amplifies eukaryotic translation initiation factor (eIF) 2α and cJun phosphorylation, resulting in transcriptional activation of stress-related genes. Signaling of ER UPR and mtUPR converges at the levels of eIF2α phosphorylation and AP1 activation. As a consequence of ER UPR and mtUPR, nuclear-encoded compartment-specific chaperones such as glucose-regulated protein 78 (GRP78) and chaperonin 60 (CPN60) are induced.

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