Sa1731 Neurotensin-HIF-1α-MicroRNA 210 Axis Orchestrates Hypoxia, Colonic Inflammation and Intestinal Angiogenesis

Abstract

G A A b st ra ct s or constructs overexpressing HA-tagged 14-3-3σ plasmids. 14-3-3σ, Rictor (an mTORC2 protein), MUC2, cleaved Notch1, p-Akt(S473), Akt1 and Akt2 protein expression was determined by Western blotting. Enterocyte differentiation was induced by treatment of HT29 cells with sodium butyrate (NaBT) and assayed by measuring intestinal alkaline phosphatase (IAP) activity, an enterocyte differentiation marker. The expression of the goblet cell differentiation marker MUC2 and the Notch1 target gene HES1 was also determined by RT-PCR. The interaction between Akt andmTORC2was determined by immunoprecipitation followed by Western blot analysis. RESULTS. Knockdown of 14-3-3 σ did not alter basal or NaBT-induced of IAP activity. However, knockdown of 14-3-3 σ significantly increased MUC2 expression associated with the inhibition of Notch1 signaling as shown by decreased cleaved Notch1 protein and decreased HES1 mRNA expression. These results suggested that 14-3-3σ plays an important role in the regulation of goblet cell differentiation without impacting enterocyte differentiation. In addition, overexpression of 14-3-3σ decreased Akt (Ser473) phosphorylation and knockdown of either Akt1 or Akt2 decreased MUC2 expression. Moreover, overexpression of 14-3-3σ decreased the interaction between Akt and mTORC2. These results suggest that 14-3-3σmay regulate Akt activity through the inhibition of mTORC2-mediated Akt (Ser473) phosphorylation. CONCLUSIONS. Our results suggest a role for 14-3-3σ in goblet cell differentiation through the alteration of TSC2, Notch and Akt signaling pathways. Importantly, our data demonstrate that 14-3-3 σ regulation of Akt phosphorylation is mediated through the altered interaction between mTORC2 and Akt.