MAPK/AP-1 activation mediates TLR2 agonist-induced SPLUNC1 expression in human lung epithelial cells

Abstract

Background: Short Palate Lung and Nasal epithelium Clone 1 (SPLUNC1) is a newly described host defense protein, primarily expressed in large airway epithelial cells. Reduced SPLUNC1 has been reported in allergic and cigarette smoke-exposed airways. We found that Mycoplasma pneumoniae increases SPLUNC1 in airway epithelium in part via activating TLR2-NF-κB pathway. However, the contribution of additional signaling pathways to TLR2-mediated SPLUNC1 expression remains unclear. In the present study, we investigated if TLR2-induced mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) signaling regulates SPLUNC1 expression in human lung epithelial cells.

Methods: Human lung epithelial NCI-H292 cells were stimulated with a TLR2 agonist Palmitoyl (3)-Cys-Ser-Lys (4)-OH (Pam(3)CSK(4)). MAPK/AP-1 activation and its role in SPLUNC1 regulation were investigated by Western blot, c-Jun activation assay, chromatin immunoprecipitation (ChIP) and real-time PCR. SPLUNC1 promoter activity was assessed by a luciferase reporter assay.

Results: Pam(3)CSK(4) increased SPLUNC1 expression in NCI-H292 cells in a dose- and time-dependent manner, and enhanced SPLUNC1 promoter activity. Pam(3)CSK(4)-treated cells demonstrated activated MAPK and c-Jun compared to untreated cells. ChIP assay indicated increased c-Jun binding to the SPLUNC1 promoter following Pam(3)CSK(4) stimulation. Inhibition of ERK1/2 significantly reduced Pam(3)CSK(4)-mediated c-Jun activation and SPLUNC1 expression.

Conclusions: Our results for the first time demonstrate that TLR2-mediated MAPK/AP-1 activation up-regulates lung epithelial SPLUNC1 expression at the transcriptional level. Understanding SPLUNC1 gene regulation should provide more specific therapeutic targets to restore deficient SPLUNC1 production in diseased airways.

FIGURE 1. Pam₃CSK₄ increases SPLUNC1 expression in NCI-H292 cells

NCI-H292 cells were stimulated with Pam₃CSK₄ after overnight growth in reduced serum medium. SPLUNC1 mRNA and protein expression was determined by real-time PCR and Western blot, respectively. Pam₃CSK₄ dose-dependently increased SPLUNC1 mRNA at 48 h (A). A time course study revealed a time-dependent increase in SPLUNC1 mRNA by Pam₃CSK₄ at 100 ng/ml (B). Pam₃CSK₄ also induced SPLUNC1 protein expression at 48 h as determined by Western blot (C) Data expressed as means ± SEM, n = 6–9.

FIGURE 2. Pam₃CSK₄ increases SPLUNC1 transcriptional activity

NCI-H292 cells were transiently co-transfected with SPLUNC1 promoter reporter construct (−943/+47bp) containing firefly luciferase (F-luc) as reporter gene and renilla luciferase (R-luc) gene for normalizing transfection efficiency. After Pam₃CSK₄ stimulation for up to 48 h, SPLUNC1 promoter activity was determined by normalizing F-luc to R-luc and expressed as % of Pam₃CSK₄–treated versus untreated cells (A). To determine the effect of Pam₃CSK₄ on SPLUNC1 mRNA stability, NCI-H292 cells were stimulated with 100 ng/ml Pam₃CSK₄ for up to 16 h, and then treated with either 0.1% DMSO or 5 μg/ml actinomycinD (ACD) to inhibit de novo mRNA synthesis. Degradation of SPLUNC1 mRNA was examined for up to 12 h after ACD addition and expressed as % of SPLUNC1 mRNA levels without ACD at indicated time points (B). Data expressed as means ± SEM, n = 3–9.

FIGURE 3. Pam₃CSK₄ activates MAPK/AP-1 in NCI-H292 cells

After overnight growth in reduced serum medium, NCI-H292 cells were stimulated with 100 ng/ml Pam₃CSK₄ for up to 24 h. Activation of ERK (A) and JNK (B) and total c-Jun expression (C) were determined by Western blot. Upper panel in Figure A, B and C shows densitometry data from 3–6 independent replicates and lower panel is a representative picture of their Western blot. Activation of c-Jun transcription factor, a downstream target of ERK activation, was also determined by using Western blot (D) as well as an ELISA-based TransAM c-Jun activation assay (E). Data expressed as means ± SEM, n = 3–5.

