Activation of Egr-1 in human lung epithelial cells exposed to silica through MAPKs signaling pathways

Abstract

The alveolar type II epithelial cell, regarded historically as a key target cell in initial injury by silica, now appears to be important in both defense from lung damage as well as elaboration of chemokines and cytokines. The molecular basis for silica-induced epithelial cell injury is poorly understood. In this study we explored the activation of nuclear factor Egr-1 and related signal pathway. Human II alveolar epithelial line A549 cells were exposed to silica for indicated time to assay the expression and activation of Egr-1 and upstream MAPKs. Immunofluorescence, western-blot techniques, RT-PCR, Electrophoretic mobility shift assay (EMSA), transient transfection assay, kinase inhibitor experiments were performed. It was found that the expression of Egr-1 at mRNA and protein level was significantly increased in A549 cells after administration with silica and the activity of Egr-1 peaked by silica treatment for 60 minutes. Furthermore, phosphorylated-ERK1/2, P38 MAPKs (the upstream kinase of Egr-1) ballooned during 15-30minutes, 30-60minutes respectively after silica exposure in A549 cells. By administration of ERK1/2, P38 inhibitor, the expression and transcription of Egr-1 were both markedly decreased. But PKC inhibitor did not prevent the increase of Egr-1. These results indicated Egr-1 played a critical role in silica-induced pulmonary fibrosis in an ERK1/2, P38 MAPKs-dependent manner, which suggests Egr-1 is an essential regulator in silicosis, and underlines a new molecular mechanism for fibrosis induced by silica.

Figure 1. Egr-1 expression and localization in lung epithelial cell line A549.

(A) 0 minute (control cells, grown in medium alone), Egr-1 expression was detected but very weak and located in cytoplasm. (B) 30 minutes, the expression of Egr-1 was increased and partly located in cytoplasm. (C) 60 minutes, robust expression of Egr-1 was located in the nuclear. (D) 120 minutes, medium expression of Egr-1 was located in cytoplasm and nuclear. (E) 240 minutes, less expression of Egr-1 was located in cytoplasm. (F) 480 minutes, Egr-1 expression almost restored to the level of control cells. Images were at ×100 magnification.

Figure 2. Silica induced the expression of Egr-1 at levels of nuclear protein and mRNA.

(a) The dynamic expression of Egr-1 nuclear protein in A549 cells, which were exposed to silica for indicated times, was determined by western-blot using anti-Egr-1 antibody. Obvious increase of Egr-1 occurred from 30 minutes of exposure, and peaked after exposure for 60 minutes followed by gradually decrease and return to baseline by 480-minute treatment (2B). Silica induced the transcription of Egr-1 and mRNA level was determined by RT-PCR. Significant differences in Egr-1 protein and mRNA expression are noted at P<0.01(**).

Figure 3. Silica increased the Egr-1 DNA binding activity in A549 cells.

The binding activity of Egr-1 in A549 cells after exposure to silica was determined by EMSA experiments, and the binding activity of Egr-1 peaked after 60-minute exposure. The binding activity of Egr-1 to specific oligonucleotides probe increased from 30-minute treatment and peaked for 60-minute exposure, then decreased. And as shown in 3B: the promoter activity increased from 30-min incubation with silica and peaked at 60-min, recovered to the level of resting control till 480-min incubation. Significant differences in binding activity and luciferase activity are noted at P<0.01(**).

Figure 4. Activation of ERK1/2, P38 MAPKs in A549 cells induced by silica.

(A) The level and localization of phosphorylated ERK1/2 and P38 were detected by immunochemistry. magnification: ×400. The expression of phosphorylated ERK1/2 (4A1) and P38 (picture not shown) was mainly in cytoplasm of untreated cells, and increased and located in nuclears (4A2-3) by silica treatment for 30 minutes. (B) The semi-quantitative expression of phosphorylated ERK1/2 (4B1) and P38 (4B2) in A549 cells was determined by western-blot, intensity of the bands on western blot were analyzed by Scion image and the relative expression of phosphorylated ERK1/2 and P38 to total ERK1/2 and P38 was calculated. ** p<0.01 compared to untreated cells.

Figure 5. Egr-1 activation by silica was mainly dependent upon activation of the MAPKs pathway.

(a) The effects of Egr-1 nuclear protein expression after administration of kinase inhibitor was determined by western-blot (5B、5C). The Egr-1 expression at nuclear protein and mRNA level was inhibited by U0126 and SB230580 respectively, but not completely disappeared by combination of both; The expression of Egr-1 was not changed by pretreatment with H7, a PKC inhibitor. The significance of Egr-1 expression is noted as P<0.05 (*), P<0.01(**).