Published ahead of print on May 25, 2006, doi:10.1164/rccm.200511-1816OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200511-1816OC
Senescence Marker Protein-30 Protects Mice Lungs from Oxidative Stress, Aging, and SmokingDepartment of Respiratory Medicine, Juntendo University, School of Medicine; Department of Molecular Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo; and Department of Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, Chiba, Japan Correspondence and requests for reprints should be addressed to Tadashi Sato, M.D., Department of Respiratory Medicine, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan. E-mail: satotada{at}med.juntendo.ac.jp
Rationale: Senescence marker protein-30 (SMP30) is a multifunctional protein providing protection to cellular functions from age-associated deterioration. We previously reported that SMP30 knockout (SMP30Y/–) mice are capable of being novel models for senile lung with age-related airspace enlargement and enhanced susceptibility to harmful stimuli. Objectives: Aging and smoking are considered as major contributing factors for the development of pulmonary emphysema. We evaluated whether SMP30Y/– mice are susceptible to oxidative stress associated with aging and smoking. Methods: Age-related changes of protein carbonyls in lung tissues from the wild-type (SMP30Y/+) and SMP30Y/– mice were evaluated. Both strains were exposed to cigarette smoke for 8 wk. Histopathologic and morphologic evaluations of the lungs, protein carbonyls and malondialdehyde in the lung tissues, total glutathione content in the bronchoalveolar lavage fluid, and degree of apoptosis of lung cells were determined. Measurements and Main Results: In the lungs of SMP30Y/– mice, protein carbonyls tended to increase with aging and were significantly higher than the age-matched SMP30Y/+ mice. Cigarette smoke exposure generated marked airspace enlargement (23.3% increase of the mean linear intercepts) with significant parenchymal destruction in the SMP30Y/– mice but not in the SMP30Y/+ mice (5.4%). The protein carbonyls, malondialdehyde, total glutathione, and apoptosis of lung cells were significantly increased after 8-wk exposure to cigarette smoke in the SMP30Y/– mice. Conclusions: Our results suggest that SMP30 protects mice lungs from oxidative stress associated with aging and smoking. The SMP30Y/– mice could be useful animal models for investigating age-related lung diseases, including cigarette smoke–induced pulmonary emphysema.
Key Words: aging • oxidative stress • pulmonary emphysema • senescence marker protein-30 • smoking
Senescence marker protein-30 (SMP30), a 34-kD protein originally identified from the rat liver, is a novel molecule that decreases with age in an androgen-independent manner, suggesting its possible role in age-related physiologic and pathologic conditions (1–3). We demonstrated that SMP30 is widely expressed in vertebrates and that the amino acid alignment is highly conserved (2, 4). In mice, SMP30 transcripts are detected in various organs, including the liver, kidney, cerebrum, testis, and lung (5). In humans, the SMP30 gene is located in the p11.3–q11.2 segment of the X chromosome (6). To clarify the physiologic role of SMP30 in age-associated organ disorders, the SMP30 knockout (SMP30Y/–) mouse was developed with gene targeting from C57BL6 mice (7). We revealed that SMP30Y/– mice are viable and fertile but have reduced weight gain and shorter life span than the wild-type (SMP30Y/+) mice (8). Hepatocytes isolated from SMP30Y/– mice were shown to be highly susceptible to tumor necrosis factor- There are many studies concerned with the relationship between aging and oxidative stress. Moderate oxidative stress may gradually develop with age because plasma levels of lipoperoxidation products and antioxidant enzyme activities in red blood cells increase with aging, whereas plasma levels of nutritional antioxidants decrease (10). The lungs are persistently exposed to oxidants generated endogenously from phagocytes and other cell types or exogenously from air pollutants or cigarette smoke (11). Pulmonary emphysema is an age-related lung disease that occurs after a prolonged period of cigarette smoking. Because cigarette smoke contains around 1017 oxidant molecules per puff and generates oxidant/antioxidant imbalance in the lungs, oxidative stress is postulated to play an important role in the pathogenesis of emphysema (11). In patients with chronic obstructive pulmonary disease, biomarkers of oxidative stress, such as protein carbonyls and lipid peroxidation products, are reported to be elevated in the lungs (12) and respiratory muscles (13). We previously reported that SMP30Y/– mice develop peripheral airspace enlargement without alveolar destruction and may thus be a novel model for senile lung (5). We hypothesized that SMP30Y/– mice may be susceptible to oxidative stress with aging. Furthermore, SMP30Y/– mice may be vulnerable to cigarette smoke exposure and generate pulmonary emphysema. In this study, we investigated the age-related changes of protein carbonyls in the lungs of SMP30Y/– and SMP30Y/+ mice and pathologically evaluated the effect of cigarette smoke exposure to the lungs and biomarkers of oxidative stress.
