Selectively decreased expression of peroxiredoxins induced by silica in pulmonary epithelial cells

Abstract

Background/aims: Peroxiredoxin (Prx) belongs to a ubiquitous family of antioxidant enzymes that regulates many cellular processes through intracellular oxidative signal transduction pathways. Silica-induced lung damage involves reactive oxygen species (ROS) that trigger subsequent toxic effects and inflammatory responses in alveolar epithelial cells resulting in fibrosis. Therefore, we investigated the role of Prx in the development of lung oxidant injury caused by silicosis, and determined the implication of ROS in that process.

Methods: Lung epithelial cell lines A549 and WI26 were treated with 1% silica for 0, 24, or 48 hours, following pretreatment of the A549 cells with N-acetyl-L-cysteine and diphenylene iodonium and no pretreatment of the WI26 cells. We transfected an HA-ubiquitin construct into the A549 cell line and then analyzed the cells via Western blotting and co-immunoprecipitation.

Results: Silica treatment induced cell death in the A549 lung epithelial cell line and selectively degraded Prx I without impairing protein synthesis in the A549 cells, even when the ROS effect was blocked chemically by N-acetyl-L-cysteine. A co-immunoprecipitation study revealed that Prx I did not undergo ubiquitination.

Conclusions: Silica treatment induces a decrease of Prx I expression in lung epithelial cell lines regardless of the presence of ROS. The silica-induced degradation of Prx does not involve the ubiquitin-proteasomal pathway.

Keywords: Epithelial cell, lung; Lung injury; Peroxiredoxins; Reactive oxygen species; Silicosis.

Figure 1

Expression of Prx isoforms in lung cells. Various cell lines (A549, Raw264.7, and WI26) were lysed, and 10 µg/lane of the total protein extract was loaded onto 12% reducing SDS-PAGE gels that were then subjected to Western blot analysis with Prx isoform-specific antibodies. All of the Prx isoforms were differentially expressed in various cell lines in this study. Prx I and Prx III-VI were all expressed in the A549 cell with minimal expression of Prx II. All Prx isoforms were expressed in the WI26 cell line. Prx I-III and Prx V, but neither Prx IV nor Prx VI, were detected in the Raw 264.7 macrophage cell line. Abbreviations : NL=normal lung tissue, A 549=A549 lung cancer cell line, WI 26=WI 26 type I epithelial cell line, RAW=RAW 264.7 cell line, Rat 2=Rat 2 fibroblast cell line.

Figure 2

Effect of silica on the abundance of Prx isoforms (Prx I - VI) in lung epithelial cell lines. (A) A549 cells were exposed to silica (1 mg/mL) for the indicated times, after which the cell lysates (10 µg or 30 µg of protein per lane) were subjected to Western blot analysis with primary antibodies. (B) WI26 cells were exposed to silica (1 mg/mL) for the indicated times. The samples were treated in the same way as in described for (A). In A549 cells, silica treatment caused a reduction in the expression of Prx I. In WI26 cells, silica treatment reduced the expression of Prx I and II while other Prx isoforms were intact.

Figure 3

Effects of NAC and DPI on silica-induced degradation of Prx I and IκB-α in A549 cells. Cells were incubated for 1 hours in the presence or absence of 1 mM NAC or 1 µM DPI, and then for 48 hours with or without silica (1 mg/mL). Cell lysates were subjected to Western blot analysis with primary antibodies. Silica treatment of the cell samples reduced the levels of Prx expression regardless of pretreatment with ROS inhibitors, whereas the control samples showed decreased expression of IκB-α, a compound that is decreased by ROS-induced protein degradation. Reduction of Prx expression was only dependent on the presence of silica treatment regardless of other effects of ROS. Abbreviations: NAC=N-acetylcysteine, DPI=diphenyleneiodo-nium.

Figure 4

Effects of MG-132 on silica-induced degradation of Prx I in A549 cells. Cells were incubated for 12 hours in the presence or absence of MG-132 (10 µM) and then incubated with or without silica (1 mg/mL) for the indicated times. Cell lysates were subjected to Western blot analysis with primary antibodies. The decreased expression of Prx I induced by treatment with 1% silica in A549 cells was affected by the addition of MG-132, a potent ubiquitin-proteasomal degradation pathway inhibitor. Abbreviations: MG-132=MG-132 proteosome inhibitor.

Figure 5

Effects of MG-132 on silica-induced degradation of Prx I in ubiquitin-A549. (A) A549 cells were transfected with the HA-ubiquitin construct. After 24 hours, the cells were cultured in the presence of silica and/or MG-132 for the indicated times. Cell lysates were subjected to Western blot analysis with primary antibodies. (B) The cells were harvested at the indicated times after being transfected with the HA-ubiquitin construct. The effects of silica were not prevented by the addition of MG-132 to cells overexpressing ubiquitin. Only LipofectamineTM 2000, which was used to obtain cells overexpressing ubiquitin, contributed to a minor decrease in the expression of Prx I. Abbreviations : HA 549=Hemaglutinin overexpressing A549, ub-549=ubquitin-overexpressing A549. Difference of beta actin size in the figure is due to partial protein degradation following silica treatment.

Figure 6

Identification of the interaction between ubiquitin and Prx I in A549 cells by co-immunoprecipitation. Ubiquitin-A549 lysates were co-immunoprecipitated with anti-HA and reacted with anti-HA and anti-Prx I antibodies. Immunoprecipitation did not detect binding between ubiquitin and Prx I indicating no ubiquitination of Prx 1 in cells. Abbreviations : HA 549=Hemaglutinin overexpressing A549, ub-549=ubquitinoverexpressing A549