The signaling pathway involved in neutrophil elastase stimulated MUC1 transcription

Abstract

We previously reported that neutrophil elastase (NE) stimulated MUC1 gene expression in A549 lung epithelial cells through binding of Sp1 to the MUC1 promoter element. The current study was undertaken to elucidate the complete signaling pathway leading to Sp1 activation. Using a combination of pharmacologic inhibitors, dominant-negative mutant, RNA interference, and soluble receptor blocking techniques, we identified a protein kinase Cdelta (PKCdelta) --> dual oxidase 1 (Duox1) --> reactive oxygen species (ROS) --> TNF-alpha-converting enzyme (TACE) --> TNF-alpha --> TNF receptor (TNFR)1 --> extracellular signal-regulated kinase (ERK)1/2 --> Sp1 pathway as responsible for NE-activated MUC1 transcription. This cascade was identical up to the point of TACE with the signaling pathway previously reported for NE-stimulated MUC5AC production. However, unlike the MUC5AC pathway, TNF-alpha, TNFR1, ERK1/2, and Sp1 were unique components of the MUC1 pathway. Given the anti-inflammatory role of MUC1 during airway bacterial infection, up-regulation of MUC1 by inflammatory mediators such as NE and TNF-alpha suggests a crucial role for MUC1 in the control of excessive inflammation during airway bacterial infection.

Figure 1.

Neutrophil elastase (NE) increases MUC1 transcript levels in primary normal human bronchial epithelial (NHBE) cells. Confluent NHBE and A549 cells were treated for 24 hours with PBS or 50, 100, or 200 nM of NE, and MUC1 mRNA levels were measured by real-time RT-PCR. MUC1 mRNA levels were normalized to GAPDH mRNA levels. Each data point represents the mean ± SEM (n = 3). *P < 0.05 compared with PBS-treated (0 nM NE) cells. The results were reproduced in separate experiments.

Figure 2.

NE-induced MUC1 transcription involves protein kinase Cδ (PKCδ), dual oxidase 1 (Duox1), reactive oxygen species (ROS), and TNF-α–converting enzyme (TACE). (A) Involvement of PKCδ. A549 cells were transfected with the pGL2b empty vector or the MUC1-pGL2b plasmid, pretreated for 30 minutes with the indicated concentrations of rottlerin, treated for 24 hours with PBS or 100 nM of NE, and relative luciferase activity (firefly/Renilla) was determined. (B) Involvement of Duox1. A549 cells were transfected with 100 nM of a Duox1 siRNA or nontargeting control RNA, the cells were incubated for 48 hours, transfected with 800 ng/well of pGL2b empty vector or MUC1-pGL2b plasmid, incubated for 24 hours, treated for 24 hours with PBS or 100 nM of NE, and relative luciferase activity was determined. (C) Involvement of ROS. A549 cells were transfected with the pGL2b empty vector or the MUC1-pGL2b plasmid, pretreated for 30 minutes with the indicated concentrations of glutathione (GSH) (C1) or 1,3-dimethyl-2-thiourea (DMTU) (C2), treated for 24 hours with PBS or 100 nM of NE, and relative luciferase activity was determined. (D) Involvement of TACE. A549 cells were transfected with the pGL2b empty vector or the MUC1-pGL2b plasmid, pretreated for 30 minutes with the indicated concentrations of TAPI-1, treated for 24 hours with PBS or 100 nM of NE, and relative luciferase activity was determined. Open bars, PBS; solid bars, NE. Each bar represents the mean ± SEM (n = 3). *P < 0.05 comparing MUC1-pGL2b–transfected cells treated with NE with the PBS control. The results were reproduced in separate experiments.

Figure 3.

