FIGURE 4.

Prostasin induced barrier formation requires endogenous matriptase. A, matriptase (siM2) depleted Caco-2 monolayers were treated on the basal side on day 4 with 5 nm recombinant prostasin (rProstasin) or left untreated, and TEER development was measured in comparison with control (control siRNA (siCtl)) cultures over the course of 5 h. Immunoblot of cell lysates prepared on day 5 shows that effective depletion of matriptase is sustained. The plot shows TEER development over time (mean ± S.E.). The low TEER of matriptase-depleted cultures is not enhanced by treatment with 5 nm recombinant prostasin. B, prostasin (siP1) depleted cultures were treated with 5 nm recombinant prostasin as in (A) or left untreated, and TEER was measured in comparison with control (control siRNA (siCtl)) cultures over the course of 5 h. The low TEER of prostasin-depleted cultures is restored to the levels of the control cultures within 3 h. C, immunoblots of cell lysates prepared from parallel cultures at the indicated times in B show that the addition of recombinant prostasin stimulates matriptase zymogen activation in prostasin-depleted cells.