Animals We used SMP30Y/– and SMP30Y/+ mice to investigate the age-related changes of mice lungs with 1-, 3-, 6-, and 12-mo-old mice (n = 5 for each group). We used 3-mo-old mice (n = 6 for each group) for the smoking experiment. Animal experimentation was approved by the Animal Care and Use Committee of Tokyo Metropolitan Institute of Gerontology and by Juntendo University, School of Medicine.
Preparation and Morphologic Evaluation of the Lungs Airspace size was assessed by determining the mean linear intercepts (MLI) according to the method previously described (15). The destructive index (DI) was determined to evaluate the severity of alveolar wall destruction (16). A DI value greater than 10% was considered to have significant destruction of the lung parenchyma (17).
Determination of Protein Carbonyls in the Lungs
Immunohistochemical Staining for Protein Carbonyls in the Lungs
Chronic Exposure to Cigarette Smoke
Determination of Malondialdehyde in the Lungs
Determination of Glutathione in the BALF
Evaluation of Apoptosis with Immunohistochemistry for Anti–Single-stranded DNA and Anti–Activated Caspase-3 Antibodies
Protein Assays
Statistical Analysis
Age-related Changes in Body Weight, Morphometry of the Lungs, and Protein Carbonyls Body weights increased with age in the SMP30Y/+ and SMP30Y/– mice, and there were no significant changes between both strains up to 6 mo of age. At 12 mo of age, the body weight of the SMP30Y/– mice were significantly less than that of the SMP30Y/+ mice (Table 1). Compared with the SMP30Y/+ mice, the SMP30Y/– mice had significantly greater MLI from 3 to 12 mo of age. There was a significant increase of MLI with aging in the lungs of the SMP30Y/– mice. On the other hand, the DI scores in both groups at every age were less than 10%, and no significant differences between the groups were recorded (Table 1). There were no inflammatory findings in the alveoli in both groups on histologic examination (data not shown).
We assessed protein oxidation in the lungs of SMP30Y/+ and SMP30Y/– mice by measuring the protein carbonyls, which are known as sensitive biomarkers of oxidative stress (21). In the lungs of SMP30Y/– mice, protein carbonyls were significantly increased in comparison with those of age-matched SMP30Y/+ mice. In addition, protein carbonyls tended to increase with age and significantly increased at 12 mo of age as compared with 1 mo of age (Figure 1). We performed immunohistochemistry with anti-DNPH antibody to detect the localization of protein carbonyls in the lungs and demonstrated that the majority of the protein carbonyls was evenly distributed and diffused in both strains but was apparently stronger in the SMP30Y/– mice (Figure 2).
Effect of Cigarette Smoke Exposure on the Morphometry of the Lungs The body weights did not change significantly between the SMP30Y/+ and SMP30Y/– mice before and after exposure to cigarette smoke for 8 wk. In addition, exposure to cigarette smoke had no influence on body weight gain in both strains as compared with the air-exposed groups (data not shown). Chronic exposure to cigarette smoke increased total cell counts in the BALF of both groups. No significant differences were observed in cell populations between air- and smoke-exposed mice of both strains. The total protein concentration in the BALF increased significantly in the SMP30Y/– mice after chronic exposure to cigarette smoke but did not increase in the SMP30Y/+ mice. The smoke-exposed SMP30Y/– mice demonstrated significantly higher levels of protein concentration in BALF than the smoke-exposed SMP30Y/+ mice (Table 2).
Chronic exposure to cigarette smoke for 8 wk generated pulmonary emphysema in the SMP30Y/– mice but not in SMP30Y/+ mice. Histologic specimens of the lung tissues of smoke-exposed SMP30Y/– mice revealed marked airspace enlargement (i.e., an increase of MLI) accompanied with disruption of alveolar wall (i.e., an increase of DI; Figures 3 and 4). Morphometric examination of the lung specimen revealed that MLI was significantly greater in the smoke-exposed SMP30Y/– mice than in the air-exposed SMP30Y/– and SMP30Y/+ mice (Figure 4A): MLI increased to 23.3% in the SMP30Y/– mice after exposure to cigarette smoke and 5.4% for the SMP30Y/+ mice. Moreover, DI increased to more than 10%, a cut-off value indicating the occurrence of significant alveolar wall destruction (17), in the lungs from smoke-exposed SMP30Y/– mice, whereas no significant increase was observed in the SMP30Y/+ mice (Figure 4B).
Effect of Cigarette Smoke Exposure on Oxidative Stress in the Lungs To investigate the effect of chronic cigarette smoke exposure on oxidative injury in the lungs, we measured protein carbonyls in the lungs of SMP30Y/+ and SMP30Y/– mice after exposure to air or cigarette smoke (Figure 5). Protein carbonyls tended to increase in the lungs of both strains after cigarette smoke exposure, although no significant statistical difference was noted (p = 0.08 and 0.07 between air and smoke exposure in SMP30Y/+ and SMP30Y/– mice, respectively). Protein carbonyls were significantly increased in the lungs of smoke-exposed SMP30Y/– mice than those of the smoke-exposed SMP30Y/+ mice. We also performed immunohistochemistry with anti-DNPH antibody on lung specimens from both strains after chronic exposure to cigarette smoke. Anti-DNPH antibody demonstrated an even and diffuse distribution of protein carbonyls in the lungs of both strains after exposure to cigarette smoke (data not shown), indicating that chronic smoke exposure seems to increase oxidative stress evenly throughout the lungs.
We assessed lipid peroxidation in the lungs by measuring MDA (Figure 6), which is known as one of the end products derived from peroxidation of polyunsaturated fatty acids and related esters (22). Baseline levels of MDA in the lung tissues from both strains (air-exposed) were almost identical. However, MDA significantly increased in the lungs of the SMP30Y/– mice after chronic cigarette smoke exposure, whereas no increase was demonstrated in the lungs of the SMP30Y/+ mice. We did not use the conventional thiobarbituric acid method to measure the MDA because of low specificity of this method. Instead, we used a method that allows us to assay MDA selectively by reacting with N-methyl-2-phenylindole in HCl, eliminating the potential inclusion of 4-hydroxylalkenals and alkanals in the measurement. The reaction produces a chromophore with maximum absorption at 586 nm, whereas the reaction with alkanals forms a product with maximum absorption at 505 nm (23).
We measured the content of total glutathione in BALF (Figure 7) because it is widely recognized as a major antioxidant in the lungs (24, 25). In SMP30Y/– mice, baseline level of total glutathione in BALF tended to be greater than that of the SMP30Y/+ mice, but no statistical significance was detected (p = 0.06).Chronic cigarette smoke exposure markedly up-regulated the amount of total glutathione in BALF from the SMP30Y/– mice (p < 0.05), whereas no effect of smoke exposure on the glutathione level was revealed in the SMP30Y/+ mice (p = 0.79). We were able to detect oxidized glutathione in BALF from the cigarette smoke–exposed SMP30Y/– mice group but not from the other groups (i.e., less than the detection limit). The amount of oxidized glutathione detected in smoke-exposed SMP30Y/– mice was 7.91 nmol/mg protein (7.95% of total glutathione content).
Effect of Cigarette Smoke Exposure on Apoptosis of Lung Cells Apoptosis of lung cells was analyzed to investigate the mechanisms of cigarette smoke-induced emphysema in the SMP30Y/– mice. Immunohistochemical examination using anti–single-stranded DNA antibody revealed that apoptosis was widely detected in bronchial and bronchiolar epithelial cells and alveolar septal cells and was significantly increased in all areas of the lungs of the cigarette smoke–exposed SMP30Y/– mice compared with the other groups (Figures 8A and 8B). On the other hand, there was no significant difference in the ratio of apoptotic nuclei detected between the air- and smoke-exposed SMP30Y/+ mice (p = 0.09) and between the air-exposed SMP30Y/+ and SMP30Y/– mice (p = 0.07). Chronic cigarette smoke–induced increase in apoptosis was further confirmed with immunohistochemical analysis of caspase-3: Activated caspase-3 was detected in the lungs of cigarette smoke–exposed SMP30Y/– mice (Figure 8C) but not in the other groups (data not shown).
