Phosphoinositide 3-kinase signaling mediates beta-catenin activation in intestinal epithelial stem and progenitor cells in colitis

Abstract

Background & aims: Mechanisms responsible for crypt architectural distortion in chronic ulcerative colitis (CUC) are not well understood. Data indicate that serine/threonine protein kinase Akt (Akt) signaling cooperates with Wingless (Wnt) to activate beta-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-beta-catenin(552)). We investigated whether phosphoinositide 3-kinase (PI3K) is required for Akt-mediated activation of beta-catenin during intestinal inflammation.

Methods: The class IA subunit of PI3K was conditionally deleted from intestinal epithelial cells in mice named I-pik3r1KO. Acute inflammation was induced in mice and intestines were analyzed by biochemical and histologic methods. The effects of chemically blocking PI3K in colitic interleukin-10(-/-) mice were examined. Biopsy samples from patients were examined.

Results: Compared with wild-type, I-pik3r1KO mice had reduced T-cell-mediated Akt and beta-catenin signaling in intestinal stem and progenitor cells and limited crypt epithelial proliferation. Biochemical analyses indicated that PI3K-Akt signaling increased nuclear total beta-catenin and P-beta-catenin(552) levels and reduced N-terminal beta-catenin phosphorylation, which is associated with degradation. PI3K inhibition in interleukin-10(-/-) mice impaired colitis-induced epithelial Akt and beta-catenin activation, reduced progenitor cell expansion, and prevented dysplasia. Human samples had increased numbers of progenitor cells with P-beta-catenin(552) throughout expanded crypts and increased messenger RNA expression of beta-catenin target genes in CUC, colitis-associated cancer, tubular adenomas, and sporadic colorectal cancer, compared with control samples.

Conclusions: PI3K-Akt signaling cooperates with Wnt to increase beta-catenin signaling during inflammation. PI3K-induced and Akt-mediated beta-catenin signaling are required for progenitor cell activation during the progression from CUC to CAC; these factors might be used as biomarkers of dysplastic transformation in the colon.

Figure 1. Loss of p85α protein expression in IECs of I-pik3r1 KO mice

Western blot analyses of total p85, p85α and p110α subunits of PI3K in cytosolic fractions from purified crypt epithelial cells at 0 and 3 hours after T cell activation with anti-CD3 mAb in pik3r1^lox/lox and I-pik3r1 KO mice. h, hours.

Figure 2. Epithelial deletion of pik3r1 attenuates T cell-mediated Akt and β-catenin signaling in SB crypts

(A) Western blot analyses of P-Akt, FoxO1, total (T) β-catenin, P-β-catenin/,, P-β-catenin⁵⁵², and cyclin D1 in nuclear or cytoplasmic extracts of purified SB crypt epithelial cells from pik3r1^lox/lox and I-pik3r1 KO mice following T cell activation. Fibrillarin and α1-Tubulin are subcellular fraction controls. (B) Cyclin D1 and cMyc in SB crypts detected by immunohistochemistry (arrows). Bar graphs show mean ± standard deviation (±SD) of positive cells/crypt in indicated groups. Scale bar size is 50μm. p-value represents the results of student t-test.

Figure 3. PI3K is required for T cell mediated Akt and β-catenin signaling in SB crypts

Immunohistochemistry staining for (A) P-Akt, (B) P-β-catenin⁵⁵² (arrows) performed in ileal sections of control and anti-CD3 treated pik3r1^lox/lox or I-pik3r1 KO mice. Insets indicate P-Akt and P-β-catenin⁵⁵² positive epithelial cells with enlarged nuclei within the crypt. (C) BrdU incorporation was examined 18 hrs after anti-CD3 treatment. (D) Bar graphs display mean ±SD of positively stained cells/crypt for P-Akt, P-β-catenin⁵⁵², and BrdU.

Figure 4. PI3K is required for epithelial cell proliferation induced by T cell activation

(A) Single crypt picture indicating enumeration of crypt cells. (B) P-β-catenin⁵⁵² and (C) BrdU labeled cells per position of SB crypt determined 3 or 18 hours after T cell activation, respectively. (D) TUNEL staining of control pik3r1^lox/lox or pik3r1^lox/lox and I-pik3r1 KO mice 18hr after anti-CD3 mAb injection. (E) Bar graph quantifying D shows mean ±SD of TUNEL-positive cells/crypt. Scale bar size is 50μm. p-value represents results of student t- test. (F) Representative image and quantification of a P-β-catenin⁵⁵² positive CBC (block arrow) with 2 neighboring paneth cells (small arrows) in the SB. 100 well-oriented SB crypts were counted 3hrs after control or anti-CD3 treatment.

Figure 5. PI3K is required for progression of chronic CID

(A) Timeline of colitis induction with Px in IL10^−/− mice and LY294002 treatment. (B) Gross findings in colons of WT, or vehicle only and LY294002 D42 treated IL10^−/− mice. (C) Ulcerating masses (red arrows) in methylene blue-stained colonic mucosa. (D) Prevalence of dysplasia in indicated group over time. (E) Western blot data showed cytosolic P-Akt and nuclear P-β-catenin in IECs. Bar size=500μm.

Figure 6. Immunohistochemical evidence that PI3K is required for progression of CID

(A) Immunohistochemistry for P-Akt, P-β-catenin⁵⁵², cyclin D1, or BrdU incorporation in colonic sections of control, D42-colitis, D42-dysplasia, D42-LY294002-treated IL10^−/− mice. (B) Bar graph shows mean ±SD of positively stained cells of P-Akt, P-β-catenin⁵⁵², cyclin D1, and BrdU in each group. *p<0.01, compared to control; **p<0.01, compared to D42-colitis, #p<0.01, compared to D42-dysplasia. Bar size=50μm. p-value represents results of student t-test.

Figure 7. β-catenin activation and Ki67 levels are increased in CUC, UCD, and CAC patients

(A) Human colonic specimens stained for P-β-catenin⁵⁵² and Ki67. Arrows indicate positively stained cells in representative control (C), active non-treated CUC, UCD, and CAC samples. Block arrow shows P-β-catenin⁵⁵² stained cells at points of crypt branching (dotted line). Scale bar size=50μm. (B) Bar graphs show mean ±SD of P-β-catenin⁵⁵²- and (C) Ki67-stained cells/100 epithelial cells in each group. The number of patients in each group in B is 20 C, 13 CUC, 10 UCD, and 2 CAC, and in C is 8 C, 10 CUC, 4 UCD, and 2 CAC. (D) Real-time RT-PCR for β-catenin target genes in human colonic tissues. Bar graphs show mean ±SD of mRNA fold-induction of cMyc (Myc), COX2 (PTGS2), and GPR49 (LGR5) in the indicated groups. Data represents specimens from 11–13 C, 19–24 CUC, 4–5 UCD, and 5–7 CAC patients. p-value represents the results of a Mann-Whitney U test. (E) Wnt signaling inhibits the destruction complex, impairs N-terminal phoshorylation (degradation), and stabilizes β-catenin which accumulates in the nucleus. In the inflammatory setting PI3K/Akt signaling is activated and β-catenin is phosphorylated at Ser552. The location for this phosphorylaton event is unknown, but P-β-catenin⁵⁵² is only detected in the nucleus in mice. Nuclear P-β-catenin⁵⁵² binds to the Tcf4 to induce transcription.