Analysis of proteinase-activated receptor 2 and TLR4 signal transduction: a novel paradigm for receptor cooperativity

Abstract

Proteinase-activated receptor 2 (PAR2), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR2, often measured by Ca2+ release. PAR2 contributes to inflammation through activation of NF-kappaB-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR2 in HEK293T cells resulted in concentration-dependent, PAR2 AP-inducible NF-kappaB reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple Gi proteins or sequester intracellular Ca2+. Because previous studies described synergistic PAR2- and TLR4-mediated cytokine production, we hypothesized that PAR2 and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR2-induced NF-kappaB activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR2 in HEK293T cells led to a synergistic increase in AP-induced NF-kappaB signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR2 and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4(-/-) and PAR2(-/-) macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.

FIGURE 1.

HEK293T/PAR₂ transfectants respond in a specific manner independent of species of origin of AP. A, HEK293T cells were co-transfected with either the control pcDNA or the untagged human PAR₂ construct, together with ELAM (NF-κB)-luciferase and pCMV1-β-galactosidase reporter constructs. Transfected cells were treated with TNF-α (50 ng/ml), 200 μm huPAR₁ AP (TFLLR-NH₂), or its inactive scrambled peptide or 200 μm huPAR₂ AP (SLIGKV-NH₂) or its inactive scrambled peptide for 16 h. Reporter activities were measured in cell lysates as described under “Experimental Procedures.” A representative experiment is shown (n = 3). B, HEK293T cells, transfected as described in A, were treated with huPAR₂ AP at the indicated concentrations or with human rIL-1β for 5 or 16 h. Cell lysates were assayed for ELAM-luc and β-galactosidase reporter activities. A representative experiment is shown (n = 8). C, HEK293T cells, transfected as described in A, were treated with medium only, rat/mouse PAR₂ AP (SLIGRL-NH₂), or huPAR₂ AP for 16 h. ELAM-luc and β-galactosidase reporter activities were measured in cell lysates. A representative experiment is shown (n = 5). RLU, relative luciferase units; M, medium.

FIGURE 2.

PAR₂ activation by AP in HEK293T/huPAR₂ transfectants is inhibited by PT and BAPTA-AM. HEK293T cells were co-transfected with huPAR₂ construct together with the ELAM-luc and β-galactosidase reporter constructs. Transfectants were pretreated with medium (M) or the indicated concentrations of PT (A) or BAPTA-AM (100 μm)(B) for 1 h. Cells were then further treated with indicated concentrations of huPAR₂ AP or TNF-α (TNF, 50 ng/ml) for an additional 16 h. Cell lysates were prepared and reporter activities measured. A representative experiment is shown (n = 5). *, p < 0.001 versus medium-pretreated controls. RLU, relative luciferase units.

FIGURE 3.

Co-expression of TLR4 and PAR₂ results in synergistic, MyD88-dependent NF-κB activation in response to PAR₂ AP. A and B, HEK293T cells were co-transfected with TLR4 signaling complex (pFLAG-huTLR4, pEF-BOS-HA-huMD-2, pcDNA3-huCD14) and/or huPAR₂ constructs, without or with DN-MyD88 construct (200 ng/well). Cells were also co-transfected with the NF-κB-luciferase and β-galactosidase reporter constructs. A, cells were treated with medium only or LPS, although in B the cells were treated with medium only or AP. A representative experiment is shown for A and B (n = 6). C, HEK293T cells were transfected as described above in the absence of reporter constructs. Levels of IL-8 in the supernatants were determined by enzyme-linked immunosorbent assay. A representative experiment is shown (n = 2). RLU, relative luciferase units. *, P < 0.001, #, P < 0.01, and +, P, < 0.05.

FIGURE 4.

Synergy between PAR₂ and TLR4 induced by PAR₂ AP requires MD-2 and a signaling-competent TLR4. A, HEK293T cells were co-transfected with the huPAR₂ construct only or huPAR₂, pFLAG-huTLR4, and huCD14, without or with the huMD-2 expression construct. All wells were co-transfected with ELAM-lucandβ-galactosidase reporter constructs. Transfected cells were treated with huPAR₂ AP or LPS, and reporter activities were measured. A representative experiment is shown (n = 4). B, HEK293T cells were co-transfected with the huPAR₂ construct only, or PAR₂ co-transfected with YFP-TLR4-WT or YFP-TLR4-P714H mutant (200 ng/well each) constructs either individually or co-transfected with huPAR₂ construct. All wells were also co-transfected with the reporter constructs. Transfected cells were treated with TNF-α, LPS, or huPAR₂ AP, and reporter activities were measured. A representative experiment is shown (n = 3). *, p < 0.001, between the PAR₂ and PAR₂ + YFP-TLR4-WT transfectants; **, p < 0.001, between PAR₂ + YFP-TLR4-WT and PAR₂ + YFP-P714H-TLR4 transfectants treated with huPAR₂ AP. RLU, relative luciferase units.

