Loss of PHLPP protects against colitis by inhibiting intestinal epithelial cell apoptosis

Abstract

A common feature of inflammatory bowel disease (IBD) is the loss of intestinal epithelial barrier function due to excessive apoptosis of intestinal epithelial cells (IECs). However, the molecular mechanism underlying increased IEC apoptosis remains unclear. Here, we investigated the role of PHLPP, a novel family of protein phosphatases, in regulating inflammation-induced IEC apoptosis in mouse models of colitis. Both Phlpp1 and Phlpp2 genes were deleted in mice. Compared with wild-type mice, PHLPP double knockout (DKO) mice were protected from colitis induced by DSS as demonstrated by lower histopathological scores, and this reduced susceptibility to colitis was associated with decreased apoptosis and increased Akt activity in IECs in vivo. In addition, epithelial organoids derived from PHLPP DKO mice were more resistant to inflammation-induced apoptosis while inhibition of Akt activity abolished the protective effect of PHLPP-loss. Furthermore, we found that PHLPP expression was significantly reduced in IECs following the induction of colitis by DSS and in human IBD patient samples. This inflammation-induced downregulation of PHLPP was partially blocked by treating cells with a proteasome inhibitor. Taken together, our results indicated that proteasome-mediated degradation of PHLPP at the onset of inflammation plays an important role in protecting IEC injury by inhibiting apoptosis.

Keywords: Akt; Inflammatory bowel disease; Intestine epithelial cell apoptosis; Knockout mouse; PHLPP.

Figure 1. Suppression of DSS-induced colitis in PHLPP-DKO mice

Colitis was induced in wild-type (WT) and PHLPP1/2 double knockout (DKO) mice by giving 2% DSS in drinking water for 7 days. The clinical progression of the colitis was determined by the percentage change in body weight (A) and disease activity index (DAI) (B). The daily body weight after the administration of DSS was normalized to that of before the treatment. DAI of WT and DKO mice was calculated as described previously [20]. Data represent means ± SEM (n = 12 for WT group including 5 male and 7 females; and 13 animals for DKO group including 5 male and 8 females. *, P<0.0001 WT vs. DKO group by overall comparison of trend over time). Note that DKO mice of both genders were equally resistant to DSS-induced colitis compared to WT mice. (C) Colon tissues were collected from WT and DKO mice treated with 2% DSS for 7 days and analyzed using hematoxylin and eosin (H&E) staining. Scale bar for images on the left = 500μm; and for images on the right = 200μm. (D) Histopathological analysis of DSS-induced colitis. Histological damage after DSS treatment was scored based on H&E staining as shown in C. Data represent means ± SEM (n = 5 animals/group; *, P<0.005 by the two-sample t-test). (E) Colon length of WT and DKO mice exposed to DSS for 7 days. Data represent means ± SEM (n = 12 and 13 animals for WT and DKO group, respectively; *, P<0.0001 WT vs. DKO group by the two-sample t-test). (F) Analysis of the inflammatory cell infiltrate in DSS-induced colitis of WT and DKO mice. Colon from WT and DKO mice exposed to DSS for 7 days were analyzed using F4/80 staining. Scale bar for images on the left = 200μm; and for images on the right = 100μm.

Figure 2. PHLPP-loss inhibits DSS-induced IEC apoptosis in mice

(A) TUNEL staining of colon tissues from DSS-treated WT and DKO mice. Scale bar, 100μm. (B) Apoptosis index was expressed as the number of TUNEL positive cells per 100 total cells counted. Colon tissues from 5 animals were analyzed in each group and cell counts were averaged from 4 randomly chosen areas. Data represent means ± SEM (n = 5; *, P<0.0001 by two-sample t-tests). (C) Western blot analysis of colonic epithelial cells isolated from control and DSS-treated WT and DKO mice. The relative levels of p-Akt and cleaved caspase-3 expression were quantified by normalizing ECL signals of p-Akt (p-S473) antibody to that of total Akt and the cleaved caspase-3 antibody to that of tubulin, respectively.

Figure 3. PHLPP-loss protects colonic organoids from DSS-induced apoptosis

Crypts isolated from WT and DKO colon tissues were seeded into Matrigel and cultured for 4 days to obtain organoids. (A) Phase contrast image of a representative colon organoid grown in 3D Matrigel and immunofluorescence staining of an organoid labeled with 5-ethynyl-2′-deoxyuridine (EdU) (green), β-catenin (red) and DAPI (blue). Scale bar, 50μm. (B) WT and DKO colon organoids grown in Matrigel were first recovered and resuspended in culture medium then treated with 2% DSS or 2% DSS plus LY294002 (10 μM) for 4 hours. Organoid lysates were analyzed for the expression of PHLPP1, PHLPP2, and cleaved caspase-3 as well as the phosphorylation status of Akt. The relative levels of p-Akt and cleaved caspase-3 expression were quantified by normalizing ECL signals of p-Akt (p-S473) antibody to that of total Akt and the cleaved caspase-3 antibody to that of tubulin, respectively.

