Proteomic analysis of radiation-induced changes in rat lung: Modulation by the superoxide dismutase mimetic MnTE-2-PyP(5+)

Abstract

Purpose: To identify temporal changes in protein expression in the irradiated rat lung and generate putative mechanisms underlying the radioprotective effect of the manganese superoxide dismutase mimetic MnTE-2-PyP(5+).

Methods and materials: Female Fischer 344 rats were irradiated to the right hemithorax with a single dose of 28 Gy and killed from day 1 to 20 weeks after irradiation. Proteomic profiling was performed to identify proteins that underwent significant changes in abundance. Some irradiated rats were administered MnTE-2-PyP(5+) and changes in protein expression and phosphorylation determined at 6 weeks after irradiation.

Results: Radiation induced a biphasic stress response in the lung, as shown by the induction of heme oxygenase 1 at 1-3 days and at 6-8 weeks after irradiation. At 6-8 weeks after irradiation, the down-regulation of proteins involved in cytoskeletal architecture (filamin A and talin), antioxidant defense (biliverdin reductase and peroxiredoxin II), and cell signaling (β-catenin, annexin II, and Rho-guanosine diphosphate dissociation inhibitor) was observed. Treatment with MnTE-2-PyP(5+) partially prevented the apparent degradation of filamin and talin, reduced the level of cleaved caspases 3 and 9, and promoted Akt phosphorylation as well as β-catenin expression.

Conclusion: A significant down-regulation of proteins and an increase in protein markers of apoptosis were observed at the onset of lung injury in the irradiated rat lung. Treatment with MnTE-2-PyP(5+), which has been demonstrated to reduce lung injury from radiation, reduced apparent protein degradation and apoptosis indicators, suggesting that preservation of lung structural integrity and prevention of cell loss may underlie the radioprotective effect of this compound.

Fig. 1

Temporal changes in protein abundance in irradiated rat lungs. (A) Rat lung homogenates were prepared at the indicated times after radiation, resolved by one-dimensional SDS-PAGE and proteins visualized by silver staining. Numbered arrows denote proteins that were excised and subjected to mass spectrometry with protein identity shown in Table 1. Abbreviations: “-” indicates non-irradiated control; “d” = days, “w”= weeks. The amount of hemoglobin was quantitated by densitometry, normalized to actin, and shown as a percent of the non-irradiated control. (B) Validation of mass spectrometry results by Western analysis. Total cell lysates from rat lung homogenates were immunoblotted with the indicated antibodies. Actin served as a loading control. (C) A separate group of rats were treated identically and lung tissues harvested and analyzed by Western analysis. For all samples, the amount of each protein was quantitated by densitometry, normalized to actin levels, and presented as a percent of the non-irradiated control above each lane.

Fig. 2

Temporal changes in Heme oxygenase 1 and β-catenin protein levels in the irradiated rat lung. (A) Rats were exposed to a single dose of 28 Gy to the right hemithorax and whole lung homogenates immunoblotted with HO-1 or β-catenin antibodies at the indicated times post-irradiation. Abbreviations: “-” indicates non-irradiated control; “d” = days, “w”= weeks. MnSOD served as a loading control. (B) A separate group of rats were treated as described in panel A. The amount of each protein was quantitated by densitometry, normalized to MnSOD levels, and presented as a percent of the non-irradiated control above each lane.

Fig. 3

Reduction of apparent protein degradation and protein indicators of apoptosis in the irradiated lung by MnTE-2-PyP⁵⁺. (A) Western analysis of rat lung samples prepared from 3 separate rats for each condition including: mock irradiation, irradiation alone, MnTE-2-PyP⁵⁺ treatment, and MnTE-2-PyP⁵⁺ plus irradiation at 6 weeks post-irradiation. Samples were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Actin served as a loading control. (B) Quantitation of the Western blot data shown in Fig. 3A. The amount of each protein was quantitated by densitometry, normalized to actin levels, and the mean presented as a percent of the non-irradiated control along with standard error. One-way ANOVA and post-hoc tests were performed to determine significance. ‘*’ indicates significance with a p-value <0.05. ns= not significant.

Fig. 3

Reduction of apparent protein degradation and protein indicators of apoptosis in the irradiated lung by MnTE-2-PyP⁵⁺. (A) Western analysis of rat lung samples prepared from 3 separate rats for each condition including: mock irradiation, irradiation alone, MnTE-2-PyP⁵⁺ treatment, and MnTE-2-PyP⁵⁺ plus irradiation at 6 weeks post-irradiation. Samples were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Actin served as a loading control. (B) Quantitation of the Western blot data shown in Fig. 3A. The amount of each protein was quantitated by densitometry, normalized to actin levels, and the mean presented as a percent of the non-irradiated control along with standard error. One-way ANOVA and post-hoc tests were performed to determine significance. ‘*’ indicates significance with a p-value <0.05. ns= not significant.

Fig. 4

A model for the mechanism of action of MnTE-2-PyP⁵⁺, in reducing lung injury from radiation. Radiation induces the generation of ROS/RNS, which in turn contributes to protein down regulation. Loss of proteins involved in ROS/RNS metabolism may contribute to further increases in ROS/RNS levels, generating a cycle of chronic inflammation. The SOD mimetic, through reduction of ROS/RNS, interrupts the cycle of chronic inflammation, reduces the level of protein down regulation and apoptosis and acts to preserve structural integrity of the lung.