Peroxisome Proliferator-activated Receptor-γ Coactivator 1-α (PGC1α) Protects against Experimental Murine Colitis

Abstract

Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is the primary regulator of mitochondrial biogenesis and was recently found to be highly expressed within the intestinal epithelium. PGC1α is decreased in the intestinal epithelium of patients with inflammatory bowel disease, but its role in pathogenesis is uncertain. We now hypothesize that PGC1α protects against the development of colitis and helps to maintain the integrity of the intestinal barrier. We selectively deleted PGC1α from the intestinal epithelium of mice by breeding a PGC1α(loxP/loxP) mouse with a villin-cre mouse. Their progeny (PGC1α(ΔIEC) mice) were subjected to 2% dextran sodium sulfate (DSS) colitis for 7 days. The SIRT1 agonist SRT1720 was used to enhance PGC1α activation in wild-type mice during DSS exposure. Mice lacking PGC1α within the intestinal epithelium were more susceptible to DSS colitis than their wild-type littermates. Pharmacologic activation of PGC1α successfully ameliorated disease and restored mitochondrial integrity. These findings suggest that a depletion of PGC1α in the intestinal epithelium contributes to inflammatory changes through a failure of mitochondrial structure and function as well as a breakdown of the intestinal barrier, which leads to increased bacterial translocation. PGC1α induction helps to maintain mitochondrial integrity, enhance intestinal barrier function, and decrease inflammation.

Keywords: bacterial pathogenesis; inflammatory bowel disease (IBD); intestinal epithelium; mitochondria; oxidative stress.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

Human tissues from patients with ulcerative colitis and control patients were analyzed for PGC1α protein levels via Western blot with densitometry (A and B, n = 9–10/group), and PGC1α expression was analyzed using qRT-PCR (C). Rag1−/− mice received CD4+CD45RBhigh cells obtained from the spleens of C57BL/6 mice via intraperitoneal injection (n = 4/group). Over 6 weeks, these mice lost weight and demonstrated inflammatory changes on H&E (D and E, scale bars, 100 μm). They demonstrated a dramatic increase in the expression of inflammatory tissue cytokines. As seen in humans with ulcerative colitis, there was an overall decrease in RNA expression of PGC1α as well as PGC1α protein levels (F and G). *, p ≤ 0.05.

FIGURE 2.

C57BL/6 mice were subjected to 3% DSS colitis for 7 days (n = 8/group). There was an overall decrease in PGC1α, Nrf-1, Nrf-2, and Tfam protein in intestinal tissue after DSS exposure (A). PGC1α expression was decreased in mice subjected to DSS and assessed using qRT-PCR. DSS-treated mice demonstrated a 30% reduction in mtDNA content relative to nDNA content as compared with control mice. Relative quantification of mtDNA/nDNA was determined by real time PCR (B). Immunofluorescence for PGC1α (C, panels i and ii; Cy3, red), E-cad (C, panels iii and iv; Cy5, cyan), and nuclear stain (DAPI, blue) illustrates a decrease in PGC1α within the intestinal epithelium after 7 days of 3% DSS exposure (C, panels v and vi, merge; scale bars, 100 μm). Quantification of immunofluorescence reveals a statistically significant decrease in PGC1α staining. Mice were then subjected to 3% DSS over 7 days followed by a transition to normal drinking water for 2 days and sacrificed at 2-day intervals (n = 4/group, 6 groups). mRNA expression of PGC1α demonstrates an early increase, followed by a decrease over time with levels decreasing significantly as compared with control mice at day 7 (D). Protein levels demonstrate a similar but delayed trend with levels peaking at day 3 before declining. BALB/c mice were treated with TNBS or ethanol (EtOH) vehicle and euthanized on day 4 after treatment (n = 4/group). Mice treated with EtOH demonstrated a non-significant decrease in PGC1α protein in intestinal tissue, whereas mice treated with TNBS demonstrated a significant decrease (E). PGC1α protein was immunoprecipitated from the intestinal tissue of C57BL/6 mice subjected to DSS colitis (n = 8). Lysine residues on PGC1α protein in mice treated with DSS were highly acetylated as compared with PGC1α from control mice (F). PGC1α protein in mice treated with DSS was highly ubiquitinated as compared with that from control mice. Densitometry confirms statistical significance. *, p ≤ 0.05.

FIGURE 3.

