Mucin 3 is involved in intestinal epithelial cell apoptosis via N-(3-oxododecanoyl)-l-homoserine lactone-induced suppression of Akt phosphorylation

Abstract

N-acyl-homoserine lactones (AHL) are quorum-sensing molecules in bacteria that play important roles in regulating virulence gene expression in pathogens such as Pseudomonas aeruginosa. The present study compared responses between undifferentiated and differentiated Caco-2 cells to N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL). A low concentration of 3-oxo-C12-HSL (30 μM) is sufficient to reduce viability accompanied by apoptosis via the suppression of phosphorylation by Akt in undifferentiated Caco-2 cells. The suppression of Akt phosphorylation appears specific in 3-oxo-C12-HSL, because other AHLs did not influence the phosphorylation status of Akt. The reduced viability induced by 3-oxo-C12-HSL was partially recovered by constitutively active Akt overexpression in undifferentiated Caco-2 cells. Since mucin is considered a vital component of the gut barrier, we investigated whether mucin protects cellular functions induced by 3-oxo-C12-HSL in undifferentiated Caco-2 cells. The results showed that mucin protected undifferentiated Caco-2 cells from apoptosis induced by 3-oxo-C12-HSL. 3-Oxo-C12-HSL did not induce cell death in differentiated Caco-2 cells that expressed higher levels of mucin 3 (MUC3) than undifferentiated Caco-2 cells. In addition, 3-oxo-C12-HSL promoted cell death in undifferentiated Caco-2 cells transfected with MUC3 siRNA and reduced MUC3 expression in undifferentiated Caco-2 cells. Therefore, MUC3 might be responsible for the survival of undifferentiated intestinal epithelial cells in the presence of 3-oxo-C12-HSL through regulating Akt phosphorylation. In conclusion, 3-oxo-C12-HSL might influence the survival of undifferentiated intestinal epithelial cells as well as interactions between these cells and pathogens.

Activity of ERK regulates mucin 3 expression and is involved in undifferentiated Caco-2 cell death induced by 3-oxo-C12-homoserine lactone

“…7) However, we previously found that relatively low concentrations of 3-oxo-C 12 -HSL between 10 and 30 μM can induce the apoptosis of undifferentiated Caco-2 cells, and increase the mucosal permeability of the isolated rat colon. 8,9) Similar levels of 3-oxo-C 12 -HSL can be generated in culture supernatants of the P. aeruginosa PT149 strain without biofilm formation. 10) Therefore, 3-oxo-C 12 -HSL can affect intestinal epithelial cells at much lower concentrations than those found in biofilms.…”

“…Cell lysates were fractionated by SDS-PAGE and the proteins were transferred to Immobilon-P polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) for immunoblotting as described. 8) Phospho-ERK1/2 and phospho-Akt were normalized to total-ERK1/2 and total-Akt, respectively. Protein bands were visualized using the enhanced Chemi-Lumi One Super system (Nacalai Tesque Inc., Kyoto, Japan) and densitometrically analyzed using Lumi Vision PRO 400EX (Aisin, Kariya, Aichi, Japan).…”

“…Quantitative real-time PCR proceeded as described. 8) In brief, total RNA was isolated using Sepasol-RNA I Super G (Nacalai Tesque). First-strand cDNAs were synthesized from 3-oxo-C 12 3-oxo-C 12 3-oxo-C 12 3-oxo- Preparation of small interfering RNAs specific to MUC3.…”

“…We knocked down MUC3 as described using small interfering RNAs (siRNAs) specific to MUC3 and a negative control (Nippon EGT, Tokyo, Japan). 8) The siRNAs (final concentration, 50 nM) were transfected into undifferentiated Caco-2 cells using Lipofectamine RNAiMAX (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. The sense sequence of MUC3 siRNA-2906 was 5′-CCACAGCUCUCACUGAAAUdTdT-3′.…”

“…8) Thus, the present study aimed to determine the involved mechanism(s) and whether upregulated MUC3 expression attenuates the 3-oxo-C 12 -HSL-induced death of undifferentiated Caco-2 cells.…”