Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell’s energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GRα and ERβ in lung tissue. Allergic airway inflammation caused reduction in mtGRα, mtERβ, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GRα and ERβ in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease

Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell’s energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease

Investigating the Role of the microRNA-34/449 Family in Male Infertility: A Critical Analysis and Review of the Literature

There is a great body of evidence suggesting that in both humans and animal models the microRNA-34/449 (miR-34/449) family plays a crucial role for normal testicular functionality as well as for successful spermatogenesis, regulating spermatozoa maturation and functionality. This review and critical analysis aims to summarize the potential mechanisms via which miR-34/449 dysregulation could lead to male infertility. Existing data indicate that miR-34/449 family members regulate ciliogenesis in the efferent ductules epithelium. Upon miR-34/449 dysregulation, ciliogenesis in the efferent ductules is significantly impaired, leading to sperm aggregation and agglutination as well as to defective reabsorption of the seminiferous tubular fluids. These events in turn cause obstruction of the efferent ductules and thus accumulation of the tubular fluids resulting to high hydrostatic pressure into the testis. High hydrostatic pressure progressively leads to testicular dysfunction as well as to spermatogenic failure and finally to male infertility, which could range from severe oligoasthenozoospermia to azoospermia. In addition, miR-34/449 family members act as significant regulators of spermatogenesis with an essential role in controlling expression patterns of several spermatogenesis-related proteins. It is demonstrated that these microRNAs are meiotic specific microRNAs as their expression is relatively higher at the initiation of meiotic divisions during spermatogenesis. Moreover, data indicate that these molecules are essential for proper formation as well as for proper function of spermatozoa per se. MicroRNA-34/449 family seems to exert significant anti-oxidant and anti-apoptotic properties and thus contribute to testicular homeostatic regulation. Considering the clinical significance of these microRNAs, data indicate that the altered expression of the miR-34/449 family members is strongly associated with several aspects of male infertility. Most importantly, miR-34/449 levels in spermatozoa, in testicular tissues as well as in seminal plasma seem to be directly associated with severity of male infertility, indicating that these microRNAs could serve as potential sensitive biomarkers for an accurate individualized differential diagnosis, as well as for the assessment of the severity of male factor infertility. In conclusion, dysregulation of miR-34/449 family detrimentally affects male reproductive potential, impairing both testicular functionality as well as spermatogenesis. Future studies are needed to verify these conclusions

mtGR and mtERβ are reduced in autopsies from fatal asthma patients.

<p>Confocal laser scanning microscopy images from control cases and fatal asthma patients were analysed using Leica Las-AF image system. Representative overlay and co-localized pixels (white pixels) images of human lung sections immune-stained with antibodies against (A) COX-I (green) and GR (red) and (B) COX-I (green) and ERβ (red) are depicted. Sections were also stained with Hoeschst nuclear fluorescence stain (blue). (C) Mean percentages of co-localization rate were calculated at ROIs placed at epithelial cell layer in the human autopsies as described in Methods section. Results are presented as means ± SEM; mtGR and mtERβ percentage of co-localization rate set at 100% for control subjects. n = 12–20 bronchi from autopsies per group; *<i>p</i><0.05 from control; **<i>p</i><0.001 from control. Scale bars, 25 µm.</p

Allergic airway inflammation increases pro-apoptotic signalling from mitochondria.

<p>Cytosol from control and allergic mice were isolated during the subcellular fractionation procedure as described in Methods section. (A) Expression of the uncleaved caspase-9 and cytochrome c were studied by Western blot in the cytosol. Representative Western blots are shown. (B) Quantitation of band intensity by densitometry from blots after normalization against actin is shown. (C) Expression of the PARP and cleaved caspase-3 were studied by Western blot in total lung homogenate. Representative Western blots are shown. (D) Quantitation of band intensity by densitometry from blots after normalization against actin is shown. (E) Hoechst and anti-cleaved caspase-3 (red) staining in representative confocal images from control and allergic mice lung sections. White arrow heads points to cleaved caspase-3 shown as red pixels in overlaid cleaved caspased-3-Hoechst images and as white pixels in cleaved caspase-3 grey scale images. Scale bars, 25 µm. Results are presented as means ± SEM; *<i>p</i><0.05; **<i>p</i><0.001 from control.</p

Allergic airway inflammation reduces GR and ERβ in highly purified mitochondrial fraction.

<p>Highly purified mitochondria were isolated from total lung homogenates from allergic and control mice as described in Methods section. (A) Representative Western blot images showing mtGR and mtERβ in isolated mitochondrial fraction. (B) Quantitation of band intensity by densitometry from blots after normalization against the succinate-ubiquinol oxidoreductase subunit of the mitochondrial Complex II OXPHOS enzyme (SDH). (C) Western blot analysis of GR, COX-I, and β-actin protein levels in cytosolic (Cytosol) and mitochondrial (Mito) fractions from control and allergic mice. (D) Quantitation of mtGR band intensity by densitometry after normalization against the expression levels of COX-I protein. (E) Western blot analysis of GR, ERβ, and β-actin, in total homogenates (TH) and cytosolic fractions from control and allergic mice. Results are presented as means ± SEM; relative measurements of GR and ERβ were set at 100% for control mice. n = 10–12 mice per group; **<i>p</i><0.001; ***<i>p</i><0.0001 from control.</p