Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway

Abstract

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway.

FIGURE 1:

NFAT5 regulates REDD1 expression in HT29 cells. (A and B) HT29 cells were transfected with NFAT5 or NTC siRNA. After 48-h incubation, transfected cells were lysed, total RNA was extracted, and real-time RT PCR was performed for analysis of REDD1 (A) and NFAT5 and NFATc1, NFATc2, NFATc3, and NFATc4 mRNA expression (B). (Data represent mean ± SD; *, p < 0.01 vs. NTC siRNA as determined by ANOVA.) (C) HT29 cells were transfected with NFAT5 or NTC siRNA. After 48-h incubation, transfected cells were lysed, and Western blot analysis was performed using antibodies against REDD1, p-Ser-6, Ser-6, NFAT5, and β-actin. (D) HT29 cells were transfected with NFAT5 or NTC siRNA. After 24-h incubation, transfected cells were treated with 100 mM NaCl for an additional 24 h and subjected to Western blot analysis using antibodies against NFAT5, REDD1, p-Ser-6, total Ser-6, and β-actin. REDD1 signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin.

FIGURE 2:

NFAT5 regulates REDD1 expression in HCT116, SW480 and Caco-2 cells. (A) HCT116, SW480, and Caco-2 cells were transfected with NFAT5 or NTC siRNA. After 48-h incubation, transfected cells were lysed, and Western blot analysis was performed using antibodies against REDD1, p-Ser-6, Ser-6, NFAT5, and β-actin. (B) HCT116 and Caco-2 cells were transiently transfected with empty vector or a construct expressing Myc-NFAT5. Forty-eight hours after transfection, cells were lysed, and Western blot analysis was performed using antibodies against REDD1, Myc-tag, and β-actin. REDD1 signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin.

FIGURE 3:

Regulation of MUC2 mRNA expression by mTORC1/Notch signaling pathway. (A) HT29 cells were transfected with NTC siRNA or siRNA targeting REDD1. (B) HT29 cells were treated with 100 nM rapamycin for 24 h. Total protein was extracted, and Western blotting was performed using anti-NICD, REDD1, anti–p-Ser-6, anti-Ser-6, Hes1, and anti–β-actin antibodies. NICD and Hes1 signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin. (C) HT29 cells were treated with 100 nM rapamycin for 24 h; total RNA was extracted and MUC2 mRNA levels were determined by real-time RT-PCR. (Data represent mean ± SD; *, p < 0.05 vs. control as determined by ANOVA.)

FIGURE 4:

NFAT5 inhibits Notch signaling. (A) HT29 or HCT116 cells were transfected with NTC siRNA or siRNA targeting NFAT5. (B) Caco-2 cells were transfected with control vector or NFAT5 plasmid. After 48-h incubation, total protein was extracted and subjected to Western blotting using anti-NICD, anti–p-Ser-6, anti-Ser-6, Hes1, and anti–β-actin antibodies. NICD and Hes1 signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin.

FIGURE 5:

NFAT5 regulation of MUC2 mRNA expression. (A) HT29 and Caco-2 cells were transfected with NTC siRNA or siRNA targeting NFAT5. (B) HCT116 and Caco-2 cells were transfected with control vector or NFAT5 plasmid. After 48-h incubation, total RNA was extracted, and MUC2 mRNA levels were determined by real-time RT-PCR. (Data represent mean ± SD; *, p < 0.01 vs. control siRNA as determined by ANOVA.)

FIGURE 6:

NFAT5/REDD1/mTOR/Notch pathway model. REDD1 is proposed to inhibit mTORC1 by displacing TSC2 from the 14-3-3 binding protein, allowing TSC2 to inhibit mTORC1. Activation of NFAT5 increases REDD1 expression resulting in inhibition of mTORC1 signaling, leading to decreased Notch signaling and an increase of goblet cell differentiation.