The unfolded protein response and chemical chaperones reduce protein misfolding and colitis in mice

Abstract

Background & aims: Endoplasmic reticulum (ER) stress has been associated with development of inflammatory bowel disease. We examined the effects of ER stress-induced chaperone response and the orally active chemical chaperones tauroursodeoxycholate (TUDCA) and 4-phenylbutyrate (PBA), which facilitate protein folding and reduce ER stress, in mice with colitis.

Methods: We used dextran sulfate sodium (DSS) to induce colitis in mice that do not express the transcription factor ATF6α or the protein chaperone P58(IPK). We examined the effects of TUDCA and PBA in cultured intestinal epithelial cells (IECs); in wild-type, P58(IPK-/-), and Atf6α(-/-) mice with colitis; and in Il10(-/-) mice.

Results: P58(IPK-/-) and Atf6α(-/-) mice developed more severe colitis following administration of DSS than wild-type mice. IECs from P58(IPK-/-) mice had excessive ER stress, and apoptotic signaling was activated in IECs from Atf6α(-/-) mice. Inflammatory stimuli induced ER stress signals in cultured IECs, which were reduced by incubation with TUDCA or PBA. Oral administration of either PBA or TUDCA reduced features of DSS-induced acute and chronic colitis in wild-type mice, the colitis that develops in Il10(-/-) mice, and DSS-induced colitis in P58(IPK-/-) and Atf6α(-/-) mice. Reduced signs of colonic inflammation in these mice were associated with significantly decreased ER stress in colonic epithelial cells.

Conclusions: The unfolded protein response induces expression of genes that encode chaperones involved in ER protein folding; these factors prevent induction of colitis in mice. Chemical chaperones such as TUDCA and PBA alleviate different forms of colitis in mice and might be developed for treatment of inflammatory bowel diseases.

Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Loss of P58IPK exacerbates DSS-induced colitis in mice due to a hyperactivated proapoptotic UPR. P58IPK+/− and P58IPK−/− littermates with wild-type bone marrow cells were fed 2.5% DSS in drinking water for 5 days, followed by 2 days of fresh water. (A) Body weight and (B) rectal bleeding were measured over 7 days. (C) After administration of DSS, the colons were isolated and fixed for H&E staining. Representative images are shown (original magnification 100×). (D) Histologic scores were measured in mice with DSS-induced colitis. (E) The proapoptotic transcription factor CHOP was induced whereas antiapoptotic Bcl2 was reduced in P58IPK−/− IECs. (F) IHC shows CHOP is induced in P58IPK−/− colonic epithelium with DSS-induced colitis. n = 7 for each group. *P < .05, **P < .01, ***P < .001.

Figure 2

Loss of ATF6α exacerbates DSS-induced colitis in mice due to defective ER chaperone induction and a hyperactivated proapoptotic UPR. Atf6α+/+, Atf6α+/−, and Atf6α−/− littermates with wild-type bone marrow cells were fed 3% DSS in drinking water for 5 days, followed by 2 days of fresh water. (A) Body weight and (B) rectal bleeding were measured over 7 days. After administration of DSS, the colons were isolated and fixed for H&E staining. Representative images are shown (C; original magnification 100×). (D) Histologic scores were measured in mice with DSS-induced colitis. (E) The expression of ER chaperones BiP, GRP94, and P58IPK is reduced whereas the proapoptotic IRE1α-JNK pathway and caspase-3 are activated in Atf6α−/− IECs with DSS-induced colitis. (F) IHC shows that expression of the ER chaperone BiP is impaired whereas the proapoptotic transcription factor CHOP is increased in Atf6α−/− colonic epithelium with DSS-induced colitis. n = 14 or 15. *P < .05, **P < .01, ***P < .001.

Figure 3

TUDCA and PBA alleviate inflammatory stimuli-induced ER stress in IECs in vitro; TUDCA ameliorates DSS-induced colitis by reducing ER stress in colonic epithelium in vivo. (A) IEC-6 cells were treated with a combination of inflammatory cytokines (TNF-α, MCP-1, and IL-1β) for 8 hours or pretreated with 5 mmol/L TUDCA or PBA for 4 hours, followed by treatment with the same inflammatory signals with 5 mmol/L TUDCA or PBA for 8 hours. The cells were then collected for RNA extraction and quantitative reverse-transcription polymerase chain reaction. The messenger RNA levels were normalized to the expression of 18S ribosomal RNA. Wild-type mice were fed 2.5% DSS in drinking water and received 500 mg/kg body wt TUDCA or the same amount of phosphate-buffered saline (PBS) without TUDCA daily by gavage (n = 8 or 10 per group). (B) Body weight and (C) rectal bleeding were measured over 8 days. (D) After administration of DSS, the colons were isolated and fixed for H&E staining. Representative images are shown (original magnification 40×). (E) Histologic scores are shown from TUDCA-treated and control mice with DSS-induced colitis. (F) Expression of genes associated with inflammation, oxidative stress, and apoptosis in colonic mucosa as well as ER stress markers in colonic IECs is shown (normalized to the expression of Gapdh). n = 8 for each group; *P < .05, **P < .01, ***P < .001.