C57BL/6 mice were subjected to 3% DSS colitis for 7 days. Activity of the electron transport chain complexes I and IV was significantly decreased in intestinal tissue, although complex II, III, and citrate synthase activities showed non-significant decreases in activity (A, n = 4/group). Immunofluorescence was performed in mice subjected to DSS to evaluate the mitochondrial outer membrane proteins Mtco2 and Tom20 (B, panels i–x, n = 8/group, scale bars, 100 μm). E-cad (Cy5, cyan) stained intestinal epithelium in both control and DSS tissue (B, panels i and ii). Tom20 (FITC, green) is decreased in mice subjected to DSS colitis as compared with WT mice (B, panels ii–iv). E-cad demonstrates colocalization with Tom20 (B, panels v and vi). Similarly, Mtco2 (Cy3, red) is significantly decreased in mice subjected to DSS colitis as compared with WT mice (B, panels vii and viii). E-cad demonstrates colocalization with Mtco2 (B, panels ix and x). All images contain nuclear stain (DAPI, blue). Mitochondrial structure was deranged in the same mice after DSS treatment as seen on TEM (C, panel i and ii, scale bars, 1 μm). DHE staining demonstrated a significant increase in superoxide ions in the intestinal tissue of mice subjected to DSS, which was confirmed by quantification (C, panels iii and iv, blue to red indicates increase in superoxide ions). Intestinal tissue from BALB/c mice subjected to TNBS colitis was similarly analyzed for DHE staining, and an increase in superoxide ions is seen in mice with colitis (D, panels i and ii). *, p ≤ 0.05.

FIGURE 4.

Intestinal epithelium-specific PGC1α knock-out mouse (PGC1αΔIEC) was created by mating a PGC1αloxP/loxP mouse with a villin-cre mouse. Mice were genotyped using RT-PCR (A). Immunofluorescence for PGC1α (Cy3, red), E-cad (FITC, green), and nuclear stain (DAPI, blue) demonstrates a significant decrease of PGC1α protein within the intestinal epithelium of PGC1αΔIEC mice (B, n = 8/group, scale bars, 100 μm). Staining in both the colon and ileum was quantified. Representative images are provided from the colon of PGC1αloxP/loxP mice (B, panel i) and PGC1αΔIEC mice (B, panels ii and iii) as well as the ileum of PGC1αloxP/loxP mice (B, panel iv) and PGC1αΔIEC mice (B, panels v and vi). qRT-PCR of intestinal tissue demonstrates PGC1α expression in the colon, ileum, and liver of PGC1αΔIEC and PGC1αloxP/loxP mice. Expression is decreased in the intestines of PGC1αΔIEC mice as compared with PGC1αloxP/loxP mice but not in the liver (C). No differences in intestinal morphology were noted between strains (D, panels i and ii). Immunofluorescence was performed for colonic villin (D, panels iii and iv, scale bars, 100 μm), colonic Muc2 (D, panels v and vi), ileal lysozyme (D, panels vii and viii), and colonic BrdU (D, panels ix and x). When quantification was performed, no differences were noted between strains. Alcian blue staining for goblet cells was similar between strains (D, xi and xii). β diversity comparisons of microbial communities within fecal samples from PGC1αΔIEC mice and their PGC1αloxP/loxP littermates are not significant. Shown are the observed species indices for each sample group (E). Displayed are principal coordinate analyses of unweighted Unifrac distances between all samples analyzed. Axis labels indicate the proportion of variance explained by each principal coordinate axis. Fecal samples of the two strains do not cluster separately within the principal coordinate space. Diversity within each group is as great as the diversity between sample types. α diversity comparisons of microbial communities within fecal samples from PGC1αΔIEC mice and their PGC1αloxP/loxP littermates are not significant. Shown are the observed species indices for each sample group (F). *, p ≤ 0.05.

FIGURE 5.

Intestinal epithelium-specific PGC1α knock-out mouse (PGC1αΔIEC) was subjected to 2% DSS for 7 days along with PGC1αloxP/loxP littermates (n = 8/group). When the % body weight was evaluated, significant differences were noted between PGC1αΔIEC control versus PGC1αΔIEC DSS-treated mice (#, p = ≤0.05), PGC1αΔIEC DSS versus PGC1αloxP/loxP DSS-treated mice (*, p ≤ 0.05), and PGC1αloxP/loxP control versus PGC1αloxP/loxP DSS-treated mice ($, p ≤ 0.05) (A). DAI score for PGC1αΔIEC mice was significantly higher than that for PGC1αloxP/loxP mice subjected to DSS colitis (B). Histologically, PGC1αΔIEC mice developed a more dramatic colitis, as quantified by average histology grade (C, scale bars, 100 μm). PGC1αΔIEC mice also demonstrated a significant increase in pro-inflammatory tissue cytokine release as compared with PGC1αloxP/loxP mice (D). Activity of the electron transport chain complexes I and IV were significantly decreased in both strains of mice after DSS exposure. However, complex IV activity was significantly decreased in PGC1αΔIEC mice subjected to DSS as compared with PGC1αloxP/loxP mice similarly treated (E). PGC1αΔIEC mice and PGC1αloxP/loxP mice were subjected to a chronic model of 2% DSS exposure (n = 16/group). Significant differences in DAI were noted in the first two cycles of DSS treatment but not the third cycle (F). As seen after acute DSS colitis, PGC1α is decreased in the intestines of PGC1αloxP/loxP mice after the completion of the chronic DSS model. *, p ≤ 0.05.

