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

The mitochondrion as a primary site of action of glucocorticoids: Mitochondrial nucleotide sequences, showing similarity to hormone response elements, confer dexamethasone inducibility to chimaeric genes transfected in LATK(-) cells

The hypothesis of a primary action of steroid hormones on mitochondrial gene expression has been supported by the detection of the glucocorticoid receptor in liver mitochondria and the demonstration of the interaction of the receptor with putative mitochondrial HREs. We now show that two putative mitochondrial glucocorticoid response elements present within the cytochrome oxidase subunit I gene (GREI and GREII), linked to a thymidine kinase promoter and to the CAT gene, transfected to LATK(-) cells, confer dexamethasone inducibility to the CAT gene. As the plasmids were stably transfected, hormone induction was analysed in the nuclear background. This effect is dose dependent and is abolished by the glucocorticoid antagonist RU38486. (C) 1997 Academic Press

The dynamic localization of the glucorticoid receptor in rat C6 glioma cell mitochondria

Glucocorticoids modify gene expression via the translocation of receptors from the cytosol to the nucleus following agonist-associated receptor activation. In this study, we have characterized mitochondrial glucocorticoid (GR) localization and associated translocation kinetics in the C6 mouse glioma cell line. Treatment of the cells, which were cultured in steroid-depleted culture medium, with the GR agonist dexamethasone (dex) resulted in a dramatic decrease in mitochondrial GR levels in parallel with those of the cytosolic receptor. The effect was not observed in isolated intact mitochondria suggesting that the effect is unlikely to be direct but is rather a component of the combined cellular response to GR activation. A marked stimulation of the expression of the mitochondrially-encoded cytochrome oxidase-1 (COX-1) gene was found following GR activation and its export from mitochondria. The effects were inhibited by RU486. Therefore, GR is likely to have a functional role at the level of the mitochondria within intact cells. (C) 2003 Elsevier Ireland Ltd. All rights reserved

Mutation detection of the human glucocorticoid receptor alpha gene area coding for the hormone-binding domain by denaturing gradient gel electrophoresis

Mutations in the hormone-binding domain of the human glucocorticoid receptor alpha (hGRalpha) gene have been detected in a variety of glucocorticoid resistance syndromes. Using the denaturing gradient gel electrophoresis technique, we developed a sensitive method for the detection of alterations in the gene area coding for the whole hormone-binding domain and part of the DNA-binding domain of the hGRalpha. This method can be applied for screening of glucocorticoid receptor gene alterations in glucocorticoid-dependent diseases. (C) 2002 Elsevier Science B.V. All tights reserved

Application of denaturing gradient gel electrophoresis (DGGE) to screen for mutations of the human glucocorticoid receptor a gene (hGR alpha)

Objectives: In a previous publication, we had presented a sensitive method to detect mutations of the segment of the human glucocorticoid. receptor alpha (hGRalpha) gene encoding the ligand binding domain (LBD) and part of the DNA binding domain (DBD) of hGRalpha, as several types of glucocorticoid resistance syndromes have been correlated with mutations in the respective nucleotide sequences. However, mutations affecting various regions covering the whole length of hGRalpha are increasingly reported in a variety of disease states. We now present an expanded screening methodology to detect mutations covering the whole length of hGRa. Design and Methods: We developed a sensitive, simple screening PCR-DGGE method to detect mutations in the aminoterminal domain and DNA-binding domain of the hGRa. Wild type hGRa cDNA and mutant samples were included in the analysis to ensure the accuracy and sensitivity of the method. Results: The PCR-DGGE method identified the mutant samples and discriminated them from wild type hGRa. Conclusions: The method described is accurate, sensitive, simple, cheap and fulfills the critera for a screening method which will be useful in delineating possible involvement of hGRalpha mutations in the aetiopathology of diseases correlated to derangements of glucocorticoid action. (C) 2003 The Canadian Society of Clinical Chemists. All rights reserved

Tumour-associated trypsin inhibitor, carcinoembryonic antigen and acute-phase reactant proteins CRP and alpha 1-antitrypsin in patients with gastrointestinal malignancies

Objectives: Elevated serum tumour-associated trypsin inhibitor (TATI) levels have been observed in association with malignancy or inflammation. The aim of our study was to evaluate the role of TATI in gastric and colorectal cancer. Design and methods: In preoperative serum samples, we measured TATI, carcinoembryonic antigen (CEA), C-reactive protein (CRP) and alpha(1)-antitrypsin (AAT). Results: Elevated levels of TATI were observed in 50% and 41.7% of patients with gastric and colorectal cancer. Elevated levels of TATI were observed only in 8% of patients with benign gastrointestinal malignancies (92% specificity). Elevated levels of CEA were observed in 25% and 24.4% of patients, respectively. The total positivity of CEA and TATI (with at least one marker positive) was 62.5% and 57%, respectively. Spearman’s test has shown a statistically significant correlation among serum TATI, CRP and AAT levels (P < 0.01). Conclusions: In gastrointestinal cancer, TATI can be used as a complementary tumour marker in addition to CEA. Regulation of TATI synthesis resembles that of acute-phase reactant proteins. (C) 2003 The Canadian Society of Clinical Chemists. All rights reserved

The oestrogen receptor codon 10 polymorphism detected in breast cancer is also present in non-malignant cells

The effect of oestrogens on oestrogen-receptive organs and cells is mediated via intracellular receptors (ER alpha and ER beta). Oestrogen receptor gene polymorphisms in the region encoding the N-terminal portion of the protein are reportedly associated with pathological conditions including breast cancer, hypertension, spontaneous abortion and coronary heart disease. A silent mutation in codon 10 of exon 1, detected in ER-negative and ER-positive human breast cancer cell lines, in breast tumors and blood DNA from breast cancer patients, has been recognized as a polymorphic site. In this study we examined. by denaturing gradient-gel electrophoresis and DNA sequence analysis, the possible presence of a codon 10 polymorphic site in normal oestrogen target organs and cells such as the uterus (myometrium and endometrium), in the placenta and peripheral blood mononuclear cells and in a benign uterus tumour (leiomyoma). We have detected ER codon 10 polymorphism in these samples and, have compared them to those observed in breast cancer samples. All tissues and cells studied were homozygous for the wild-type gene, and were heterozygous as well as homozygous for the codon-10-variant type. These results indicate that the presence of the codon-10-variant type is not a characteristic of breast cancer. Out current findings suggest that further investigations are warranted to elucidate the possible linkage of ER codon 10 polymorphism to physiological and pathological conditions

Glucocorticoid receptor alpha and beta isoforms are not mutated in bipolar affective disorder

The periodically hyperactive hypothalamic-pituitary-adrenal (HPA) axis in bipolar affective disorders, as well as the reported changes in the binding characteristics of the glucocorticoid receptor (GR), suggest the possible involvement of the GR in the aetiopathology of this disease. This was investigated by screening the coding sequences of both GR isoforms, GR alpha and GR beta, for the presence of mutations. As a genetic predisposition has been implicated, we included in this study bipolar patients who were siblings. By RT-PCR of peripheral blood mononuclear cells from patients suffering from bipolar illness, using primers spanning the whole length of the GR alpha and GR beta coding region and subsequent agarose gel electrophoresis, heteroduplex and sequence analyses, no GR mutations could be detected. Since glucocorticoid receptor activity can be modulated by agents other than the respective ligand leg by growth factors, cytokines and stress signals), our results favor derangements in the modulation of GR activity by such agents and not in the primary structure of the receptor as aetiopathologic factors of bipolar disease