Figure 4
Figure 4. Matriptase mediates epithelial signaling induced by the coagulation initiation complex. (A) HaCaT cells were incubated for 1 hour in the presence of anti-TF (4.5 µg/mL), GB83 (20 µM), or FabE2 (500 nM), and changes in TEER were recorded after the addition of FVIIa (50 pM)/X (100 nM). (Left) Representative tracings normalized to prestimulation values and vehicle control. (Right) Area under the curve (AUC) (mean ± SD of 4 independent experiments). Note that all inhibitors blunted the ability of coagulation proteases to increase TEER. (B) TF-transfected MCF7 cells were stimulated with the matriptase activator prostasin (5 nM), PAR2-AP (50 μM), FVIIa (10 nM), FXa (20 nM), or thrombin (5 nM), and TEER was recorded. Note that the PAR2-AP and all proteases but thrombin induce a prolonged increase in barrier function. (C) MCF7 cells were incubated with matriptase (0.5 nM) or PAR2-AP (50 μM), and TEER was recorded. When TEER peaked in response to matriptase, the serine protease inhibitor ecotinRR (50 nM) or vehicle control was added (arrow). Note the rapid return to baseline on inhibition of matriptase, suggesting that continuous receptor activation sustains high resistance. The graph is representative of 3 independent experiments (quantification in supplemental Figure 7B). Tracings in B and C are normalized to prestimulation values and vehicle control. (D) TF-transfected MCF7 cells were incubated with camostat (1 μM), FabE2 (500 nM), or vehicle for 15 min before the addition of agonists PAR2-AP (50 μM), FVIIa (10 nM), FXa (20 nM), FVIIa (50 pM)/FX (100 nM), or prostasin (5 nM), and TEER was recorded during 4 hours. The AUC was quantified and expressed as a percentage of the unrestrained agonist response. Camostat and FabE2 significantly inhibited responses to the matriptase-activator prostasin and to coagulation proteases, but not to PAR2-AP. (E) TF-transfected MCF7 or HaCaT cells were incubated with camostat (1 μM) or vehicle for 15 min before addition of agonists, and TEER was recorded during 4 hours. The AUC was quantified and expressed as a percentage of the unrestrained agonist response. Responses are more sensitive to camostat at low concentrations of coagulation proteases and are more sensitive in HaCaT than MCF7. (F) HaCaT cells were incubated with camostat (1 μM), FabE2 (500 nM), or vehicle for 10 min before stimulation for 10 min with FVIIa (50 pM) and/or FX (100 nM), hepsin (10 nM), thrombin (αT, 5 nM), or PAR2-AP (50 µM). Total ERK and phospho-ERK levels were determined by western blotting. (Left) Representative experiment. (Right) Pooled ratiometric analyses of pERK/total ERK (mean ± SD; n = 3). Note that all agonists except thrombin induced an increase in ERK phosphorylation and that activation by proteases, but not peptide agonist, was sensitive to matriptase inhibitors. After 1-way ANOVA, a Newman-Keuls multiple comparison test was used for statistical analysis in A and for PAR2-AP, FVIIa, and FXa stimulations in D; Student t test was used for analysis of the data elsewhere. ***P < .001 relative to vehicle control; #P < .05, ##P < .01, ###P < .001 relative to uninhibited.

Matriptase mediates epithelial signaling induced by the coagulation initiation complex. (A) HaCaT cells were incubated for 1 hour in the presence of anti-TF (4.5 µg/mL), GB83 (20 µM), or FabE2 (500 nM), and changes in TEER were recorded after the addition of FVIIa (50 pM)/X (100 nM). (Left) Representative tracings normalized to prestimulation values and vehicle control. (Right) Area under the curve (AUC) (mean ± SD of 4 independent experiments). Note that all inhibitors blunted the ability of coagulation proteases to increase TEER. (B) TF-transfected MCF7 cells were stimulated with the matriptase activator prostasin (5 nM), PAR2-AP (50 μM), FVIIa (10 nM), FXa (20 nM), or thrombin (5 nM), and TEER was recorded. Note that the PAR2-AP and all proteases but thrombin induce a prolonged increase in barrier function. (C) MCF7 cells were incubated with matriptase (0.5 nM) or PAR2-AP (50 μM), and TEER was recorded. When TEER peaked in response to matriptase, the serine protease inhibitor ecotinRR (50 nM) or vehicle control was added (arrow). Note the rapid return to baseline on inhibition of matriptase, suggesting that continuous receptor activation sustains high resistance. The graph is representative of 3 independent experiments (quantification in supplemental Figure 7B). Tracings in B and C are normalized to prestimulation values and vehicle control. (D) TF-transfected MCF7 cells were incubated with camostat (1 μM), FabE2 (500 nM), or vehicle for 15 min before the addition of agonists PAR2-AP (50 μM), FVIIa (10 nM), FXa (20 nM), FVIIa (50 pM)/FX (100 nM), or prostasin (5 nM), and TEER was recorded during 4 hours. The AUC was quantified and expressed as a percentage of the unrestrained agonist response. Camostat and FabE2 significantly inhibited responses to the matriptase-activator prostasin and to coagulation proteases, but not to PAR2-AP. (E) TF-transfected MCF7 or HaCaT cells were incubated with camostat (1 μM) or vehicle for 15 min before addition of agonists, and TEER was recorded during 4 hours. The AUC was quantified and expressed as a percentage of the unrestrained agonist response. Responses are more sensitive to camostat at low concentrations of coagulation proteases and are more sensitive in HaCaT than MCF7. (F) HaCaT cells were incubated with camostat (1 μM), FabE2 (500 nM), or vehicle for 10 min before stimulation for 10 min with FVIIa (50 pM) and/or FX (100 nM), hepsin (10 nM), thrombin (αT, 5 nM), or PAR2-AP (50 µM). Total ERK and phospho-ERK levels were determined by western blotting. (Left) Representative experiment. (Right) Pooled ratiometric analyses of pERK/total ERK (mean ± SD; n = 3). Note that all agonists except thrombin induced an increase in ERK phosphorylation and that activation by proteases, but not peptide agonist, was sensitive to matriptase inhibitors. After 1-way ANOVA, a Newman-Keuls multiple comparison test was used for statistical analysis in A and for PAR2-AP, FVIIa, and FXa stimulations in D; Student t test was used for analysis of the data elsewhere. ***P < .001 relative to vehicle control; #P < .05, ##P < .01, ###P < .001 relative to uninhibited.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal