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Heme oxygenase-1 protects corexit 9500A-induced respiratory epithelial injury across species.

Li FJ, Duggal RN, Oliva OM, Karki S, Surolia R, Wang Z, Watson RD, Thannickal VJ, Powell M, Watts S, Kulkarni T, Batra H, Bolisetty S, Agarwal A, Antony VB - PLoS ONE (2015)

Bottom Line: CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production.Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis.Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, United States of America.

ABSTRACT
The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis.

No MeSH data available.


Related in: MedlinePlus

CE exposure results in the cleavages of tight junctional proteins (ZO-1, ZO-2 and occluding) and adherens junctional proteins (E-cadherin and FAK).Cell lysates from BEAS-2B cells were analyzed by western blotting with anti-ZO-1, ZO-2 and Occludin (A) and E-cadherin and FAK (B) antibodies at different concentration of CE for 4 hours. The densities of protein bands were determined by densitometry and the data represent a fold change from the control density. The data are representative of three independent experiments. Data are shown as a mean ± SD. * p < 0.05, ** p < 0.01 vs control by a one-way ANOVA with HSD test.
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pone.0122275.g003: CE exposure results in the cleavages of tight junctional proteins (ZO-1, ZO-2 and occluding) and adherens junctional proteins (E-cadherin and FAK).Cell lysates from BEAS-2B cells were analyzed by western blotting with anti-ZO-1, ZO-2 and Occludin (A) and E-cadherin and FAK (B) antibodies at different concentration of CE for 4 hours. The densities of protein bands were determined by densitometry and the data represent a fold change from the control density. The data are representative of three independent experiments. Data are shown as a mean ± SD. * p < 0.05, ** p < 0.01 vs control by a one-way ANOVA with HSD test.

Mentions: To understand the effects of CE on the distribution and expression of the TJs proteins ZO-1, ZO-2 and occludin, we performed immunofluorescence staining and western blotting. CE induced membrane delocalization of ZO-1 (Fig. 1D) after exposure to CE for 1 hour and concentration-dependent decreases of ZO-1, ZO-2 and occludin protein expression when cells were exposure to CE for 4 hours (Fig. 3A). To examine the effects of CE on the cytoskeletal F-action network, we stained BEAS-2B cells with TRITC-Phalloidin. After 1 hour of treatment with CE, the actin cytoskeleton was disrupted and internalized into cytoplasm and outside of nuclear (Fig. 1D). To investigate the fate of E-cadherin and FAK during apoptosis, lysates of BEAS-2B cells were examined by western blotting analysis following CE exposure. Exposure to CE at concentrations of 100 ppm and 150 ppm resulted in a significant decrease in E-cadherin and FAK (Fig. 3B). This was particularly dramatic in the 150 ppm treatment, which resulted in a 72% and 94% decrease in E-cadherin and FAK, respectively, as determined by quantitative density analysis (Fig. 3B). Intercellular junctions and their cytoskeletal proteins are important in the maintenance of cell membrane integrity as well as prevention of apoptosis [10,48–52]. These data suggest that intercellular junctions and cytoskeletal actin filaments may function as substrate targets for CE-induced alterations in permeability and caspase-3-dependent apoptosis.


Heme oxygenase-1 protects corexit 9500A-induced respiratory epithelial injury across species.

Li FJ, Duggal RN, Oliva OM, Karki S, Surolia R, Wang Z, Watson RD, Thannickal VJ, Powell M, Watts S, Kulkarni T, Batra H, Bolisetty S, Agarwal A, Antony VB - PLoS ONE (2015)

CE exposure results in the cleavages of tight junctional proteins (ZO-1, ZO-2 and occluding) and adherens junctional proteins (E-cadherin and FAK).Cell lysates from BEAS-2B cells were analyzed by western blotting with anti-ZO-1, ZO-2 and Occludin (A) and E-cadherin and FAK (B) antibodies at different concentration of CE for 4 hours. The densities of protein bands were determined by densitometry and the data represent a fold change from the control density. The data are representative of three independent experiments. Data are shown as a mean ± SD. * p < 0.05, ** p < 0.01 vs control by a one-way ANOVA with HSD test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383564&req=5

pone.0122275.g003: CE exposure results in the cleavages of tight junctional proteins (ZO-1, ZO-2 and occluding) and adherens junctional proteins (E-cadherin and FAK).Cell lysates from BEAS-2B cells were analyzed by western blotting with anti-ZO-1, ZO-2 and Occludin (A) and E-cadherin and FAK (B) antibodies at different concentration of CE for 4 hours. The densities of protein bands were determined by densitometry and the data represent a fold change from the control density. The data are representative of three independent experiments. Data are shown as a mean ± SD. * p < 0.05, ** p < 0.01 vs control by a one-way ANOVA with HSD test.
Mentions: To understand the effects of CE on the distribution and expression of the TJs proteins ZO-1, ZO-2 and occludin, we performed immunofluorescence staining and western blotting. CE induced membrane delocalization of ZO-1 (Fig. 1D) after exposure to CE for 1 hour and concentration-dependent decreases of ZO-1, ZO-2 and occludin protein expression when cells were exposure to CE for 4 hours (Fig. 3A). To examine the effects of CE on the cytoskeletal F-action network, we stained BEAS-2B cells with TRITC-Phalloidin. After 1 hour of treatment with CE, the actin cytoskeleton was disrupted and internalized into cytoplasm and outside of nuclear (Fig. 1D). To investigate the fate of E-cadherin and FAK during apoptosis, lysates of BEAS-2B cells were examined by western blotting analysis following CE exposure. Exposure to CE at concentrations of 100 ppm and 150 ppm resulted in a significant decrease in E-cadherin and FAK (Fig. 3B). This was particularly dramatic in the 150 ppm treatment, which resulted in a 72% and 94% decrease in E-cadherin and FAK, respectively, as determined by quantitative density analysis (Fig. 3B). Intercellular junctions and their cytoskeletal proteins are important in the maintenance of cell membrane integrity as well as prevention of apoptosis [10,48–52]. These data suggest that intercellular junctions and cytoskeletal actin filaments may function as substrate targets for CE-induced alterations in permeability and caspase-3-dependent apoptosis.

Bottom Line: CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production.Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis.Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, United States of America.

ABSTRACT
The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis.

No MeSH data available.


Related in: MedlinePlus