Neutrophil elastase and proteinase-3 trigger G protein-biased signaling through proteinase-activated receptor-1 (PAR1)

Abstract

Neutrophil proteinases released at sites of inflammation can affect tissue function by either activating or disarming signal transduction mediated by proteinase-activated receptors (PARs). Because PAR1 is expressed at sites where abundant neutrophil infiltration occurs, we hypothesized that neutrophil-derived enzymes might also regulate PAR1 signaling. We report here that both neutrophil elastase and proteinase-3 cleave the human PAR1 N terminus at sites distinct from the thrombin cleavage site. This cleavage results in a disarming of thrombin-activated calcium signaling through PAR1. However, the distinct non-canonical tethered ligands unmasked by neutrophil elastase and proteinase-3, as well as synthetic peptides with sequences derived from these novel exposed tethered ligands, selectively stimulated PAR1-mediated mitogen-activated protein kinase activation. This signaling was blocked by pertussis toxin, implicating a Gαi-triggered signal pathway. We conclude that neutrophil proteinases trigger biased PAR1 signaling and we describe a novel set of tethered ligands that are distinct from the classical tethered ligand revealed by thrombin. We further demonstrate the function of this biased signaling in regulating endothelial cell barrier integrity.

Keywords: Biased Signaling; Calcium Signaling; Endothelial Cell; G Protein-coupled Receptors (GPCR); Intracellular Trafficking; MAP Kinases (MAPKs); Neutrophil; Proteinase-activated Receptor; Thrombin.

FIGURE 1.

Neutrophil enzymes disarm PAR1 to attenuate thrombin-stimulated calcium signaling. A, representative traces showing calcium signaling in KNRK-PAR1-eYFP cells in response to TFLLR-NH₂ (TF), thrombin (Thr), NE, and PR3. // indicates 20 min. B, histogram quantifying thrombin-dependent calcium signaling in KNRK-PAR1-eYFP cells following NE treatment for 20 min. C, histogram quantifying thrombin-dependent calcium signaling in KNRK-PAR1-eYFP cells following PR3 treatment for 20 min. Data are expressed as mean ± S.D. as a percentage of the signal triggered by 20 nm thrombin.

FIGURE 2.

Neutrophil enzymes activate MAPK signaling in KNRK-PAR1-eYFP cells. A, activation of p42/44 MAP kinase by thrombin (Thr) and TFLLR-NH₂ (TF) in KNRK-PAR1-eYFP cells but not in KNRK-pCDNA3 cells. B, concentration-dependent activation of p42/44 MAP kinase by NE in KNRK-PAR1-eYFP cells but not in KNRK-pCDNA3 cells. C, concentration-dependent activation of p42/44 MAP kinase by PR3 in KNRK-PAR1-eYFP cells but not in KNRK-pCDNA3 cells. D, concentration-dependent activation of p42/44 MAP kinase by NE in MCF7-PAR1-eYFP cells. E, concentration-dependent activation of p42/44 MAP kinase by PR3 in MCF7-PAR1-eYFP cells but not in MCF7-pCDNA3 cells. All MAPK signaling data are shown following 10 min of agonist treatment. Blots are representative of data from at least 3 experiments performed in independently cultured cells.

FIGURE 3.

Time course of neutrophil enzyme-activated MAPK signaling in KNRK-PAR1-eYFP cells. Activation of p42/44 MAP kinse by: A, thrombin (Thr); B, TFLLR-NH₂ (TF); C, NE; and D, PR3 in KNRK-PAR1-eYFP cells. E, graph showing densitometry analysis of normalized p42/44 MAP kinase activation in KNRK-PAR1-eYFP cells over 90 min. Data are expressed as mean ± S.E., n = 3.

FIGURE 4.

Confocal imaging of PAR1 dynamics following activation with TFLLR-NH₂, thrombin, NE, and PR3. A, a schematic depicting the structure of the mCherry-hPAR1-eYFP construct and the expected pseudo-color image when the receptor is intact or proteolytically cleaved. SP is signal peptide; TL is thrombin-revealed tethered ligand. B, untreated HEK-mCherry-PAR1-eYFP cell showing intact hPAR1 on the cell surface. C, HEK-mCherry-PAR1-eYFP cells treated with TFLLR-NH₂ for 3 min showing the intact receptor clustering on the cell membrane. D, HEK-mCherry-PAR1-eYFP cells treated with TFLLR-NH₂ for 30 min showing the intact receptor internalized. E, HEK-mCherry-PAR1-eYFP cells treated with thrombin for 3 min showing the N terminus-cleaved receptor on the cell surface. F, HEK-mCherry-PAR1-eYFP cells treated with thrombin for 30 min showing the N terminus-cleaved receptor internalized. G, HEK-mCherry-PAR1-eYFP cells treated with NE for 3 min showing the N terminus-cleaved receptor on the cell surface. H, HEK-mCherry-PAR1-eYFP cells treated with NE for 30 min showing the N terminus-cleaved receptor on the cell surface. I, HEK-mCherry-PAR1-eYFP cells treated with PR3 for 3 min showing the N terminus-cleaved receptor on the cell surface. J, HEK-mCherry-PAR1-eYFP cells treated with PR3 for 30 min showing the N terminus-cleaved receptor on the cell surface. Internalized red structures are presumed to represent the proteolytically released-internalized mCherry-tagged receptor N-terminal fragment.