We have demonstrated that the oxidative stress in the lungs is greater in the SMP30Y/– mice than the SMP30Y/+ mice and tends to gradually increase in the SMP30Y/– mice as determined from measuring the protein carbonyls, one of the biomarkers for oxidative stress (26, 27), in the lungs. We did not measure the age-related changes of lipid peroxidation as another biomarker for oxidative stress and glutathione as a major antioxidant in BALF. At 5 mo of age, however, there was no significant statistical difference between SMP30Y/+ and SMP30Y/– mice in lipid peroxidation of the lungs (Figure 6; p = 0.76) and glutathione in BALF (Figure 7; p = 0.06). In contrast, chronic cigarette smoke exposure for 8 wk resulted in a marked increase of lipid peroxidation and up-regulation of glutathione, with the tendency of a greater increase of protein carbonyls in the SMP30Y/– mice, but it did not generate significant influence on these biomarkers of oxidative stress in the SMP30Y/+ mice. Our results indicate that the lack of a SMP30 molecule brings forth an endogenous mild oxidative stress situation in the lungs and makes the lungs highly susceptible to oxidative stress induced by smoke exposure even if exogenous oxidative stress is so mild that it does not generate oxidative proteins and lipids and up-regulate glutathione in BALF of the SMP30Y/+ mice. In this context, SMP30Y/– mice seem to be an excellent tool for investigating the pathophysiology of the lung associated with oxidant/antioxidant imbalance like smoke-induced emphysema and aging-related conditions like the senile lung.
The precise function of SMP30 in terms of oxidant and antioxidant balance remains undetermined. We demonstrated previously that SMP30 is localized in the nuclei in addition to the cytoplasm of cultured mouse hepatocytes and is similar in its amino acid sequence to bacterial and yeast RNA polymerases (28). Accordingly, SMP30 may regulate gene expression, and the lack of SMP30 may cause down-regulation of antioxidant enzymes. It has recently been reported that with gene targeting of the Nrf2 (nuclear factor, erythroid-derived 2, like 2), a transcription factor regulating gene expression involved in antioxidant defense, inflammation, and cellular apoptosis, plays an important role in the development of smoke-induced emphysema (29): Nrf2–/– mice were demonstrated to be extremely susceptible to cigarette smoke–induced emphysema after 6 mo of exposure. Glutathione is a potent antioxidant in the lungs and is highly concentrated in the epithelial lining fluid (24, 25). The transcription of the gene for We previously reported that SMP30Y/– is a novel murine model of senile lung because senile lungs develop spontaneous airspace enlargement without parenchymal destruction (5). This was confirmed in this study because SMP30Y/– mice showed significantly greater MLI at 3 mo of age than SMP30Y/+ mice. The SMP30Y/– mouse seems to be not only a murine model of senile lung but also a murine model of cigarette smoke–induced emphysema. SMP30Y/– mice are markedly susceptible to cigarette smoke, and smoke exposure for 8 wk was sufficient to develop cigarette smoke–induced pulmonary emphysema with marked airspace enlargement and parenchymal destruction. Although some animal models have been reported to develop cigarette smoke–induced pulmonary emphysema, most animal models required a longer period of cigarette smoke exposure, generally 6 or 7 mo, to generate smoke-induced emphysema. Because cigarette smoke–induced pulmonary emphysema in humans usually occurs in the elderly population, we considered that aging of the lung can be an important factor and should be incorporated into the experimental animal model for such condition. The effect of age on lung morphometry (36) and in the development of chronic cigarette smoke–induced lung pathology (37) has been reported. In BALB/cNNia mice, alveolar multiplication seemed to be completed by 38 d of age; interalveolar pore formation increased until 9 mo of age; and lung volume, alveolar surface area, and total volume of alveolar wall increased with age between 9 and 28 mo of age, which is postulated to be attributed to aging of the lungs (36). It was reported that in C57BL/6 mice, the older mice (8–10 mo of age) developed pathologic manifestations closely resembling pulmonary fibrosis and developed peribronchiolar and perivascular accumulations of lymphocytes and macrophages in the lungs after 9 mo exposure to cigarette smoke, whereas young mice (2 mo of age) revealed accumulations of inflammatory cells without fibrosis (37). Among other mice models for emphysema, the klotho mutant mouse