Adaptive unfolded protein response attenuates alcohol-induced pancreatic damage

Abstract

Background & aims: Endoplasmic reticulum (ER) stress responses (collectively known the unfolded protein response [UPR]) have important roles in several human disorders, but their contribution to alcoholic pancreatitis is not known. We investigated the role of X-box binding protein 1 (XBP1), a UPR regulator, in prevention of alcohol-induced ER stress in the exocrine pancreas.

Methods: Wild-type and Xbp1(+/-) mice were fed control or ethanol diets for 4 weeks. Pancreatic tissue samples were then examined by light and electron microscopy to determine pancreatic alterations; UPR regulators were analyzed biochemically.

Results: In wild-type mice, ethanol activated a UPR, increasing pancreatic levels of XBP1 and XBP1 targets such as protein disulfide isomerase (PDI). In these mice, pancreatic damage was minor. In ethanol-fed Xbp1(+/-) mice, XBP1 and PDI levels were significantly lower than in ethanol-fed wild-type mice. The combination of XBP1 deficiency and ethanol feeding reduced expression of regulators of ER function and the up-regulation of proapoptotic signals. Moreover, ethanol feeding induced oxidation of PDI, which might compromise PDI-mediated disulfide bond formation during ER protein folding. In ethanol-fed Xbp1(+/-) mice, ER stress was associated with disorganized and dilated ER, loss of zymogen granules, accumulation of autophagic vacuoles, and increased acinar cell death.

Conclusions: Long-term ethanol feeding causes oxidative ER stress, which activates a UPR and increases XBP1 levels and activity. A defective UPR due to XBP1 deficiency results in ER dysfunction and acinar cell pathology.

Figure 1. Ethanol feeding activates UPR and XBP1 in rat pancreas

Rats were fed control (C) or ethanol (E) diets for 6 weeks. (A) Electron micrographs showing ultrastructure of pancreatic acinar cells. Ethanol-fed rats exhibit well preserved acinar architecture (right panel) compared to controls (left panel), but ER is dilated (closed arrows). Bars, 2 μm. (B) RT-PCR showing higher XBP1 mRNA splicing (sXBP1) in pancreas from ethanol-fed than in control-fed rats. (C) Western blot analysis of spliced (sXBP1) and unspliced (uXBP1), Grp78 and GAPDH (loading control). In B and C each lane represents an individual rat. Graph shows densitometry for XBP1 and Grp78 (means±SEM; n=5). * P<.05 vs. C diet (Student t-test).

Figure 2. Pancreatic acini isolated from Xbp1^+/− mice are prone to ER stress

(A) and (B) Pancreatic acini isolated from wild-type and Xbp1^+/− mice were incubated with and without CCK-8 for 30 min. (A) Immunoblot shows protein levels of sXBP1 and uXBP1, phospho-PERK, phospho- and total eIF2α after stimulation with 0.1 nM or 100 nM CCK-8. (B) Graph shows amylase secretion by acini stimulated with CCK-8 (mean±SEM, 4 independent studies). Immunoblot shows cellular amylase content. * P<.05 vs. wild-type (two-way ANOVA and Tukey post-tests: CCK, P<.001; genotype, P=0.002, interaction, P=0.624). (C) Immunoblots show amylase content in media, and cellular levels of sXBP1, amylase, PDI and ERK1/2 (loading control) in unstimulated acini cultured for 24 h (n=2 independent experiments). (D) Graph shows densitometry for immunoblots depicted in panel C (mean±SEM, n=3). * P<.05 vs. wild-type (Student t-test).

Figure 3. Ethanol feeding causes significant pancreatic damage in Xbp1^+/− mice

Wild-type and Xbp1^+/− mice were fed either control or ethanol diet. (A) Representative pancreatic H&E staining from ethanol-fed wild-type (panels a-b) and Xbp1^+/− mice (panels c-d) after 4-weeks on diets. Pancreas of ethanol-fed wild-type mice appeared normal. Pancreas of ethanol-fed Xbp1^+/− mice displayed patchy areas of acinar cell necrosis (c, white arrow), and areas with abundant stroma cells and tubular complexes (c, black arrows). In panel d, the typical eosin staining in apical areas of acini is decreased, suggesting loss of zymogen granules. Pancreatic sections from all control-fed mice appeared normal (not shown). Bars, 20 μm. (B) Electron micrographs from ethanol-fed wild-type (panel a) and Xbp1^+/− mice (panels b-d) demonstrate features of ER stress in Xbp1^+/− mice. Acinar cells in wild-type mice show regular ultrastructure, with a slightly dilated ER (a, closed arrows). In Xbp1^+/− mice, acinar cells often displayed extensively dilated ER (b; higher magnification in c) with occasional accumulation of dense materials (d). ZG, zymogen granules (open arrows); AV, autophagic vacuoles (arrowheads); L, acinar lumen; Bars, 1 μm. (C) Graph shows percentage of acinar cell necrosis per total pancreatic area evaluated in H&E tissue sections (median±25th and 75th percentiles; n=4–6 mice). * P<.05 vs. wild-type mice (Kruskal–Wallis test and Dunn posthoc test). (D). Graph shows number of zymogen granules per cell section measured in EM pancreatic sections (mean±SEM, n=4–6 mice). * P<.05 vs. wild-type mice (two-way ANOVA and Tukey post-tests). Criteria for quantification in C and D are explained in Supplementary Materials. (E) Immunoblots showing pancreatic levels of amylase and GAPDH (loading control). Each lane represents an individual mouse. Graph shows amylase quantification relative to control-fed wild-type mice (mean±SEM, n=5).

