Abstract
BACKGROUND: Several mouse genetic models have suggested an important role for EGF, Wnt, BMP/TGFbeta and Notch signaling pathways in the renewal and differentiation of intestinal stem cells. EGF and its orthologs TGFalpha and epiregulin are factors regulating growth in epithelial tissues through activation of the RAS/RAF/MEK/ERK MAPK signaling pathway. Accordingly, in human and mouse intestines, ERK activity is predominantly found in progenitor cells in the transit-amplifying zone of the crypts where it is thought to control the proliferation/differentiation switch (Aliaga 1999). Indeed, we and others have reported that ERKs are selectively inactivated during absorptive cell differentiation hence supporting the hypothesis that these kinases must be shut down for the initiation of this differentiation process (Lemieux 2011). However, the role of ERK signaling in differentiation of the secretory cell lineage, particularly in Goblet cell differentiation remains to be elucidated. AIMS: This study was therefore conducted to analyze the role of ERK/MAPK signaling in the differentiation of intestinal Goblet cells and to elucidate the molecular events involved in this possible regulation. METHODS: Goblet cell number and differentiation were analyzed in three mouse models exhibiting sustained activation of ERK/MAPK signaling in intestinal epithelium: mice expressing oncogenic BRAF(V600E) in intestinal epithelial cells (IECs), mice expressing activated Shp2(E76K) mutant in IECs and mice knockout for Dusp6 (an inhibitor of ERK). Western blot, qPCR analyses and luciferase assays were performed in goblet-like cells LS174T treated or not with the MEK inhibitor CI-1040 (2 uM). RESULTS: Alcian blue staining in the colon of BRaf(V600E), Shp2(E76K) and Dusp6(-/-) mice reveal a marked increase in the number of Goblet cells in comparison to their control littermates. Interestingly, inhibition of MEK/ERK signaling with CI-1040 significantly reduces MUC2 transcript levels in LS174T cells. This decrease in MUC2 expression is associated with decreased transcriptional activity of KLF4 which is involve in Goblet cell terminal differentiation. Most interestingly, we found that inhibition of the MEK/ERK signaling activates the NOTCH pathway as visualized by an increased cleavage of NICD, the NOTCH intracellular domain, and expression of HES1, a target gene. This accumulation of NICD and HES1 can be prevent by gamma-secretase complex inhibition suggesting a NOTCH-dependent mechanism. Notably, qPCR analyses demonstrated an increased expression of the NOTCH ligands Delta-like 1 and Delta-like 4 in CI-1040-treated cells. CONCLUSIONS: Taking together, our results strongly suggest that RAF/MEK/ERK signaling pathway promotes Goblet cell differentiation by inhibiting the activation of NOTCH, a pathway known to inhibit the secretory cell fate. FUNDING AGENCIES: CIHRFRQS