FIGURE 6.

C57BL/6 mice were subjected to 3% DSS colitis and SRT1720 100 mg/kg/day via oral gavage versus vehicle (water). Significant differences in % body weight change were seen in control versus DSS (*, p ≤ 0.05), control + SRT1720 versus DSS + SRT1720 (#, p ≤ 0.05), and DSS versus DSS + SRT1720 ($, p ≤ 0.05) (A, n = 8/group). Histopathology demonstrated significant manifestations of disease in DSS-treated animals with a preservation of architecture in SRT1720-treated animals (B, panels i–iv, scale bar, 100 μm). TEM demonstrated a predicted derangement of mitochondrial structure in mice subjected to DSS colitis with a partial preservation of structure in SRT1720-treated mice (B, panels v–viii, scale bar, 1 μm). The average histology grade for DSS + SRT-treated mice is significantly lower that that for DSS-treated mice (C, *, p ≤ 0.05). No changes were seen in control mice (data not shown). Interestingly, PGC1α was increased in SRT-treated DSS animals as compared with DSS-treated mice, as demonstrated by Western blot and qRT-PCR (D and E). Similarly, tissue cytokine release was significantly decreased in DSS + SRT-treated mice as compared with DSS-treated mice (E). Activity of the electron transport chain complexes I and IV were significantly decreased in both DSS and DSS + SRT-treated mice. However, complex IV activity was significantly decreased in DSS as compared with DSS + SRT-treated mice (F). PGC1αΔIEC mice treated with SRT1720 along with DSS exposure demonstrated minimal protection from DSS colitis (G and I). Significant differences in DAI were noted between PGC1αΔIEC control versus PGC1αΔIEC DSS-treated mice (^p = ≤ 0.05), PGC1αΔIEC DSS versus PGC1αloxP/loxP DSS-treated mice (&, p ≤ 0.05), PGC1αloxP/loxP control versus PGC1αloxP/loxP DSS-treated mice (#, p ≤ 0.05), and PGC1αloxP/loxP control versus PGC1αloxP/loxP DSS + SRT-treated mice ($, p ≤ 0.05) (G). A small but significant difference in DAI score was noted between PGC1αΔIEC DSS versus PGC1αΔIEC DSS + SRT-treated mice only at day 7 (*, p ≤ 0.05). Average histology grades were not significantly different between PGC1αΔIEC DSS-treated mice and PGC1αΔIEC DSS + SRT-treated mice (H, scale bar, 100 μm; *, p ≤ 0.05; ns, not significant). Similarly, tissue cytokine levels were not significant between PGC1αΔIEC DSS-treated mice and PGC1αΔIEC DSS + SRT-treated mice (I, *, p ≤ 0.05; ns, not significant).

FIGURE 7.

After 7 days of 2% DSS exposure, in situ staining for bacterial 16S rRNA using EUB338 (red) and DAPI nuclear stain (blue) was performed (A, n = 8/group, scale bar, 100 μm). A significant increase in bacterial translocation is seen in PGC1αΔIEC mice when compared with PGC1αloxP/loxP mice. Quantification of immunofluorescence demonstrates statistical significance (B). The tight junction protein occludin is mildly decreased in PGC1αloxP/loxP mice subjected to DSS colitis. Occludin levels in PGC1αΔIEC mice are decreased at baseline and are nearly absent after DSS colitis. Zo-1 levels are also decreased in PGC1αΔIEC mice subjected to DSS colitis as compared with control mice (C). qRT-PCR analysis reveals stable expression of occludin in PGC1αloxP/loxP mice subjected to DSS as compared with control mice, whereas a significant decrease is seen in PGC1αΔIEC mice subjected to colitis (D). Immunofluorescence for occludin demonstrates a diffuse decrease in staining after DSS induction in PGC1αloxP/loxP mice with a greater decrease in PGC1αΔIEC mice (E, scale bar, 100 μm). *, p ≤ 0.05.

FIGURE 8.

We propose that PGC1α is inactivated through acetylation and targeted for degradation by ubiquitination within the intestinal epithelium during intestinal inflammation. A decrease in PGC1α protein down-regulates the mitochondrial biogenesis pathway and results in an overall decrease in healthy mitochondria. This potentiates inflammation through a dysregulated release in ROS, decreased oxidative phosphorylation, and an accumulation of unhealthy mitochondria. Consequently, there is a decrease in the tight junction protein occludin, and the intestinal barrier is compromised leading to translocation of luminal bacteria.