Figure 4. Ethanol-induced XBP1 activation is blunted in pancreas of Xbp1^+/− mice

Wild-type and Xbp1^+/- mice were fed either control or ethanol diet for 4 weeks. (A) Pancreatic XBP1 splicing was assessed by RT-PCR. GAPDH was examined as a housekeeping gene. (B) Immunoblot analyses of spliced and unspliced XBP1, IRE1α, and ERK1/2 (loading control) in pancreas from wild-type and XBP+/− mice (panels A, B, each lane represents an individual mouse). (C) Graphs show quantification of sXBP1 and IRE1α protein expression relative to control-fed wild-type mice (mean±SEM, n=5 mice). * P<.05 vs. wild-type mice; # P<.05 vs. C diet (two-way ANOVA and Tukey post-tests).

Figure 5. XBP1 deficiency augments pancreatic UPR in ethanol-fed mice

Immunoblot analyses of key UPR regulators in pancreas from wild-type and Xbp1^+/− mice fed control or ethanol diet for 4 weeks. Shown are expression levels of phospho-Thr980-PERK, total and phospho-Ser51-eIF2α, Grp78 (A), and ATF4 (B). ERK1/2 was used as loading control. Each lane represents an individual mouse. (C) Densitometric quantification of phospho-PERK, phospho-eIF2α and ATF4 levels. Graphs show increased protein content in ethanol-fed Xbp1^+/− mice, relative to ethanol-fed wild-type mice. * P<.05 vs. wild-type mice (Student t-test).

Figure 6. XBP1 deficiency diminishes ER components involved in protein folding and ERAD

Pancreatic expression of XBP1 targets were determined in wild-type and Xbp1^+/− mice fed either ethanol or control diet. (A) qRT-PCR analyses of the chaperones ERdj4 and EDEM1 (mean±SEM; n=3). mRNA values were standardized to those of mouse ARP and expressed relative to control-fed wild-type mice. *P<.05 vs. wild-type mice (two-way ANOVA). Immunoblots show pancreatic protein content of EDEM1 (B) and the ER oxidoreductases PDI and ERp57 (C). ERK1/2 and GAPDH were used as loading controls. (D) Quantitation of PDI and ERp57 expression in immunoblots relative to C diet, normalized to those of GAPDH. * P<.05 vs. wild-type mice; # P<.05 vs. C diet (two-way ANOVA and Tukey post-tests). (E) Immunoblot shows selective changes in PDI electrophoretic mobility associated with reduction/alkylation of thiol groups by TCEP and AMS treatment. AMS increases MW about 500 daltons per thiol group. As judged by the relative mobility of the bands, alkylation was less in samples from ethanol-fed mice, suggesting oxidation of fewer PDI free thiol groups. In B, C and E, each lane represents an individual mouse.

Figure 7. XBP1 deficiency and ethanol feeding induce upregulation of CHOP and apoptosis in mouse pancreas

(A) CHOP mRNA expression was assessed in pancreas from control- and ethanol-fed wild-type and XBP+/− mice by RT-PCR. GAPDH was used as a housekeeping gene. (B) Immunoblots show pancreatic levels of CHOP and the antiapoptotic proteins, Bcl-2 and Bcl-xL. In A, B, each lane represents an individual mouse. (C) Apoptotic cells were assessed by TUNEL staining of pancreatic sections. Ethanol-fed wild-type mice lacked apoptotic nuclei (panel a). Similar results were found for all mice fed control diet (not shown). In Xbp1^+/− mice fed ethanol, TUNEL positive nuclei (brown staining) were found mainly in damaged areas of parenchyma displaying extensive loss of acinar cells (panel b). Apoptotic cells comprise acinar and stroma cells. Bars, 20 μm.