Botulinum toxin complex increases paracellular permeability in intestinal epithelial cells via activation of p38 mitogen-activated protein kinase

Abstract

Clostridium botulinum produces a large toxin complex (L-TC) that increases paracellular permeability in intestinal epithelial cells by a mechanism that remains unclear. Here, we show that mitogen-activated protein kinases (MAPKs) are involved in this permeability increase. Paracellular permeability was measured by FITC-dextran flux through a monolayer of rat intestinal epithelial IEC-6 cells, and MAPK activation was estimated from western blots. L-TC of C. botulinum serotype D strain 4947 increased paracellular dextran flux and activated extracellular signal-regulated kinase (ERK), p38, but not c-Jun N-terminal kinase (JNK) in IEC-6 cells. The permeability increase induced by L-TC was abrogated by the p38 inhibitor SB203580. These results indicate that L-TC increases paracellular permeability by activating p38, but not JNK and ERK.

Fig. 1.

Effect of toxins on IEC-6 cell morphology and paracellular permeability. (A) Fluorescent microscopy of IEC-6 cells treated with L-TC (200 nM). Actin filaments were visualized with Alexa Fluor 546-conjugated phalloidin. (B) Time-dependent changes in FITC-dextran flux through IEC-6 cell layers. Cells seeded in Transwells were incubated with each toxin (200 nM) and fixed with formaldehyde. FITC-dextran was allowed to pass through the cell layer for 1 hr. Values of FITC-dextran fluorescence intensities were normalized with those obtained in cells without toxin treatment (time 0). (C) Effects of neuraminidase (NDase) treatment on FITC-dextran flux. Cells were treated with NDase for 18 hr and then incubated with L-TC for 24 hr. Values were normalized with FITC-dextran flux of cells incubated with L-TC and without NDase treatment. Data represent mean ± SEM, n=3.

Fig. 2.

Toxin-induced phosphorylation of MAPKs in IEC-6 cells. (A, C and E) Time-dependent changes in phosphorylation induced by 200 nM toxin. Upper two panels show representative band patterns of phosphoryated and total MAPKs in IEC-6 cells incubated with L-TC. Open circle, control; closed circle, BoNT; open square, NTNHA/HAs; closed square, L-TC. (B, D and F) Dose-dependent phosphorylation of MAPKs after incubation with L-TC for 12 hr. After toxin incubation, cells were harvested and proteins were subjected to SDS-PAGE and western blotting using antibodies against phosphorylated and total MAPKs. Band densities of phosphorylated MAPKs were normalized to those of total MAPKs, and further normalized with those obtained in cells without toxin treatment (time 0). Data represent mean ± SEM, n=3–9.

Fig. 3.

Effect of MAPK inhibitors on FITC-dextran flux through IEC-6 cell layers treated with (A, B) or without (C, D) L-TC. PD, PD98059; SB, SB203580; SP, SP600125. Each inhibitor at 10 (A, C) or 20 (B, D) µM was added to the medium 30 min before the experiments. Cells were then incubated with or without L-TC (200 nM) in the presence of each inhibitor for 12 hr. Values were normalized with FITC-dextran flux of cells incubated without inhibitors. Data represent mean ± SEM, n=6−15. **, P<0.01 and *, P<0.05 compared to L-TC (A, B) or control (D) group (non-repeated ANOVA followed by Bonferroni correction).