TSC2/mTORC1 signaling controls Paneth and goblet cell differentiation in the intestinal epithelium

Abstract

The intestinal mucosa undergoes a continual process of proliferation, differentiation and apoptosis, which is regulated by multiple signaling pathways. Notch signaling is critical for the control of intestinal stem cell maintenance and differentiation. However, the precise mechanisms involved in the regulation of differentiation are not fully understood. Previously, we have shown that tuberous sclerosis 2 (TSC2) positively regulates the expression of the goblet cell differentiation marker, MUC2, in intestinal cells. Using transgenic mice constitutively expressing a dominant negative TSC2 allele, we observed that TSC2 inactivation increased mTORC1 and Notch activities, and altered differentiation throughout the intestinal epithelium, with a marked decrease in the goblet and Paneth cell lineages. Conversely, treatment of mice with either Notch inhibitor dibenzazepine (DBZ) or mTORC1 inhibitor rapamycin significantly attenuated the reduction of goblet and Paneth cells. Accordingly, knockdown of TSC2 activated, whereas knockdown of mTOR or treatment with rapamycin decreased, the activity of Notch signaling in the intestinal cell line LS174T. Importantly, our findings demonstrate that TSC2/mTORC1 signaling contributes to the maintenance of intestinal epithelium homeostasis by regulating Notch activity.

Figure 1

TSC2 inactivation results in altered goblet cell differentiation. (a) Immunohistochemical (IHC) staining demonstrated increased expression of phospho-S6 (some are indicated by arrows) in intestinal epithelium of TG mice compared with WT mice. (b) Mucosal protein lysates extracted from WT and TG mice were used for western blot detection of phospho-S6 and total S6 protein expression. The levels of p-S6 were quantitated densitometrically and expressed as fold change with respect to total S6. (c and d) Alcian blue (AB) staining of the intestine revealed a reduction in mucinous goblet cells in TSC2-mutant TG mice compared with WT mice (c, some are indicated by arrows). (d) Quantification of AB-positive cells in WT- and TSC2-mutant TG mice. (Data represent mean±S.D.; *P<0.05 versus WT). (e and f) (IHC) staining for MUC2 further confirmed the decrease in goblet cells in TSC2-mutant TG mice compared with WT mice (e, some are indicated by arrows). (f) Quantification of MUC2-positive cells in WT- and TSC2-mutant TG mice. (Data represent mean±S.D.; *P<0.05 versus WT). (g) Mucosal protein lysates extracted from WT and TG mice were used for western blot detection of MUC2 protein expression. Scale bars, 50 μm

Figure 2

TSC2 inactivation results in altered Paneth cell differentiation in intestine. (a) Immunohistochemical (IHC) staining of the small intestine for lysozyme showed the decrease in Paneth cells (arrow) in TSC2-mutant TG mice compared with WT mice. (b) Quantification of lysozyme-positive cells in WT and TSC2-mutant TG mice. (Data represent mean±S.D.; *P<0.05 versus WT). Scale bars, 50 μm

Figure 3

TSC2 inactivation leads to the increased Notch signaling in intestine. (a) Immunohistochemical (IHC) staining (arrow) for NICD and Hes1 demonstrated increased expression in the intestinal epithelium of TG mice compared with WT mice. (b) Mucosal protein lysates extracted from WT and TG mice were used for western blot detection of NICD and Hes1 protein expression. (c) TSC2-mutant TG mice were treated with or without DBZ for 5 day. The inhibition of Notch signaling by DBZ was demonstrated by staining with NICD (arrow). The goblet cell population, revealed by Alcian blue staining (arrow) and Paneth cells, assessed by IHC staining of lysozyme (arrow), are reduced in the small intestinal epithelium of TSC2-mutant TG mice. Treatment with DBZ restored the goblet and Paneth cell numbers. (Data represent mean±S.D.; *P<0.05 versus WT alone; ^#P<0.05 versus TG alone). Scale bars, 50 μm

Figure 4

TSC2/mTOR signaling pathway controls differentiation of the goblet and Paneth cell lineages. WT- and TSC2-mutant TG mice were treated with or without rapamycin for 6 days. The inhibition of mTORC1 signaling by rapamycin was demonstrated by staining with phospho-S6 (a, arrow). The goblet cell population, revealed by Alcian blue staining and MUC2 immunostaining (b, arrow; c), and Paneth cells, assessed by immunohistochemical staining of lysozyme (d, arrow; e), are reduced in the small intestinal epithelium of TSC2-mutant TG mice. (Data represent mean±S.D.; *P<0.05 versus WT alone; #P<0.05 versus TG alone). Treatment with rapamycin restored the goblet and Paneth cell numbers. Scale bars, 50 μm

Figure 5

TSC2/mTOR is an upstream regulator of Notch-Hes1 signaling in the human colon cancer cell line LS174T. (a) LS174T cells were transfected with non-targeting control (NTC) siRNA or siRNA-targeting TSC2. (b) LS174T cells were treated with 100 nM rapamycin for 24 h. (c) LS174T cells were transfected with NTC siRNA or siRNA-targeting mTOR. Total protein was extracted and western blotting performed using anti-NICD, anti-Hes1, anti-TSC2, anti-mTOR, anti-p-S6, anti-S6 and anti-β-actin antibodies. (d) LS174T cells were transfected with NTC siRNA or siRNA-targeting mTOR; total RNA was extracted and MUC2 mRNA levels were determined by real-time RT-PCR. (Data represent mean±S.D.; *P<0.05 versus NTC siRNA)

Figure 6

TSC2/mTOR signaling pathway regulates intestinal cell differentiation through Notch1. (a) WT and TG mice were treated with or without rapamycin for 6 days. The decreased immunohistochemical staining for NICD and Hes1 (arrow) demonstrated the inhibition of Notch signaling by rapamycin in the intestinal epithelium. (b) Mucosal protein lysates extracted from WT and TG mice treated with or without rapamycin for 6 days were used for western blot detection of NICD and Hes1 protein expression. Scale bars, 50 μm

Figure 7

Schematic representation of TSC2/mTOR/Notch pathway model. TSC2 functions as a complex with TSC1 and inhibits mTORC1 signaling, thus leading to decreased Notch signaling and increased intestinal cell differentiation