FIGURE 3.

Peptidomimetic PAR₂ agonists stimulate MAPK signaling in model epithelial cells. 16HBE14o- cells were dose-dependently treated with 2-f-LIGRLO-NH₂, 2-at-LIGRL-NH₂, and 6-an-LIGRL-NH₂ for 5 min. A, representative immunoblot showing a robust increase in ERK 1/2 phosphorylation starting at a dose of 2.5 μm 2-f-LIGRLO-NH₂. Quantitation using densitometry shows that this increase was maintained up to 10 μm (n = 4). B, representative immunoblot showing 2-at-LIGRL-NH₂ first induced ERK 1/2 phosphorylation at a dose of 2.5 μm. This increase was maintained at 5 and 10 μm. Densitometry is consistent with an initial activation at 2.5 μm agonist and peak activation by 5 μm (n = 4). C, representative immunoblot and quantitative densitometry showing that 6-an-LIGRL-NH₂-induced ERK 1/2 phosphorylation was increased in a dose-dependent manner (n = 4). *** (A–C), significant increase compared with the vehicle control, p < 0.001. D, an in-cell Western assay was performed to develop MAPK activation curves for each agonist. pERK was assayed for MAPK signaling, and DRAQ5 was assayed as a total cell control. The percent maximal pERK/DRAQ5 is graphed for each agonist (±S.E.). EC₅₀ values calculated from four experiments for each agonist are as follows: 2-f-LIGRLO-NH₂, 317 nm; 2-at-LIGRL-NH₂, 495 nm, and 6-an-LIGRL-NH₂, 864 nm. t-ERK, total ERK.