TNF receptor (TNFR) but not epidermal growth factor receptor (EGFR) is involved in NE-stimulated MUC1 transcription. (A) EGFR is not involved in NE-stimulated MUC1 transcription. A549 cells were transfected with the pGL2b empty vector or the MUC1-pGL2b plasmid, pretreated for 30 minutes with the indicated concentrations of AG1478, treated for 24 hours with PBS or 100 nM NE, and relative luciferase activity was determined. (B) TNF-α increases MUC1 transcription. A549 cells were transfected with the pGL2b empty vector or the MUC1-pGL2b plasmid, treated for 12 or 24 hours with PBS or 50 ng/ml of TNF-α, and relative luciferase activity was determined. (C) TNF-α increases MUC1 protein expression. Nontransfected A549 cells were treated for 24 hours with PBS or 50 ng/ml of TNF-α and MUC1 protein in cell lysates analyzed by immunoprecipitation/immunoblotting with MUC1 antibody (left panel). The blot in the left panel of C was stripped and reprobed with peroxidase-conjugated goat anti-mouse IgG antibody alone (i.e., no primary antibody) to demonstrate equal protein loading and membrane transfer efficiencies. (D) NE increases MUC1 transcription through TNFR1. A549 cells were transfected with the pGL2b empty vector or the MUC1-pGL2b plasmid, treated for 24 hours with PBS or 100 nM of NE in the presence of the indicated concentrations of soluble TNFR1, and relative luciferase activity was determined. Open bars, PBS; solid bars, NE. Each bar represents the mean ± SEM (n = 3). *P < 0.05 comparing MUC1-pGL2b–transfected cells treated with NE with the PBS control. The results were reproduced in separate experiments.

Figure 4.

NE increases MUC1 transcription through ERK1/2. (A) A549 cells were transfected with the pGL2b empty vector or the MUC1-pGL2b plasmid, pretreated for 30 minutes with the indicated concentrations of U0126, SB202190, or SP600125, treated for 24 hours with PBS or 100 nM of NE, and relative luciferase activity was determined. Open bars, PBS; solid bars, NE. Each bar represents the mean ± SEM (n = 3). *P < 0.05 comparing MUC1-pGL2b–transfected cells treated with NE with the PBS control. (B) A549 cells were treated for 24 hours with PBS or 100 nM of NE and cell lysates were examined by immunoblotting with antibodies against phospho-ERK1/2 or β-tubulin. The results were reproduced in separate experiments.

Figure 5.

Inhibition of Duox1, PKCδ, or ROS blocks NE-stimulated ERK1/2 activation. A549 cells were pretreated for the indicated times with PBS, 5.0 μM of rottlerin (A), 10 mM of GSH (C), or 25 μM of soluble TNFR1 (D), or were transfected with 100 nM of the PKCδ siRNA (B). After pretreatments, the cells were treated for 24 hours with PBS or 100 nM NE and cell lysates were examined by immunoblotting with antibodies against phospho-ERK1/2 or β-tubulin. The results were reproduced in separate experiments.

Figure 6.

NE increases threonine phosphorylation of Sp1, but not NF-κB activation. (A) A549 cells were treated for 24 hours with PBS or 100 nM of NE, equal protein aliquots of cell lysates were immunoprecipitated with anti-Sp1 antibody, and immunoblotted with anti–phospho-threonine antibody (upper panel) or anti-Sp1 antibody (lower panel). (B) A549 cells were pretreated for 30 minutes with PBS or 10 μg/ml of U0126, treated for 24 hours with PBS or 100 nM of NE and phospho-Sp1 levels determined as in A. (C) A549 cells were transfected with the MUC1-pGL2b alone, or cotransfected with MUC1-pGL2b plus mIκB-α-pCMV4 or pCMV4 alone, treated for 24 hours with PBS or 100 nM of NE, and relative luciferase activity was determined. (D) A549 cells were transfected with the MUC1-pGL2b plasmid, pretreated for 30 minutes with PBS or 50 μg/ml of SN-50 or the mock control peptide, treated for 24 hours with PBS or 100 nM of NE, and relative luciferase activity was determined. Open bars, PBS; solid bars, NE. Each bar represents the mean ± SEM (n = 3). *P < 0.05 comparing cells treated with NE with the respective PBS controls. The results were reproduced in separate experiments.

Figure 7.

Schematic illustration of the NE-stimulated signaling pathways leading to MUC1 and MUC5AC gene expression in airway epithelial cells. The question mark indicates the unknown identity of the cell surface receptor that binds to NE.