FIGURE 5.

Co-expression of PAR₂ results in synergistic NF-κB activation induced by PAR₂ AP with TLR4 but not with TLR2 and TLR3. HEK293T cells were co-transfected with either TLR4 signaling complex, FLAG-tagged TLR2, or FLAG-tagged TLR3 and/or huPAR₂ construct. All wells were transfected with the NF-κB-luciferase and pCMV1-β-galactosidase reporter constructs. Transfectants were treated with medium only or respective TLR agonist (LPS for TLR4, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(Lys)₄-OH, trihydrochloride (Pam3Cys) for TLR2, and poly(I:C) for TLR3), or AP. A representative experiment is shown (n = 3). *, p < 0.001, between the PAR₂ and PAR₂ + TLR4-WT transfectants. RLU, relative luciferase units.

FIGURE 6.

TLR4 co-immunoprecipitates with PAR₂ in a PAR₂ AP-dependent manner. HEK293T cells, transfected with either HA-PAR₂ vector or YFP-TLR4-WT vector along with MD-2 and CD14 expression vectors, or co-transfected together with HA-PAR₂ and YFP-TLR4 complex, were incubated for a further 72 h before washing and lysing as described under “Experimental Procedures.” Immunoprecipitation (IP) and Western blotting (IB) were carried out to analyze the physical interaction between PAR₂ and TLR4 proteins. A representative experiment is shown (n = 3). Molecular weight of proteins's represented in kDa (on right).

FIGURE 7.

PAR₂-mediated activation of NF-κB is decreased by inhibitors of TRIF and TRAM. HEK293T cells were co-transfected with huPAR₂ construct and DN-MyD88, DN-TRIF, or DN-TRAM (50 ng/well) expression vectors (A). Transfected cells were treated for 16 h with the indicated concentrations of huPAR₂ AP and ELAM-luc, and β-galactosidase reporter activities were measured. B, HEK293T cells were transfected with huPAR₂ construct, ELAM-luc, and β-galactosidase reporter constructs. Transfected cells were pretreated with medium or 40 μm CP or the indicated cell-permeable adapter blocking peptide (BP), and then treated with AP for an additional 16 h, at which time reporter activities were measured. A representative experiment is shown for each experiment (n = 3). #, p < 0.001. RLU, relative luciferase units.

FIGURE 8.

Co-transfection of TLR4 receptor complex with PAR₂ inhibits RANTES promoter activity. HEK293T cells were co-transfected with either huPAR₂ construct or huPAR₂ plus TLR4 signaling complex (pFLAG-huTLR4; pEFBOS-HA-huMD-2; pcDNA3-huCD14). The RANTES-luciferase reporter (500 ng/well) and β-galactosidase vector were also co-transfected. Transfectants were treated with the indicated concentrations of huPAR₂ AP for 16 h, and luciferase and β-galactosidase activities were measured. A representative experiment is shown (n = 5). **, p < 0.05. RLU, relative luciferase units.

FIGURE 9.

Macrophages deficient in TLR4 or PAR₂ show dysregulated responses to AP or LPS, respectively, and the SW620 intestinal epithelial cell line exhibits a synergistic response to co-stimulation with AP and LPS. A, TLR4^–/– macrophages exhibit diminished IL-1β gene induction in response to huPAR₂ AP. Macrophages from C57BL/6J or TLR4^–/– mice were stimulated with huPAR₂ AP (200 μm) or LPS (10 ng/ml) for 2 h, and steady-state IL-1β mRNA was measured. Results represent mean ± S.E. of six separate experiments. *, p < 0.001; Medium (C57BL/6J versus TLR4^–/–); #, p < 0.001 Medium versus LPS (C57BL/6J); ‡, p < 0.001 LPS (C57BL/6J versus TLR4^–/–); §, p < 0.001 Medium versus AP (C57BL/6J); and ¶, p < 0.001; AP (C57BL/6J versus TLR4^–/–). B, PAR ^–/–₂ macrophages exhibit diminished iNOS gene induction and NO• activity in response to LPS. Macrophages from C57BL/6J or PAR ^–/–₂ mice were stimulated for the indicated times with 10 ng/ml LPS for iNOS mRNA or with 100 ng/ml LPS for supernatant analysis for NO. Data are representative of three separate experiments. M, medium control; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (C). SW620 cells respond synergistically to huPAR₂ AP (200 μm) and LPS (100 ng/ml) co-stimulation to augment IL-8 secretion (t = 24 h). Data represent the mean ± S.E. of three separate experiments. M, medium control; RP, reverse peptide; ns, not significant; *, p < 0.05 (Medium versus AP); **, p < 0.01 (LPS versus LPS + AP).

FIGURE 10.

Hypothetical model of PAR₂ signaling and interaction with TLR4. See “Discussion” for description of model.