Serratia marcescens serralysin induces inflammatory responses through protease-activated receptor 2

Abstract

The Serratia marcescens-derived protease serralysin is considered to play an important role in the pathogenesis of infection. Protease-activated receptor 2 (PAR-2) is activated by trypsin and also several other trypsin-like serine proteases, leading to the modulation of inflammatory and immune responses. However, little is known about the activation of PAR-2 by bacterial proteases and its roles in bacterial infection. In this study, we investigated whether S. marcescens serralysin activates host inflammatory responses through PAR-2. Our results demonstrated that serralysin induces interleukin-6 (IL-6) and IL-8 mRNA expression in a human lung squamous cell carcinoma, EBC-l cells. In addition, serralysin activated activator protein 1 (AP-1)-, CCAAT/enhancer-binding protein (C/EBP)-, and nuclear factor-kappaB (NF-kappaB)-driven promoters in EBC-1 cells. An electrophoretic mobility shift assay showed that serralysin activates the binding of AP-1, C/EBPbeta, and NF-kappaB in the cells. Inactivation of serralysin resulted in the failure of transactivation of AP-1-, C/EBP-, and NF-kappaB-driven promoters in the cells. Furthermore, serralysin activated AP-1-, C/EBP-, and NF-kappaB-driven promoters via PAR-2 in HeLa cells. PAR-2 antagonist peptides decreased serralysin-induced transactivation of AP-1-, C/EBP-, and NF-kappaB-driven promoters in EBC-1 cells. Considered together, these results suggest that serralysin requires PAR-2 to activate the critical transcription factors AP-1, C/EBPbeta, and NF-kappaB for host inflammatory responses.

FIG. 1.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of serralysin purified by anion-exchange column chromatography. The gel was stained with Coomassie blue G-250. Lane M, molecular weight markers; lane 1, supernatant proteins were precipitated with ammonium sulfate; lane 2, flowthrough fraction from Q-Sepharose anion-exchange column; lane 3, purified serralysin.

FIG. 2.

Serralysin induces IL-6 and IL-8 mRNA expression in EBC-1 cells. EBC-1 cells were stimulated with a range of concentrations of serralysin for 2 h. Total RNA was then extracted, and quantitative real-time reverse transcription-PCR analysis was performed to determine the amounts of IL-6, IL-8, and β-actin mRNA. IL-6 and IL-8 mRNA levels, normalized to those β-actin, are expressed as the fold induction over unstimulated EBC-1 cells. Values represent the mean ± the standard error of the mean (SEM) from three independent experiments.

FIG. 3.

Serralysin as well as trypsin activates AP-1-, C/EBP-, and NF-κB-driven promoters in EBC-1 cells. EBC-1 cells were transfected with 95 ng of appropriate plasmids pAP-1-Luc (A and D), pC/EBP-Luc (B and E), and pNF-κB-Luc (C and F) as reporter plasmids and 5 ng of phRG-TK as an internal control plasmid. After 32 h, transfected cells were stimulated at the indicated concentrations of serralysin or trypsin. After a further 16 h of incubation, the cells were lysed and assayed for luciferase activity. The data are presented as the relative luciferase activity. The results for each set of transfections were normalized for Renilla luciferase activity and to the unstimulated samples. Values represent the mean ± the SEM from three independent experiments.

FIG.4.

EMSA of AP-1, C/EBPβ, and NF-κB in nuclear extract prepared from EBC-1 cells stimulated with serralysin. The following oligonucleotides were used: AP-1, 5′-GGATAGCCTGAGTCAGGGGATA-3′ (A); C/EBP, 5′-GGATAGGCTTGCGCAATGGATA-3′ (B); and NF-κB, 5′-GGATCCGGGGACTTTCCGCGGAT-3′ (C). The consensus sequences for the binding of these transcription factors are underlined. A ³²P-labeled, double-stranded oligonucleotide probe was incubated with nuclear extract from EBC-1 cells stimulated with or without 1 nM serralysin for 2 h as described in Materials and Methods. The specificity of the binding is shown by competition with unlabeled double-stranded oligonucleotides. The competitors were used in a 100-fold molar excess over labeled probes. Supershift assays were performed with 1 μg of the appropriate antibodies: rabbit anti-c-Jun/AP-1 antibody (lane A), rabbit anti-C/EBPβ antibody (lane β), rabbit anti-NF-κB p50 antibody (lane p50), and rabbit anti-NF-κB p65 antibody (lane p65), or normal rabbit IgG as a control (lanes C). Lane P indicates the probe-only control. The data are presented from a representative experiment, and similar results were obtained in three independent experiments.