Figure 4

PBA alleviates DSS-induced colitis by reducing ER stress in colonic epithelium. Wild-type mice were fed 2% DSS in drinking water and received 500 mg/kg body wt PBA or the same amount of PBS without PBA daily by gavage. (A) Body weight and (B) rectal bleeding were measured over 10 days. (C) Colon lengths were measured after induction of DSS colitis. (D) After administration of DSS, the colons were isolated and fixed for H&E staining. Representative images are shown (original magnification 40×). (E) Histologic scores are shown from control and PBA-treated mice with DSS-induced colitis. (F) Expression of genes associated with inflammation, oxidative stress, and ER stress in colonic mucosa as well as ER stress markers in colonic IECs is shown (normalized to the expression of Gapdh). n = 8 for each group; *P < .05, **P < .01, ***P < .001.

Figure 5

Either TUDCA or PBA mitigates inflammation in mice with chronic colitis. (A) Wild-type mice with established chronic DSS-induced colitis received 300 mg/kg body wt TUDCA or the same amount of PBS without TUDCA (control) daily by gavage for 10 days. (a) Colon from a mouse with chronic colitis fed with PBS alone (control), showing severe epithelial ulceration (arrowheads), loss of goblet cell morphology (mucodepletion; bracket), and transmural inflammatory infiltrate (arrow). Inset shows transmural inflammatory infiltrate at the edge of an ulcer (bracket). (b) Mice with chronic colitis treated with TUDCA display reduced mucosal damage and inflammation (original magnification 40×; 100× for inset). (B) Histologic scores, including damaged area involved, mucodepletion of glands, and inflammatory cell infiltration, are shown from TUDCA-treated and control mice with chronic DSS-induced colitis. n = 6 or 8 for each group. (C) Wild-type mice with established chronic DSS-induced colitis received 500 mg/kg body wt PBA or the same amount of PBS without PBA (control) daily by gavage for 10 days. (a) Cecum from a mouse with chronic colitis fed with PBS alone (control) showing severe ulceration (arrowheads), inflammatory infiltrates (arrows), and loss of goblet cell morphology (bracket). (b) Cecum from a mouse with chronic colitis treated with PBA showing reduced mucosal damage and inflammation (original magnification 40×). (D) Histologic scores are shown from PBA-treated and control mice with chronic DSS-induced colitis. n = 9 or 13 for each group. *P < .05, **P < .01.

Figure 6

Feeding of TUDCA or PBA corrects the defects of P58IPK−/− and Atf6α−/− mice in response to DSS-induced colitis. P58IPK+/+, P58IPK+/−, and P58IPK−/− mice and Atf6α+/+, Atf6α+/−, and Atf6α−/− mice were reconstituted with wild-type bone marrow cells and fed 3% DSS in drinking water for 5 days, followed by 2 days of fresh water. During the same period, the animals received 500 mg/kg body wt TUDCA, PBA, or the same amount of PBS without TUDCA/PBA daily by gavage. (A and C) Body weight was measured over the 7-day period. (B and D) After treatment, the colons were isolated and fixed for H&E staining and histologic scoring. n = 7–14 for each group; *P < .05, **P < .01, ***P < .001.

Figure 7

Either TUDCA or PBA alleviates chronic colitis in Il10−/− mice. Il10−/− mice with piroxicam-induced colitis received 2 mg/mL PBA or TUDCA in the drinking water for 3 weeks. (A) Feeding of TUDCA or PBA reduces signs of chronic colitis in Il10−/− mice. Periodic acid–Schiff (PAS) staining shows mucin in goblet cells, and trichrome staining indicates collagen deposition in the colon. (B) Histologic scores are shown using the standard of Otuska et al. (C) Histologic scores are shown using the standard of Berg et al. (D) Histologic scores are shown for colonic fibrosis. n = 10 –11 for each group. (E) The mice were killed after the experiment, and the colonic IECs were isolated for protein extraction and Western blotting. Representative immunoblots are shown. 1, no piroxicam-induced colitis; 2, colitis; 3, colitis → TUDCA; 4, colitis → PBA. *P < .01, **P < .001, ***P < .0001.