FIGURE 3. Pam₃CSK₄ activates MAPK/AP-1 in NCI-H292 cells

After overnight growth in reduced serum medium, NCI-H292 cells were stimulated with 100 ng/ml Pam₃CSK₄ for up to 24 h. Activation of ERK (A) and JNK (B) and total c-Jun expression (C) were determined by Western blot. Upper panel in Figure A, B and C shows densitometry data from 3–6 independent replicates and lower panel is a representative picture of their Western blot. Activation of c-Jun transcription factor, a downstream target of ERK activation, was also determined by using Western blot (D) as well as an ELISA-based TransAM c-Jun activation assay (E). Data expressed as means ± SEM, n = 3–5.

FIGURE 3. Pam₃CSK₄ activates MAPK/AP-1 in NCI-H292 cells

After overnight growth in reduced serum medium, NCI-H292 cells were stimulated with 100 ng/ml Pam₃CSK₄ for up to 24 h. Activation of ERK (A) and JNK (B) and total c-Jun expression (C) were determined by Western blot. Upper panel in Figure A, B and C shows densitometry data from 3–6 independent replicates and lower panel is a representative picture of their Western blot. Activation of c-Jun transcription factor, a downstream target of ERK activation, was also determined by using Western blot (D) as well as an ELISA-based TransAM c-Jun activation assay (E). Data expressed as means ± SEM, n = 3–5.

FIGURE 4. Pam₃CSK₄ enhances c-Jun binding to the SPLUNC1 promoter

Several putative AP-1/c-Jun binding motifs were identified in the −2.5kb flanking region of SPLUNC1 gene including SPLUNC1 promoter sequence. Six different primer sets were designed to map 9 putative AP-1/c-Jun binding motifs across −2.5kb flanking region of SPLUNC1 gene (A). After overnight growth in reduced serum medium, NCI-H292 cells were stimulated with Pam₃CSK₄ (100 ng/ml) for up to 2 h. Binding of c-Jun to SPLUNC1 promoter region (−833/−1036 and −1733/−2004 bases) containing putative Ap-1/c-Jun DNA binding motifs was determined by ChIP assay (B). Upper panel shows a representative picture of agarose gels; lower panel shows densitometric analysis of PCR products visualized on agarose gel electrophoresis. Data expressed as means ± SEM, n = 3.

FIGURE 5. Pam₃CSK₄-mediated ERK1/2 and c-Jun activation up-regulates SPLUNC1 expression

NCI-H292 cells were pre-treated with DMSO (0.1%) or MEK1/2 inhibitor PD98059 (10 μM). An hour later, cells were stimulated with Pam₃CSK₄ for up to 24 h. ERK1/2 inhibition by PD98059 resulted in decreased ERK1/2 and c-Jun activation as determined by Western blot (A) and ELISA-based TransAM c-Jun activation assay (B). Effect of PD98059 on Pam₃CSK₄-induced SPLUNC1 mRNA was determined by real-time quantitative PCR (C). Data expressed as means ± SEM, n = 3–5.

FIGURE 5. Pam₃CSK₄-mediated ERK1/2 and c-Jun activation up-regulates SPLUNC1 expression

NCI-H292 cells were pre-treated with DMSO (0.1%) or MEK1/2 inhibitor PD98059 (10 μM). An hour later, cells were stimulated with Pam₃CSK₄ for up to 24 h. ERK1/2 inhibition by PD98059 resulted in decreased ERK1/2 and c-Jun activation as determined by Western blot (A) and ELISA-based TransAM c-Jun activation assay (B). Effect of PD98059 on Pam₃CSK₄-induced SPLUNC1 mRNA was determined by real-time quantitative PCR (C). Data expressed as means ± SEM, n = 3–5.

FIGURE 6. Summary of our research findings

Pam₃CSK₄-mediated TLR2 activation results in ERK1/2 and c-Jun activation, which enhances c-Jun binding to SPLUNC1 promoter region to up-regulate SPLUNC1 expression in lung epithelial cells.