and senescence-accelerated mouse (SAM) are unique due to their biological aging. The homozygous mutant klotho mice demonstrate a shorter life span and exhibit pulmonary emphysema, arteriosclerosis, osteoporosis, skin atrophy, and ectopic calcifications. However, the klotho mutant mice are distinct in developing pulmonary emphysema spontaneously without smoking (38). On the other hand, the SAM mice are the naturally occurring animal models for accelerated aging after normal development and maturation (39). We have recently reported that the SAMP1 mouse is capable of developing smoke-induced emphysema after 8 wk of cigarette smoke exposure. We also demonstrated that SAMP1 mice can be used for experiments involving therapeutic intervention because the development of smoke-induced emphysema was successfully prevented with concomitant administration of tomato juice, which contains a potent nutritional antioxidant, lycopene (14). In this context, SMP30Y/– mice further illustrate the significance of biological aging of the lungs in the development of cigarette smoke–induced pulmonary emphysema and may be considered as valuable animal models for smoke-induced emphysema. Several mechanisms are likely to be involved in the development of cigarette smoke–induced pulmonary emphysema. An increase of oxidative stress to the lungs may be associated with many of the pathogenic processes, such as direct injury to lung cells, mucus hypersecretion, inactivation of antiproteases, enhancing lung inflammation through activation of redox-sensitive transcription factors, and apoptosis of lung cells (11). In the present study, chronic smoke exposure increased total cell count in SMP30Y/+ and SMP30Y/– mice. On the other hand, SMP30Y/– mice showed total protein level in BALF increased twofold from the baseline level after smoke exposure, whereas no change was detected in SMP30Y/+ mice. These findings may suggest that the inflammation in the lungs induced by chronic smoke exposure may be more pronounced in SMP30Y/– mice than in SMP30Y/+ mice, although we did not measure any other parameters of inflammation. Pulmonary emphysema can be generated without apparent inflammation, and it has recently been recognized that alveolar cell apoptosis could be one of the crucial process in emphysema: Direct instillation of activated caspase-3 (40) or vascular endothelial cell apoptosis resulting from the blockade of the vascular endothelial growth factor receptors (41) has been demonstrated to result in emphysema. As we reported previously that hepatocytes from SMP30Y/– mice are susceptible to apoptosis (7), we confirmed in this study that lungs cells are also susceptible to apoptosis triggered by oxidative stress. Accordingly, SMP30Y/– mice may be ideal animal models for cigarette smoke–induce emphysema in terms of investigating mutual interactions among apoptosis, oxidative stress, and inflammation, which is proposed as the mechanism for irreversible progression of parenchymal destruction (42, 43). Recently, up-regulation of lung ceramide, a second messenger lipid, has been reported to be a key pathogenic element in these mutual interactions (44). Because we have previously demonstrated that abnormal lipid metabolism occurs in the liver of the SMP30Y/– mice (8), we need to examine whether SMP30 may be involved in the regulation of lung ceramide as the next step of our study.
The authors thank Dr. Toshio Kumasaka, Department of Pathology, Juntendo University, School of Medicine, for advice and technical help.
Supported by Grant-in-Aid for Scientific Research No. 13470130 (Y.F.) and No. 15390259 (Y.F.); by the High Technology Research, Center Grant from the Ministry of Education, Culture, Sports, Science, and Technology, Smoking Science Foundation No. FP00404086 (N.M.); by a grant to the Respiratory Failure Research Group from the Ministry of Health, Labor and Welfare, Japan (K.S.); and by the Institute for Environmental and Gender-Specific Medicine, Juntendo University, Graduate School of Medicine (K.S.). This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200511-1816OC on May 25, 2006 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form November 28, 2005; accepted in final form May 24, 2006
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TOP
ABSTRACT
METHODS
and Fas-mediated apoptosis (
p < 0.05, compared with 1 mo of age) and were significantly greater than those of the age-matched SMP30Y/+ mice (*p < 0.05). Values are presented as mean ± SD (n = 5 for each group).
-glutamylcysteine synthetase has been reported to be mediated by the redox-sensitive transcription factor, activating protein-1 (