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Major Effect of Oxidative Stress on the Male SP-A1 Type II Cell miRNome
Description
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A7681 - Major Effect of Oxidative Stress on the Male SP-A1 Type II Cell miRNome
Author Block: G. Noutsios, N. Thorenoor, X. Zhang, T. Umstead, F. Durrani, D. S. Phelps, J. Floros; Pediatrics, Penn State College of Medicine, Hershey, PA, United States.
Rationale
Pulmonary surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. In humans there are two different proteins, SP-A1 and SP-A2, which are produced by the alveolar type II cells (T2C). We have previously shown that microRNAs (miRNAs) not only affect SP-A expression, but they also contribute to the maintenance of T2C phenotype, and regulate anti-oxidant pathways. In the present study using humanized transgenic mice (hTG), we sought to investigate the differential influence of SP-A1 and SP-A2 in T2C miRNome under oxidative stress (OxS).
Methods
SP-A knock out (KO) and hTG mice expressing SP-A1 and SP-A2 (3 mice/group, n=36) were exposed to 2 ppm O3 or filter air for 3 h, sacrificed 4 h post OxS, and T2C isolated. RNA was extracted, miRNAs purified, cDNA generated and used to quantify levels of 372 most abundantly expressed miRNAs.
Results
Ozone exposure in SP-A1 females and SP-A2 mice (males and females) did not result in significant miRNome changes. However, in SP-A1 males, we found significant changes in miRNome in terms of sex and sex-OxS effects, with 24 miRNAs differentially expressed under OxS. A two-way ANOVA test for the sex and treatment effects showed that the F-stat for the interaction between the two factors (sex and treatment) was F=16.69 with F crit=3.84, and p=4.63 x 10-5. The mRNA targets of these miRNAs included PLCG2, IRS1, JAK2 and MAP2K4. We have found several connections with the MAPK/ERK pathway: PLCG2 transmits signals across the cell membrane and IRS1 mediates actin-cytoskeleton signaling. Also, JAK2 is involved in actin filament polymerization, which provides structure and shape to the cells.
Conclusions
The data indicate that SP-A1 in response to OxS has a major effect on the male T2C miRNome and this may be mediated via the MAP/ERK pathway. Of note, our recent publication showed that SP-A2, in response to OxS, had a major effect on male alveolar macrophage miRNome1, indicating a differential role of SP-A1 and SP-A2. This is consistent with previous data where SP-A2 exhibits a higher innate immune activity and SP-A1 plays an important role on surfactant structural organization2. Thus, dysregulation of either SP-A1 or SP-A2 may affect innate immunity and/or surfactant structure and potentially surfactant function. Both processes are essential for normal lung function and derangement of either has been identified as a problem in most pulmonary diseases.
1. Noutsios, Biol Sex Differ 2017,8(1),37
2. Lopez-Rodriguez, Biophys J 2016,111(3),524-536
Author Block: G. Noutsios, N. Thorenoor, X. Zhang, T. Umstead, F. Durrani, D. S. Phelps, J. Floros; Pediatrics, Penn State College of Medicine, Hershey, PA, United States.
Rationale
Pulmonary surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. In humans there are two different proteins, SP-A1 and SP-A2, which are produced by the alveolar type II cells (T2C). We have previously shown that microRNAs (miRNAs) not only affect SP-A expression, but they also contribute to the maintenance of T2C phenotype, and regulate anti-oxidant pathways. In the present study using humanized transgenic mice (hTG), we sought to investigate the differential influence of SP-A1 and SP-A2 in T2C miRNome under oxidative stress (OxS).
Methods
SP-A knock out (KO) and hTG mice expressing SP-A1 and SP-A2 (3 mice/group, n=36) were exposed to 2 ppm O3 or filter air for 3 h, sacrificed 4 h post OxS, and T2C isolated. RNA was extracted, miRNAs purified, cDNA generated and used to quantify levels of 372 most abundantly expressed miRNAs.
Results
Ozone exposure in SP-A1 females and SP-A2 mice (males and females) did not result in significant miRNome changes. However, in SP-A1 males, we found significant changes in miRNome in terms of sex and sex-OxS effects, with 24 miRNAs differentially expressed under OxS. A two-way ANOVA test for the sex and treatment effects showed that the F-stat for the interaction between the two factors (sex and treatment) was F=16.69 with F crit=3.84, and p=4.63 x 10-5. The mRNA targets of these miRNAs included PLCG2, IRS1, JAK2 and MAP2K4. We have found several connections with the MAPK/ERK pathway: PLCG2 transmits signals across the cell membrane and IRS1 mediates actin-cytoskeleton signaling. Also, JAK2 is involved in actin filament polymerization, which provides structure and shape to the cells.
Conclusions
The data indicate that SP-A1 in response to OxS has a major effect on the male T2C miRNome and this may be mediated via the MAP/ERK pathway. Of note, our recent publication showed that SP-A2, in response to OxS, had a major effect on male alveolar macrophage miRNome1, indicating a differential role of SP-A1 and SP-A2. This is consistent with previous data where SP-A2 exhibits a higher innate immune activity and SP-A1 plays an important role on surfactant structural organization2. Thus, dysregulation of either SP-A1 or SP-A2 may affect innate immunity and/or surfactant structure and potentially surfactant function. Both processes are essential for normal lung function and derangement of either has been identified as a problem in most pulmonary diseases.
1. Noutsios, Biol Sex Differ 2017,8(1),37
2. Lopez-Rodriguez, Biophys J 2016,111(3),524-536
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