FIG.4.

EMSA of AP-1, C/EBPβ, and NF-κB in nuclear extract prepared from EBC-1 cells stimulated with serralysin. The following oligonucleotides were used: AP-1, 5′-GGATAGCCTGAGTCAGGGGATA-3′ (A); C/EBP, 5′-GGATAGGCTTGCGCAATGGATA-3′ (B); and NF-κB, 5′-GGATCCGGGGACTTTCCGCGGAT-3′ (C). The consensus sequences for the binding of these transcription factors are underlined. A ³²P-labeled, double-stranded oligonucleotide probe was incubated with nuclear extract from EBC-1 cells stimulated with or without 1 nM serralysin for 2 h as described in Materials and Methods. The specificity of the binding is shown by competition with unlabeled double-stranded oligonucleotides. The competitors were used in a 100-fold molar excess over labeled probes. Supershift assays were performed with 1 μg of the appropriate antibodies: rabbit anti-c-Jun/AP-1 antibody (lane A), rabbit anti-C/EBPβ antibody (lane β), rabbit anti-NF-κB p50 antibody (lane p50), and rabbit anti-NF-κB p65 antibody (lane p65), or normal rabbit IgG as a control (lanes C). Lane P indicates the probe-only control. The data are presented from a representative experiment, and similar results were obtained in three independent experiments.

FIG. 5.

Inactivation of serralysin leads to the abrogation of inducible AP-1-, C/EBP-, and NF-κB-driven promoters activity in response to simulation with serralysin in EBC-1 cells. EBC-1 cells were transfected with 95 ng of appropriate plasmids: pAP-1-Luc, pC/EBP-Luc, and pNF-κB-Luc as reporter plasmids and 5 ng of phRG-TK as an internal control plasmid. After 32 h, transfected cells were stimulated with EDTA- or PBS-treated serralysin at a final concentration of 1 nM. After a further 16 h of incubation, the cells were lysed and assayed for luciferase activity. The data are presented as in the legend to Fig. 2. *, P < 0.05; **, P < 0.01 (compared to unstimulated cells). ##, P < 0.01 (compared to PBS-treated serralysin-stimulated cells).

FIG. 6.

Serralysin activates AP-1-, C/EBP-, and NF-κB-driven promoters in HeLa cells transfected with human PAR-2. HeLa cells were transfected with 95 ng of appropriate plasmids: pAP-1-Luc (A), pC/EBP-Luc (B), and pNF-κB-Luc (C) as reporter plasmids and 5 ng of phRG-TK as an internal control plasmid, in addition to human PAR-2 expression plasmid. The pcDNA3.1 was used to ensure that all transfection mixtures had a total of 350 ng of DNA. After 48 h, transfected cells were stimulated with or without 1 nM serralysin. After a further 6 h of incubation, the cells were lysed and assayed for luciferase activity. The data are presented as in the legend to Fig. 2.

FIG. 7.

Human PAR-2 antagonist peptides reduce serralysin-induced transactivation of AP-1-, C/EBP-, and NF-κB-driven promoters. EBC-1 cells were transfected with 95 ng of the appropriate plasmids: pAP-1-Luc (A), pC/EBP-Luc (B), and pNF-κB-Luc (C) as reporter plasmids and 5 ng of phRG-TK as an internal control plasmid. After 32 h, transfected cells were incubated with the indicated antagonists at a final concentration of 200 μM for 1 h prior to stimulation with or without 1 nM serralysin. After a further 16 h of incubation, the cells were lysed and assayed for luciferase acitivity. The data are presented as in the legend to Fig. 2. *, P < 0.05; **, P < 0.01 (compared to unstimulated cells). #, P < 0.05; ##, P < 0.01 (compared to stimulated cells in the absence of the antagonists).