Wnt signaling suppresses MAPK-driven proliferation of intestinal stem cells

Abstract

Intestinal homeostasis depends on a slowly proliferating stem cell compartment in crypt cells, followed by rapid proliferation of committed progenitor cells in the transit amplifying (TA) compartment. The balance between proliferation and differentiation in intestinal stem cells (ISCs) is regulated by Wnt/β-catenin signaling, although the mechanism remains unclear. We previously targeted PORCN, an enzyme essential for all Wnt secretion, and demonstrated that stromal production of Wnts was required for intestinal homeostasis. Here, a PORCN inhibitor was used to acutely suppress Wnt signaling. Unexpectedly, the treatment induced an initial burst of proliferation in the stem cell compartment of the small intestine, due to conversion of ISCs into TA cells with a loss of intrinsic ISC self-renewal. This process involved MAPK pathway activation, as the proliferating cells in the base of the intestinal crypt contained phosphorylated ERK1/2, and a MEK inhibitor attenuated the proliferation of ISCs and their differentiation into TA cells. These findings suggest a role for Wnt signaling in suppressing the MAPK pathway at the crypt base to maintain a pool of ISCs. The interaction between Wnt and MAPK pathways in vivo has potential therapeutic applications in cancer and regenerative medicine.

Keywords: Gastroenterology; Mouse stem cells; Signal transduction; Stem cells.

Figure 1. Wnt inhibition enhances proliferation of intestinal stem cells.

(A) C59 induced proliferation in the crypt base. Representative images of jejunal samples from mice treated with 1 dose of vehicle or C59 (100 mg/kg) 1 day prior to euthanasia. Proliferative cells were marked with EdU given 2 hours prior to euthanasia and DAPI stained the nuclei of cells. Arrows indicate EdU⁺ cells in crypt base. Scale bar, 25 μm. Image on right is higher magnification of outlined area of crypt (scale bar, 10 μm). (B) Quantification of EdU⁺ cells within 10 counted cells in the crypt base. Fifteen to twenty crypts were counted for each region of each mouse intestine (vehicle, n = 9 mice; C59, n = 8 mice; 3 experimental replicates). (C) Representative images of Ki67 staining in the vehicle- or C59-treated mice. Scale bar, 20 μm. Arrows indicate Ki67⁺ cells in the crypt base. (D) Enrichment of Ki67⁺ cells in the crypt base of vehicle- versus C59-treated mice. Twenty crypts were counted for each region of intestine per mouse (vehicle, n = 4; C59, n = 7; 2 experimental replicates). (E) C59 does not induce apoptosis in intestinal crypts. Representative images of cleaved-caspase 3 (CAS3) staining in jejunal sections of mice treated as described above. Arrows mark the apoptotic cells in villi as an internal positive control. Scale bar, 50 μm. ***P < 0.001, Mann-Whitney U test.

Figure 2. Passive lineage commitment of Lgr5 stem cells is intact after Wnt inhibition.

(A and C) Drug dosing protocol. Lgr5-EGFP-IRES-CreER^T2/Rosa-LSL-tdTomato mice were treated with tamoxifen and C59 according to the time line. (B) Wnt inhibition (C59 treatment with 100 mg/kg, once daily [QD]) for 3 days does not block Lgr5⁺ lineage tracing. Representative images of positive lineage–traced cells from Lgr5 cells, which are marked by endogenous Rosa-tdTomato (red), are shown for both vehicle- and C59-treated mice. (D) More intensive Wnt inhibition for 2 days still does not block Lgr5⁺ lineage tracing. Representative images of lineage tracing in vehicle- and C59-treated mice (50 mg/kg; BID, twice daily) are shown. Scale bar, 100 μm.

Figure 3. Wnt inhibition upregulates MAPK signaling in the crypt base.

(A) PORCN inhibition suppresses expression of Wnt target genes and stem cell markers. Mice were administered C59 at the indicated dose and frequency (+, 50 mg/kg twice daily [BID]; ++, 100 mg/kg once daily [QD]), and quantitative RT-PCR was performed on RNA from duodenal samples. Expression of Axin2, Lgr5, Ascl2, Olfm4, Hopx, and Bmi1 was normalized to Pgk and Actb (actin). Each dot represents 1 mouse. (B) C59 treatment leads to increased ERK1/2 phosphorylation at the crypt base. Representative images of p-ERK1/2 staining in jejunum samples. Scale bar, 50 μm. (C) Quantification of p-ERK1/2⁺ cells (cytoplasmic or nuclear staining) in crypt base of vehicle- or C59-treated mice (100 mg/kg, QD). NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001, Mann-Whitney U test.

Figure 4. Suppressing MAPK signaling inhibits C59-induced proliferation of intestinal stem cells.

(A) Schematic time line of trametinib, C59, and vehicle (Veh) administration in mice. Trametinib was administered (3 mg/kg, QD) 32 hours prior to administration of vehicle or C59 (100 mg/kg, QD). A total of 4 doses of trametinib and 2 doses of C59 or vehicle were gavaged according to hourly time point (indicated by arrows) prior to euthanasia. (B) The combination of trametinib and C59 treatment blocked proliferation and impaired intestinal homeostasis. EdU was injected 2 hours prior to euthanasia into 4 groups of mice treated with either vehicle or C59 alone, or in combination with trametinib (Tram). Left panels show representative images of total EdU⁺ staining in jejunal crypts. Right panels show higher magnification of crypt as indicated. Scale bar, 25 μm. (C) Top graph shows the number of counts for EdU⁺ cells within 10 cells in the base of the crypt. NS, nonsignificant; *P < 0.05; ***P < 0.001, Kruskal-Wallis 1-way ANOVA test. (D) Trametinib blocks PORCN inhibition–induced burst of proliferation. The number of EdU⁺ cells per 10 cells of the crypt base was determined. C59 treatment increased the number of crypts with 4 or more EdU⁺ cells, and this was reversed by coadministration of trametinib. Vehicle, n = 7. Trametinib plus vehicle, n = 6. C59, n = 8. Trametinib plus C59, n = 6. Error bar indicates SEM; 2 experimental replicates.