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Cigarette Smoke Associated Carbon Black and Cadmium Are Synergistic for the Development of Pulmonary Fibrosis Through Oxidative Stress-Dependent Phosphorylation of AKT and Vimentin
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A1053 - Cigarette Smoke Associated Carbon Black and Cadmium Are Synergistic for the Development of Pulmonary Fibrosis Through Oxidative Stress-Dependent Phosphorylation of AKT and Vimentin
Author Block: F. Li, R. Surolia, H. Li, Z. Wang, G. Liu, V. J. Thannickal, V. B. Antony; University of Alabama at Birmingham, Birmingham, AL, United States.
Rationale: Up to 65% of patients with IPF have a history of smoking. Cigarettes contain 2-3 μg of Cadmium (Cd) which can induce parenchymal fibrosis. However, the effects of carbon black (CB), also a constituent of cigarette smoke generated from the incomplete combustion of organic materials, on Cd-induced fibrosis have not been addressed. Methods: Carbon black particles and/or CdCl2 were administered intratracheally to C57BL/6 mice and fibrosis was evaluated by immunohistochemistry and sircol collagen assay on day 7 through day 28. Oxidative stress and kinases-related signaling proteins expression and secretion were monitored by western blotting and ELISA. Intracellular reactive oxygen species (ROS) production from alveolar macrophages was examined by flow cytometry. Results: We found that CdCl2 caused parenchymal fibrosis in mice. CB alone caused minimal fibrosis but clearly enhanced Cd-induced fibrosis, indicated by the increased expression of α-SMA and collagen. Cd and CB exposure increased the number of total cell count, neutrophils, especially macrophages in bronchoalveolar lavage (BAL) compared to either alone. Signaling studies showed that CB prolonged Cd-induced phosphorylation of AKT and vimentin, but not Cdc2 for up to 24 h. CB and Cd also synergistically induced intracellular ROS production in alveolar macrophages. Inhibition of ROS production by HO-1 inducer, carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited Cd-induced AKT and vimentin phosphorylation, indicating that Cd-mediated protein phosphorylation was mediated by oxidative stress. Interestingly, the presence of citrullinated vimentin (Cit-Vim) could be detected in plasma and BAL obtained from Cd and CB treated mice and Cit-Vim production was significantly inhibited by pretreatment with AKT inhibitor or phosphatase inhibitor, suggesting that Cit-Vim secretion was dependent on AKT kinase activity and vimentin phosphorylation status. Conclusion: These data indicate that CB may function as a cofactor in the pathogenesis of Cd-induced pulmonary fibrosis in regulating citrullinated vimentin secretion by a mechanism involving ROS production and prolonged activation of both AKT and vimentin.
Author Block: F. Li, R. Surolia, H. Li, Z. Wang, G. Liu, V. J. Thannickal, V. B. Antony; University of Alabama at Birmingham, Birmingham, AL, United States.
Rationale: Up to 65% of patients with IPF have a history of smoking. Cigarettes contain 2-3 μg of Cadmium (Cd) which can induce parenchymal fibrosis. However, the effects of carbon black (CB), also a constituent of cigarette smoke generated from the incomplete combustion of organic materials, on Cd-induced fibrosis have not been addressed. Methods: Carbon black particles and/or CdCl2 were administered intratracheally to C57BL/6 mice and fibrosis was evaluated by immunohistochemistry and sircol collagen assay on day 7 through day 28. Oxidative stress and kinases-related signaling proteins expression and secretion were monitored by western blotting and ELISA. Intracellular reactive oxygen species (ROS) production from alveolar macrophages was examined by flow cytometry. Results: We found that CdCl2 caused parenchymal fibrosis in mice. CB alone caused minimal fibrosis but clearly enhanced Cd-induced fibrosis, indicated by the increased expression of α-SMA and collagen. Cd and CB exposure increased the number of total cell count, neutrophils, especially macrophages in bronchoalveolar lavage (BAL) compared to either alone. Signaling studies showed that CB prolonged Cd-induced phosphorylation of AKT and vimentin, but not Cdc2 for up to 24 h. CB and Cd also synergistically induced intracellular ROS production in alveolar macrophages. Inhibition of ROS production by HO-1 inducer, carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited Cd-induced AKT and vimentin phosphorylation, indicating that Cd-mediated protein phosphorylation was mediated by oxidative stress. Interestingly, the presence of citrullinated vimentin (Cit-Vim) could be detected in plasma and BAL obtained from Cd and CB treated mice and Cit-Vim production was significantly inhibited by pretreatment with AKT inhibitor or phosphatase inhibitor, suggesting that Cit-Vim secretion was dependent on AKT kinase activity and vimentin phosphorylation status. Conclusion: These data indicate that CB may function as a cofactor in the pathogenesis of Cd-induced pulmonary fibrosis in regulating citrullinated vimentin secretion by a mechanism involving ROS production and prolonged activation of both AKT and vimentin.
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