PKR protects colonic epithelium against colitis through the unfolded protein response and prosurvival signaling

Abstract

Background: The dsRNA-activated protein kinase (PKR) phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2α), a global regulator of protein synthesis in mammals. In addition, PKR activates several signal transduction pathways including STAT3 and AKT. PKR is activated by a number of inflammatory stimuli that are induced in the inflamed intestine. In this study we intended to determine the role of PKR in colonic epithelial cells during experimental colitis in mice.

Methods: Age- and sex-matched PKR(+/+,+/-) and PKR(-/-) littermate mice were reconstituted with wildtype bone marrow cells and subjected to dextran sodium sulfate (DSS)-induced colitis.

Results: PKR(-/-) mice displayed more severe clinical and histological manifestations upon DSS colitis compared with their PKR(+/+,+/-) littermates. In response to DSS colitis, the colonic epithelial cells of PKR(-/-) mice exhibited impaired activation of the unfolded protein response (UPR) signaling, including eIF2α phosphorylation, endoplasmic reticulum (ER) chaperone response, and ER-associated degradation (ERAD) components, as well as antioxidative stress response. In addition, the phosphorylation of STAT3 and AKT, which are protective against epithelial cell death and colonic inflammation, was also impaired in the colonic epithelial cells of PKR(-/-) mice upon DSS colitis.

Conclusions: These data demonstrate that PKR is a physiologically relevant transducer of inflammatory response signaling in colonic epithelial cells. PKR may promote the homeostasis and survival of intestinal epithelial cells (IECs) through eIF2α-mediated UPR activation, as well as the activation of STAT3 and AKT pathways. In the absence of PKR, the survival and proliferation of IECs was impaired, thus exacerbating intestinal inflammation.

Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.

Figures

FIGURE 1

PKR−/− mice are more sensitive to DSS-induced colitis. PKR−/− and PKR+/+,+/− littermates were reconstituted with wildtype bone marrow cells. After 8 weeks the mice were fed 3% DSS in drinking water for 7 days; their body weight and rectal bleeding were monitored daily. (A) Colonic epithelial cells isolated from PKR−/− mice after bone marrow transplantation show very low levels of Pkr mRNA. (B) PKR−/− mice show more significant body weight loss during DSS colitis. (C) PKR−/− mice show more severe rectal bleeding during DSS colitis. (D) PKR−/− mice show more severe colon shortening after 7-day of DSS administration. PKR+/+,+/− : n = 13; PKR−/− : n = 11; *P < 0.05, **P < 0.01. (E) Two-month-old C57BL/6J wildtype mice were reconstituted with PKR+/+ or PKR−/− bone marrow cells. After 8 weeks the mice were fed 3% DSS in drinking water for 5 days followed by 2 days of free water; their body weight was monitored daily. PKR+/+ : n = 8; PKR−/−: n = 7.

FIGURE 2

PKR−/− mice show more severe histological manifestation after 7 days of DSS administration. PKR−/− and PKR+/+,+/− littermates reconstituted with wildtype bone marrow cells were fed 3% DSS in drinking water for 7 days. (A) After DSS administration the colons were isolated and fixed for H&E staining. Representative images are shown (100×). (B) Histological scores including damage area involved and inflammatory cell infiltration taken from PKR−/− and PKR+/+,+/− littermates with DSS colitis. PKR+/+,+/− : n = 8; PKR−/− : n = 10; *P < 0.05, **P < 0.01.

FIGURE 3

PKR−/− mice show hyperactivated inflammatory response in the colon upon DSS administration. PKR−/− and PKR+/+,+/− litter-mates with wildtype bone marrow cells were fed 3% DSS in drinking water for 7 days. After DSS administration the mice were euthanized and colonic epithelial cells were collected for RNA extraction, cDNA synthesis, and Q-RT-PCR. The mRNA levels were normalized to the expression of Gapdh. PKR+/+,+/− : n = 6; PKR−/− : n = 5; *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 4

Activation of the UPR signaling and prosurvival pathways is impaired in PKR−/− colonic epithelial cells upon DSS colitis. PKR−/−and PKR+/+,+/− littermates with wildtype bone marrow cells were fed 3% DSS in drinking water for 6 days. (A) After DSS administration the mice were euthanized and the colonic epithelial cells were isolated for protein extraction and immunoblotting. (B) After DSS administration the mice were euthanized and the colonic epithelial cells were isolated for RNA extraction, cDNA synthesis, and Q-RT-PCR. The mRNA levels were normalized to the expression of Gapdh. (C) After DSS administration the mice were euthanized and the colons were isolated, fixed, and paraffin-embedded for immunohistochemical staining of BiP, ATF4, and phospho-STAT3 (200× or 400×). PKR+/+,+/− : n = 6; PKR−/− : n = 6; *P < 0.05, **P < 0.01.