Cigarette smoke-induced Egr-1 upregulates proinflammatory cytokines in pulmonary epithelial cells

Abstract

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide and is a progressive and irreversible disorder. Cigarette smoking is associated with 80-90% of COPD cases; however, the genes involved in COPD-associated emphysema and chronic inflammation are poorly understood. It was recently demonstrated that early growth response gene 1 (Egr-1) is significantly upregulated in the lungs of smokers with COPD (Ning W and coworkers, Proc Natl Acad Sci 2004;101:14895-14900). We hypothesized that Egr-1 is activated in pulmonary epithelial cells during exposure to cigarette smoke extract (CSE). Using immunohistochemistry, we demonstrated that pulmonary adenocarcinoma cells (A-549) and primary epithelial cells lacking basal Egr-1 markedly induce Egr-1 expression after CSE exposure. To evaluate Egr-1-specific effects, we used antisense (alphaS) oligodeoxynucleotides (ODN) to knock down Egr-1 expression. Incorporation of Egr-1 alphaS ODN significantly decreased CSE-induced Egr-1 mRNA and protein, while sense ODN had no effect. Via Egr-1-mediated mechanisms, IL-1beta and TNF-alpha were significantly upregulated in pulmonary epithelial cells exposed to CSE or transfected with Egr-1. To investigate the relationship between Egr-1 induction by smoking and susceptibility to emphysema, we determined Egr-1 expression in strains of mice with different susceptibilities for the development of smoking-induced emphysema. Egr-1 was markedly increased in the lungs of emphysema-susceptible AKR/J mice chronically exposed to cigarette smoke, but only minimally increased in resistant NZWLac/J mice. In conclusion, Egr-1 is induced by cigarette smoke and functions in proinflammatory mechanisms that likely contribute to the development of COPD in the lungs of smokers.

Figure 1.

Exposure of A-549 cells and SAECs to CSE upregulates Egr-1 expression. Cells at 40–50% confluence were incubated in media alone, 25% CSE, or 50% CSE for 2 h, immediately fixed with 4% paraformaldehyde, and immunostained for Egr-1 protein expression. Egr-1 expression was not detected in control cells grown in medium alone. Egr-1 was markedly induced in cells exposed to 25% and 50% CSE. Experiments without primary antibodies revealed no immunoreactivity after exposure to 50% CSE. Images are at ×80 magnification.

Figure 2.

CSE induces Egr-1 expression in human pulmonary adenocarcinoma cells (A-549) and Primary SAECs. (A and B) Representative Western blot analysis reveals no Egr-1 expression in cells grown in medium alone (control). Significant upregulation of Egr-1 was observed in serum-starved cells and in cells exposed to 50% CSE for 2 h. (C) Relative densitometry was quantified by using NIH Image-J software. Significant differences in Egr-1 protein expression as compared with control are noted at P ⩽ 0.01 (**).

Figure 3.

Egr-1 αS ODNs inhibit Egr-1 mRNA and protein expression. (A) A-549 cells were transfected with Egr-1 sense (S) or antisense (αS) oligonucleotides (10 μM) and serum starved or exposed to CSE 12 h after transfection for 2 h. Total RNA was isolated, 1.0 μg was reverse transcribed, and the resulting cDNA was subjected to PCR analysis. αS oligonucleotides significantly decreased Egr-1 mRNA expression. (B) Cells manipulated as outlined above were lysed and subjected to Western blot analysis. Significant αS ODN-mediated decreases in Egr-1 expression were detected after serum starvation or exposure to CSE. Relative densitometry was quantified by using NIH Image-J software. Significant differences in Egr-1 protein expression are noted at P ⩽ 0.05 (*) and P ⩽ 0.01 (**).

Figure 4.

CSE exposure induces proinflammatory cytokine secretion. A-549 cells were serum starved or exposed to 50% CSE for 2 h and medium was immediately assayed for secreted IL-1β (shaded bars) and TNF-α (solid bars) by ELISA. Compared with basal levels observed from cells grown in DMEM-10% FCS (control), cells exposed to media lacking serum or 50% CSE had significantly elevated levels of secreted IL-1β and TNF-α. SAECs exposed to 50% CSE also induced cytokine secretion. Data presented are from two experiments, each performed in triplicate. *P ⩽ 0.05.

Figure 5.

Egr-1 αS ODNs inhibit CSE-induced IL-1β secretion. A-549 cells were transfected with Egr-1 sense (S) or antisense (αS) oligonucleotides (10 μM) and serum starved or exposed to CSE 12 h after transfection for 2 h. Medium was isolated and subjected to ELISA. Secreted IL-1β was significantly decreased by Egr-1 αS oligonucleotides after serum starvation or CSE exposure. Data presented are from two experiments, each performed in triplicate. *P ⩽ 0.05.

Figure 6.

Egr-1 αS ODNs inhibit CSE-induced TNF-α secretion. A-549 cells were transfected with Egr-1 sense (S) or antisense (αS) oligonucleotides (10 μM) and serum starved or exposed to CSE 12 h after transfection for 2 h. Medium was isolated and subjected to ELISA. Secreted TNF-α was not significantly decreased by Egr-1 αS ODNs after serum starvation. TNF-α secretion was significantly inhibited by Egr-1 αS ODNs in cells exposed to 50% CSE. Data presented are from two experiments, each performed in triplicate. *P ⩽ 0.05.

Figure 7.

Exogenous Egr-1 induces proinflammatory cytokine secretion. In the absence of CSE, A-549 cells were transfected with Egr-1 (pCMV-Egr-1), and secreted IL-1β or TNF-α in the medium was assessed by ELISA. While transfection experiments with 200–400 ng Egr-1 significantly induced IL-1β secretion, TNF-α secretion was significantly elevated only after 800 ng of Egr-1 was transfected. Data presented are from two experiments, each performed in triplicate. *P ⩽ 0.05.

Figure 8.

Egr-1 is induced in the lungs of mice exposed to cigarette smoke. Cigarette smoke–induced emphysema hypersensitive (AKR/J) and resistant (NZWLac/J) mice were maintained in environments either lacking (nonsmokers) or including cigarette smoke (smokers) for 6 mo. Immunohistochemistry reveals minimal Egr-1 expression in nonsmoker lungs of both strains; however Egr-1 expression was markedly increased in AKR/J smoker lungs. Egr-1 was minimally increased in the lungs of smoker NZWLac/J mice resistant to cigarette smoke–induced emphysema. Control sections incubated in serum alone had no immunoreactivity (not shown). Figures are at ×100 magnification.