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.