Figure 4. PHLPP-loss prevents TNFα-induced cell death in small intestinal organoids

Crypts isolated from WT and DKO small intestine were seeded into Matrigel and cultured for 4 days to obtain organoids. (A) The WT and DKO organoids were treated with TNFα (0 or 20 ng/ml) plus cycloheximide (2.5 μg/ml) for 3 hours and stained with propidium iodide (PI). The appearance of PI penetrating into unfixed cells indicates apoptosis. Images shown are merged bright field and PI fluorescence images. Scale bar, 100μm. (B) The percentage of PI positive areas were quantified using Nikon Element AR software. Data represent means ± SEM (n = 5; *, P<0.001 by two-sample t-tests). (C) The WT and DKO organoids were treated with TNFα for 3 hours as described in (A). Organoid lysates were analyzed for the expression of PHLPP1, PHLPP2, and cleaved caspase-3 as well as the phosphorylation status of Akt. The relative levels of p-Akt and cleaved caspase-3 expression were quantified by normalizing ECL signals of p-Akt (p-S473) antibody to that of total Akt and the cleaved caspase-3 antibody to that of tubulin, respectively.

Figure 5. PHLPP expression is downregulated in DSS-induced colitis in mice

(A) WT mice were treated with 2% DSS for 4 days, and colonic epithelial cells were isolated from control and DSS-treated mice. Cell lysates were analyzed for the expression of PHLPP1, PHLPP2, and the phosphorylation level of Akt. (B) Western blots as shown in (A) were quantified and the relative levels of PHLPPs were obtained by normalizing ECL signals of PHLPP1 or PHLPP2 antibodies to that of tubulin. The expression level in untreated control mice was set to 1. Data represent the mean ± SEM (n=3, * p<0.01 by two-sample t-tests). (C) Total RNA was extracted from isolated colonic epithelial cells as described in (A). Real-time RT-PCR analysis was performed using probes specific for the mouse PHLPP1 or PHLPP2 gene. Data represent the mean ± SD (n=3).

Figure 6. Inflammatory stimuli decrease PHLPP expression by promoting protein degradation

(A–B) Time course of TNFα- and LPS-induced PHLPP downregulation in Caco2 cells. Cells were treated with TNFα (20 ng/ml) (A) or LPS (50 μg/ml) (B) for the indicated time, and cell lysates were prepared and analyzed for the expression of PHLPP1, PHLPP2 and tubulin using Western blotting. The relative expression of PHLPP isoforms was quantified by normalizing ECL signals generated by the PHLPP antibodies to that of tubulin, and this number for the untreated cells was set to 1. (C–D) Caco2 cells were treated with TNFα for 24 hours as described above. Proteasome inhibitor MG132 (20 μM) or DMSO was added to the cells for the last 3 hours of the TNFα treatment. (C) Representative Western blots showing the expression of PHLPP1, PHLPP2 and tubulin in cell lysates after different treatments. (D) The relative expression of PHLPP isoforms was quantified by normalizing ECL signals generated by the PHLPP antibodies to that of tubulin. The expression level in the control cells was set to 1. Data represent the mean ± SEM (n=3). (E–F) Caco2 cells were treated with LPS for 24 hours as described above. Proteasome inhibitor MG132 (20 μM) or DMSO was added to the cells for the last 3 hours of the LPS treatment. (E) Representative Western blots showing the expression of PHLPP1, PHLPP2 and tubulin in cell lysates after different treatments. (F) The relative expression of PHLPP isoforms was quantified by normalizing ECL signals generated by the PHLPP antibodies to that of tubulin. The expression level in the control cells was set to 1. Data represent the mean ± SEM (n=3). (G) Colon organoids of WT mice grown in Matrigel were treated with LPS and MG132 as described above. Protein lysates were prepared from organoids after removing Matrigel. The expression of PHLPP1, PHLPP2 and tubulin was analyzed using Western blotting. (H) The relative expression of PHLPP isoforms was quantified by normalizing ECL signals generated by the PHLPP antibodies to that of tubulin. The expression level under control condition was set to 1. Data represent the mean ± SEM (n=3).

Figure 7. The expression of PHLPP isoforms is downregulated in IBD patient samples

(A) Biopsy specimens from normal controls or IBD patients were lysed and analyzed for the expression of PHLPP1, PHLPP2, and the phosphorylation level of Akt using Western blotting. (B) Western blots as shown in (A) were quantified and the relative levels of PHLPPs were obtained by normalizing ECL signals of PHLPP1 or PHLPP2 antibodies to that of tubulin. The expression level in control samples was set to 1. Data represent the mean ± SEM (n=5, * p<0.05 by two-sample t-tests). (C) Diagram showing the effect of inflammation-induced downregulation of PHLPP on intestinal epithelial cell (IEC) apoptosis. Our study demonstrates that PHLPP expression is downregulated by inflammatory factors that are associated with IBD through increased protein degradation. As a consequence, loss of PHLPP expression promotes the activation of Akt and protects IECs from inflammation-induced apoptosis.