C391 inhibits Alternaria alternata-induced signaling in airway epithelial and HEK293 cells
Proteinases contained in A. alternata activate PAR2 to promote Ca2+ mobilization, β-arrestin recruitment and activation of downstream signaling events (Boitano et al., 2011; Yee et al., 2018). We have shown that A. alternata -induced Ca2+ mobilization in human airway epithelial (16HBE14o-) cells requires PAR2 (Boitano et al., 2011). To determine whether C391, which can block both peptidomimetic-induced and trypsin-induced PAR2 signaling (Boitano et al., 2015), can effectively block A. alternata -induced signals, we examined Ca2+ mobilization, MAPK phosphorylation and β-arrestin-2 recruitment. Treatment of 16HBE14o- cells with A. alternata (~0.65 U/mL trypsin activity) raised [Ca2+]i from an average resting concentration of ~35 nM to ≥ 150 nM, within the 3 min experimental protocol, with 59.0 ± 24.0% of cells showing a Ca2+ response ([Ca2+]i >100nM) (Figure 1A, C, E - H ; n = 3). Pre-incubation of cells with 3 µM C391 for 2 min prior to addition of A. alternata significantly reduced the percentage of cells exhibiting a Ca2+ ( 6.4 ± 2.7%), similar to that observed with 3 µM C391 alone (4.2 ± 1.3%), and resulted in an average [Ca2+]i of less than 75 nM (Figure 1A, D, I-L ; n = 3, p < 0.05).
A. alternata has been shown to increase Ca2+signaling, independent of PAR2, via ATP (O’Grady, Patil, Melkamu, Maniak, Lancto & Kita, 2013). We thus repeated the C391 experiments following exposure of 16HBE14o- cells to either 1 µM (data not shown) or 2.5 µM ATP (Figure 1M - O, n = 3) , which induced a Ca2+ response in 15.2 ± 3.8% and 62.3 ± 11.5% of cells, respectively. This Ca2+ response was unaffected by pre-treatment with 3 µM C391 which resulted in a response in 21.3 ± 96.8% (1 µM ATP) and 53.8 ± 9.4% (2.5 µM ATP) of cells. Due to the inherent similarities of PAR2 with other PARs, we also tested the effects of C391 on thrombin-induced Ca2+ signaling in 16HBE14o- cells (Figure 1M, P-Q ). High concentrations of thrombin (100 µM) induced a low level of Ca2+ response in 16HBE14o- cells (13.3 ± 2.7%; n = 6) that was not statistically different when cells were pre-incubated with 3 µM C391 (11.0 ± 3.6%; n = 7).
Activation of PAR2 results in MAPK signalling, in part through a β-arrestin-dependent pathway, that can be measured via phosphorylation of the 42kD and 44kD MAPK family members (p-MAPK) and we have demonstrated previously that C391 inhibits peptidomimetic-induced MAPK activation (Boitano et al., 2015). Treatment of 16HBE14o- cells with increasing concentrations of A. alternata for 5 minutes resulted in a 1.4-fold increase in MAPK phosphorylation, with an EC50 of 35 µg/mL (Figure 2A ; n = 4 for all concentrations), similar to that observed with the peptidomimetic agonist 2-at-LIGRL-NH2 (600 nM; Figure 2B ; n = 4 for all concentrations). However, a 95% confidence interval for pMAPK could not be constructed with A. alternata due to non-proteinase activation of MAPK pathways (O’Grady, Patil, Melkamu, Maniak, Lancto & Kita, 2013; Yee et al., 2018). Pre-treatment with increasing concentrations of C391 (30 nM - 100 μM) for 15 min inhibitedA. alternata ( 20 µg/mL) -induced p-MAPK with an IC50 of 12.6 μM (CI: 3.2 - 50.6 μM). C391 alone did not alter p-MAPK at any of the concentrations tested (not shown;Figure 2C ).
We have previously shown that A. alternata -induced activation of PAR2 leads to recruitment of β-arrestin-2, and many of the indicators of allergic asthma involving PAR2 activation are abolished in β-arrestin-2-/- mice (Nichols et al., 2012; Yee et al., 2018). To determine whether C391 inhibits β-arrestin-2 recruitment to PAR-2 in response to A. alternata , we used bioluminescence resonance energy transfer (BRET) between PAR2-YFP and β-arrestin2-RLuc. We have previously used this technique to monitor recruitment of β-arrestin-1 and 2 to PAR2 in response to trypsin, the potent peptidomimetic agonist 2-f-LIGRLO-NH2 (which is an equivalent agonist to 2-at-LIGRL-NH2 used in the Ca2+ and MAPK signaling assays), and A.alternata (Flynn et al., 2011; McGuire, Saifeddine, Triggle, Sun & Hollenberg, 2004; Nichols et al., 2012; Yee et al., 2018). C391 blocked β-arrestin-2 recruitment in response to 2-f-LIGRLO-NH2 (Imax = 88±4%; Log IC50=-7.8±0.07, CI: 7-21nM, n=6; Figure 3A ), or A. alternata(Imax = 88±5%; Log IC50 =-7.9±0.17, CI 1 – 27 nM; n=3; Figure 3B) . We conclude that C391 can inhibit A. alternata -induced Ca2+ mobilization, MAPK phosphorylation and β-arrestin-2 recruitment, with similar efficacy to that seen when PAR2 is activated by peptidomimetic agonists or trypsin.