Fig. 6.

Tight junction barrier defect in T84 cells due to Shigella infection is regulated by extracellular signal-regulated kinase (ERK) signaling pathway for claudin-2 and -4 phosphorylation status. Effects of apical (Ap) Zn²⁺and basolateral (Bl) PD98059 (MAPK inhibitor) on transepithelial electrical resistance (TER; A), 4-kDa FITC-dextran flux (B) in uninfected (Control) and Shigella-infected (NY962/92) T84 monolayers. Anthrapyrazolone (SP600125, 10 μM) failed to inhibit Shigella-mediated decrease in TER and increase permeability of 4-kDa FITC dextran in T84 monolayers (top A and B, respectively). C: claudin-2 (top) and -4 (bottom) localization in uninfected and Shigella-infected T84 monolayers treated with or without PD98059 or SP600125. Bar graph represents quantification of multiple images based on continuity of claudin-2 and -4 stains (broken vs. unbroken) in uninfected (Control) and Shigella-infected T84 cell monolayers in presence of PD98059 and SP600125. D: effect of PD98059 on Shigella adhesion (top) and invasion (bottom) in infected T84 cell monolayers. Both adhesion and invasion of Shigella are not inhibited by PD98059 in T84 cell monolayers. Data are means ± SE of 3 independent experiments. NS, not significant. E: effect of Shigella infection on ERK phosphorylation in presence or absence of Zn²⁺ in T84 cell monolayers as assessed by Western blot of phospho (p)-ERK. Densitometric quantification (ratio of ratios) of p-ERK levels relative to total GAPDH [(p-ERK/GAPDH)/(total ERK/GAPDH)]in triplicate experiments. F: immunoprecipated (IP) proteins with anti-claudin-2 (top) and anti-claudin-4 (bottom) antibody from T84 cell lysate of uninfected and Shigella-infected in presence or absence of Zn²⁺ were examined for Ser/Thr phosphorylation by Western blotting using phosphor-antibody. Whole cell lysate was used as loading control. Representative blots represent 3 independent experiments. G: effects of PD98059 on IL-6 and IL-8 production in uninfected (Control) and Shigella-infected T84 monlayers in presence (NY962/92 + PD98059) or absence (NY962/92) of PD98059. Results are expressed as means ± SE. NS, P > 0.05, which is considered not statistically significant.