Pro-apoptotic Noxa is involved in ablative focal irradiation-induced lung injury

Abstract

Although lung injury including fibrosis is a well-documented side effect of lung irradiation, the mechanisms underlying its pathology are poorly understood. X-rays are known to cause apoptosis in the alveolar epithelial cells of irradiated lungs, which results in fibrosis due to the proliferation and differentiation of fibroblasts and the deposition of collagen. Apoptosis and BH3-only pro-apoptotic proteins have been implicated in the pathogenesis of pulmonary fibrosis. Recently, we have established a clinically analogous experimental model that reflects focal high-dose irradiation of the ipsilateral lung. The goal of this study was to elucidate the mechanism underlying radiation-induced lung injury based on this model. A radiation dose of 90 Gy was focally delivered to the left lung of C57BL/6 mice for 14 days. About 9 days after irradiation, the mice began to show increased levels of the pro-apoptotic protein Noxa in the irradiated lung alongside increased apoptosis and fibrosis. Suppression of Noxa expression by small interfering RNA protected cells from radiation-induced cell death and decreased expression of fibrogenic markers. Furthermore, we showed that reactive oxygen species participate in Noxa-mediated, radiation-induced cell death. Taken together, our results show that Noxa is involved in X-ray-induced lung injury.

Keywords: Noxa; apoptosis; lung injury; radiation.

Figure 1

Morphologic observation in control and irradiation (IR) groups. (A) Representative gross findings. Mice were killed at the indicated time‐points after irradiation, and the lungs were immersed in fixation solution for several days. Lungs were photographed after complete fixation. Hematoxylin and eosin‐stained (B) and Masson's trichrome‐stained (C) irradiated lung sections from a minimum of three mice were examined at each time‐point. Representative images of the major findings are shown (magnification: × 400). Quantification of inflammation and fibrosis score is shown in each right panel. **P < 0.01, ***P < 0.001 versus control.

Figure 2

Effect of irradiation on Noxa mRNA and protein expression. Quantitative RT‐PCR analysis (A and C) and western blotting (B and D) showed that irradiation increased Noxa expression in the mouse lungs and MLE12 cells. cDNA was synthesized from the total RNAs extracted from irradiated mouse lungs (A) and MLE12 cells (C) exposed to X‐rays and subjected to RT‐PCR analysis. The Noxa expression level of the control was arbitrarily defined as 1. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. Cell lysates (20 μg) extracted from X‐ray‐irradiated mouse lungs (B) and MLE‐12 cells (D) were subjected to western blotting analysis using polyclonal anti‐Noxa and β‐actin antibodies.

Figure 3

Activation of Noxa promoter by X‐rays. MLE12 cells were transiently transfected with 1 μg luciferase reporter plasmid. After 24 hrs of transfection, the cells were subjected to X‐rays for the indicated time periods and luciferase activity was determined. The means ± S.D. of three independent experiments are shown.

Figure 4

Noxa mediates cell death in response to X‐rays. (A) L132 human lung epithelial cells were infected with Ad‐Noxa or a control virus. After 18 hrs of infection, cells were treated with X‐rays at doses of 0 or 10 Gy at the indicated time‐points. Cell death was investigated using the WST‐1‐based cytotoxicity assay. Ad‐Noxa or control virus infected cells were treated with 10 Gy and harvested at 24 hrs for following analysis. The cell lysates were analysed using Western blot to detect total or cleaved form of PARP (B). The cells were double stained with Annexin V‐FITC/PI and then analysed by flow cytometry (C). (D) Cells were treated with Noxa siRNA for 48 hrs, and treated with X‐rays at doses of 0 or 10 Gy at the indicated time‐points. Cell death was investigated using the WST‐1‐based cytotoxicity assay (left). Representative western blotting of Noxa expression after transfection with siRNA (right). *P < 0.05, **P < 0.01, ***P < 0.001. (E) After 24 hrs of 10 Gy irradiation, L132 cells were double stained with anti‐Noxa antibody (green) and Mito Tracker (Red) or ER‐tracker (Red). The images were merged to investigate of the subcellular localization of Noxa.

Figure 5

Reactive oxygen species (ROS) mediate Noxa‐induced cell death. L132 cells were treated with X‐rays at doses of 0 or 10 Gy for 6 hrs after infection with Ad‐Noxa (A) or transfection with Noxa siRNA (B). Cells were stained with 2ʹ,7ʹ‐dichlorofluorescein diacetate and subjected to flow cytometric analysis. L132 cells were infected with Ad‐Noxa for 18 hrs, and then treated with or without N‐acetylcysteine for an additional 3 hrs. Cells were treated with X‐rays at doses of 10 Gy for 24 hrs, and then cell death was investigated using the WST‐1‐based cytotoxicity assay (C) and analysed by western blot using anti‐phospho‐ASK1 (Thr845) and phosphor‐JNK antibodies (D).

Figure 6

Knockdown of Noxa expression suppressed X‐ray‐induced fibrogenic markers. The si‐Noxa and si‐cont L132 cells were treated with X‐rays at a dose of 10 Gy for 48 hrs. mRNA was extracted from those cells, and RT‐PCR was performed for the indicated fibrogenic markers. **P < 0.01, ***P < 0.001 versus control; #P < 0.05, ##P < 0.01 versus control+irradiation (IR).

Figure 7

Immunohistochemistry images of Noxa from TUNEL and 8‐OHdG staining in the X‐ray‐induced mouse lung fibrosis model. Mice were killed at days 0, 5, 9 and 14 after irradiating with X‐rays at a dose of 90 Gy. Sections from irradiated lungs were immunostained with anti‐Noxa antibody (A). The terminal deoxynucleotidyl transferase‐mediated deoxyuridine‐5‐triphosphate‐biotin nick end labelling (TUNEL) assay was performed (B) and oxidative stress for 8‐OHdG (C) was evaluated in irradiated lung slices adjacent to those used for the analysis of Noxa expression.