FIGURE 5.

Mapping neutrophil proteinase cleavage of PAR1 N terminus. Five overlapping synthetic peptides covering the entire first extracellular domain of human PAR1 (PAR1^29–41, PESKATNATLDPR; PAR1^35–53, NATLDPRSFLLRNPNDKYE; PAR1^46–65, RNPNDKYEPFWEDEEKNESG; PAR1^64–83, SGLTEYRLVSINKSSPLQKQ; and PAR1^82–102, KQLPAFISEDASGYLTSSWLT) (Table 1) were synthesized and subject to proteolysis by NE and PR3. A, HPLC trace showing peaks corresponding to the major cleavage products for PAR1^35–53 subject to NE proteolysis. Peptide identities corresponding to each peak were obtained by mass spectrometry. B, HPLC trace showing peaks corresponding to the major cleavage products for PAR1^29–41 subject to PR3 proteolysis. Peptide identities corresponding to each peak were obtained by mass spectrometry. C, schematic depicting the PAR1 N-terminal region showing the major identified cleavage sites for NE and PR3 as well as known cleavage sites for MMP-1, thrombin, and APC. No significant cleavage of PAR1^35–53 was seen with PR3 and NE failed to cleave PAR1^29–41. Neither proteinase cleaved PAR1^46–65, PAR1^64–83, nor PAR1^82–102.

FIGURE 6.

Confocal imaging of PAR1 cleavage by thrombin and NE in mCherry-PAR1L⁴⁴E,L⁴⁵E-eYFP transfected cells. A, untreated HEK-mCherry-PAR1L44E,L45E-eYFP cell showing intact receptor on the cell surface (yellow pseudocolour from overlay of N-terminal mCherry (red) and C-terminal eYFP (green) tags. B, thrombin-treated (3 min) HEK-mCherry-PAR1L44E,L45E-eYFP cell showing removal of the N-terminal mCherry tag (image showing green pseudocolor from C-terminal eYFP tag). C, 3-min NE-treated HEK-mCherry-PAR1L44E,L45E-eYFP cell showing intact receptor on the cell surface (yellow pseudocolour from overlay of N-terminal mCherry (red) and C-terminal eYFP (green) tags. D, 20-min NE-treated HEK-mCherry-PAR1L44E,L45E-eYFP cell showing intact receptor on the cell surface (yellow pseudocolour from overlay of N-terminal mCherry (red) and C-terminal eYFP (green) tags.

FIGURE 7.

Peptides derived from neutrophil enzyme-revealed tethered ligands activate PAR1-dependent MAP kinase signaling in a Gα_i-dependent manner. A, neutrophil elastase-tethered ligand (NE-TL-AP, RNPNDKYEPF-NH₂), and B, proteinase-3-tethered ligand (PR3-TL-AP, TLDPRSF-NH₂) peptides activate KNRK-PAR1-eYFP cell MAPK in a concentration-dependent manner. MAPK activation is not seen in empty vector (EV)-transfected KNRK-pCDNA3 cells treated with 20 μm NE-TL-AP or PR3-TL-AP. C, MAPK activation by NE-TL-AP, and D, PR3-TL-AP in endogenous PAR1-expressing HEK-293 cells, but not in HEK-293 cells treated with the PAR1 antagonist SCH79797 (2 μm). E, histogram quantifying the PTX inhibition of MAPK signaling caused by thrombin, NE-TL-AP, and PR3-TL-AP in KNRK-PAR1-eYFP cells. Data are expressed as mean ± S.E. * indicates significant difference (p < 0.05, n = 3) from non-PTX-treated cell MAPK signal.

FIGURE 8.

Induction of F-actin stress fiber formation and modulation of permeability in HUVECs by NE-TL-AP and PR3-TL-AP. Confocal microscopy image of phalloidin-labeled F-actin (red) and DAPI-labeled nucleus (blue) in HUVECs treated with no agonist (A), thrombin (5 nm) (B), NE-TL-AP (20 μm) (C), and PR3-TL-AP (4 μm) (D). Integrity of the endothelial barrier was monitored by tracking the flux of FITC-dextran across HUVEC monolayers cultured on transwell supports following treatment with thrombin (5 nm) in combination with NE-TL-AP (20 μm) (E), PR3-TL-AP (4 μm) (F), APC-TL-AP (100 μm) (G), and MMP-TL-AP (20 μm) (H) as indicated. Permeability (% control) was normalized to the fluorescent reading obtained in the lower chamber for cells that had not been treated with agonists (expressed as 100%). Data are expressed as mean ± S.E. * indicates significant difference (p < 0.05) compared with agonist-untreated cells and # indicates significant difference (p < 0.05) compared with thrombin-treated cells (n = 6 for E and F; n = 3 for G and H).