Loss of PACS-2 delays regeneration in DSS-induced colitis but does not affect the ApcMin model of colorectal cancer

Abstract

PACS-2 is a multifunctional sorting protein that mediates cell homeostasis. We recently identified PACS-2 in a functional genome-wide siRNA screen for novel regulators of the metalloproteinase ADAM17, the main sheddase for ligands of the ErbB receptor family. Of note, we showed that Pacs2^-/- mice have significantly reduced EGFR activity and proliferative index in the intestinal epithelium. As EGFR signaling is highly mitogenic for intestinal epithelial stem cells, and plays essential roles in intestinal epithelial regeneration and tumor development, we have now examined the role of PACS-2 in these processes. Specifically, we analyzed the role of Pacs2-deficiency in a DSS-induced colitis model as well as in the genetic Apc^Min colon cancer model. We now report that loss of PACS-2 delays tissue regeneration after colonic injury with little effect on key inflammatory parameters. We did however not observe any apparent effects on tumor formation driven by excessive proliferative signaling downstream from APC-deficiency. Our findings reveal that the role of PACS-2 in regulating ADAM17-mediated shedding is not an obligate requirement for the epithelium to respond to the strong inflammatory or tumorigenic inducers in the models assessed here.

Keywords: ADAM17; ApcMin model; DSS-induced colitis; PACS-2; colon cancer.

Figure 1. Pacs2^-/- mice show no significant changes in susceptibility to DSS-induced colitis

A. Schematic of the DSS treatment regimen and data collection. Littermate Pacs2^+/+ (control, n = 21) and Pacs2^-/- (n = 17) mice received 2.3% DSS in their drinking water for five days, followed by five days on regular water. Mice were weighed on days 0, 2, and 5-10, and stool consistency and blood content additionally monitored from days 5-10. B. Quantitative real-time PCR analysis of Pacs2 and Pacs1 mRNA levels in the colon of control (n = 5) and Pacs2^-/- mice (n = 4). Gapdh served as a control for normalization. C. Quantitative real-time PCR analysis of Pacs2 mRNA levels in colonic tissue isolated from Pacs2^+/+ mice at Day 0, 6 and 10 during DSS-induced colitis. D. Kaplan-Meier plot depicting the percentage survival of control (61 %) versus Pacs2^-/- (35 %) mice. Survival was evaluated by log-rank (Mantel-Cox) test (control, n = 21; Pacs2^-/-, n = 17). E. Changes in colon length in control and Pacs2^-/- mice on day 6 of the DSS protocol (control, n = 9; Pacs2^-/-, n = 4). F. Daily changes in body weight during DSS-induced colitis. Changes in body weight percentage were calculated by normalizing body weight at the specific day to the body weight at day 0 (control, n = 21; Pacs2^-/-, n = 17). G. The Disease Activity Index (DAI) was calculated as the average of the weight loss score, stool score, and bleeding scores (control, n = 21; Pacs2^-/-, n = 17). All data were pooled from three independent experiments. Graphs represent the mean ± S.E.M; ***p < 0.005 analyzed by two-way ANOVA.

Figure 2. Pacs2^-/- mice show comparable inflammation and tissue damage during DSS-induced colitis

Histological sections were scored blindly using the criteria outlined in Table 2. A. Representative images of colonic specimens from control and Pacs2^-/- mice isolated at day 6 and day 10. Scale bar overview pictures = 1 mm. Scale bar detail figures = 0.1 mm. B. Dot plots show the histology score, calculated as the sum of the epithelial damage score and the inflammation score. The individual scores were calculated as described in Table 2. All data represent the mean ± S.E.M. and were analyzed by unpaired Student’s t-test (n = 6-13 as indicated). Data were pooled from three independent experiments.

Figure 3. Evaluation of the regenerative response of intestinal epithelial cells in DSS-treated Pacs2^-/- mice

A. Representative images of colonic specimens from control and Pacs2^-/- mice isolated at day 6 and day 10 and stained for phosphorylated EGFR (Tyr1068) and nuclear DAPI stain. B. The graph shows the pEGFR fluorescence intensity quantified along a line across colonic crypts (represented by the dotted line in A) on 5 equally positioned, random confocal images from each of 5 control and 5 Pacs2-/- mice. C. Representative images of colonic specimens from control and Pacs2^-/- mice isolated at day 6 and day 10 and stained for Ki67 positive proliferating cells. D. The graph shows Ki67 positive cells represented as the percentage diaminobenzidine (DAB) positive area per total hematoxylin stained nuclear area from 5 equally positioned, random images from each of 5 control and 5 Pacs2-/- mice. E. Representative images of colonic specimens from control and Pacs2^-/- mice isolated at day 6 and day 10 and stained for apoptotic cells by ApopTag. F. The graph shows the number of apoptotic cells per mm² counted from 3 equally positioned, random images from each of 5 control and 5 Pacs2-/- mice. Scale bars = 50 µm. All graphs show mean values ± SEM analyzed by unpaired two-tailed Student’s t-test. **p < 0.01.

Figure 4. Characterization of Apc^Min/+ Pacs2^-/- mice

A. Representative cropped western blots showing expression of PACS-2 (top panel) and β-catenin (middle panel) in tumor (T) and non-tumor (NT) colonic tissue samples from littermate Apc^Min/+ Pacs2^+/+ (control) and Apc^Min/+ Pacs2^-/- mice. Actin served as loading control. B. Quantitative real-time PCR analysis of Pacs2 (left) and Pacs1 (right) mRNA levels in the small intestine of control and Apc^Min/+ Pacs2^-/- mice (control, n = 7; Apc^Min/+ Pacs2^-/-, n = 3). Gapdh served as a control for normalization. C. Quantitative real-time PCR analysis of Pacs2 mRNA levels in tumor (T) and non-tumor (NT) tissue isolated from small intestine of Apc^Min/+ Pacs2^+/+ mice (NT, n = 5; T, n = 4). All data represent the mean ± S.E.M and were analyzed by unpaired Student’s t-test; ***p < 0.001.

Figure 5. Pacs2-deficiency has no apparent effects on intestinal tumor growth in the Apc^Min mouse model of colorectal cancer

A. Representative gross images of ileum specimens from control and Apc^Min/+ Pacs2^-/- mice. White arrowheads indicate tumors. TM = tunica muscularis. Scale bar = 500 µm. Analysis of tumor number B., tumor distribution in the proximal, middle, and distal small intestine C. and colon D., tumor area E. and tumor size F. in control (n = 21) and Apc^Min/+ Pacs2^-/- (n = 19) mice. All data represent the mean ± S.E.M (B, E, F) or the mean ± the minimum/maximum data points (C, D) and were analyzed by unpaired Student’s t-test.

Figure 6. PACS-2 loss does not affect tumor morphology, ß-catenin expression or cell proliferation in the Apc^Min model of colorectal cancer

Representative histological images of ileal adenomas from Apc^Min/+ Pacs2^+/+ (control) and Apc^Min/+ Pacs2^-/- mice. A. depicts H&E staining (scale bars = 100 µm), B. immunohistochemistry for ß-catenin (upper panel 10x, scale bars = 100 µm; lower panels scale bars = 50 µm; white arrowhead = nuclear staining; black arrow = membrane staining), and C. shows staining for the proliferation marker Ki67 (upper panel 10x, scale bars = 100 µm; lower panel scale bars = 50 µm).