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 ERß in lung tissue. Allergic airway inflammation caused reduction in mtGRα, mtERß, 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 ERß 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.

Glucocorticoids induce mitochondrial gene transcription in HepG2 cells: role of the mitochondrial glucocorticoid receptor.

Glucocorticoids are major regulators of a plethora of cellular functions, acting on target cells through glucocorticoid receptors (GR) and modulation of gene transcription, among other mechanisms. One main site of action of glucocorticoids is the hepatocyte, which responds to the hormonal stimulus with induction of several proteins among them enzymes of oxidative phosphorylation (OXPHOS), both nuclearly and mitochondrially encoded. The induction of OXPHOS is regarded as a result of a nuclear action of the receptor on the respective nuclear genes and on genes encoding mitochondrial transcription factors. The presence of GR in mitochondria and of sequences in the mitochondrial genome similar to glucocorticoid responsive elements, suggested a direct action of GR on mitochondrial transcription. We demonstrate in HepG2 hepatocarcinoma cells specific binding of GR to the regulatory D-loop region of the mitochondrial genome and show that dexamethasone induces the mitochondrial transcription factors A, B1, and B2, the mitochondrial ribosomal RNA, and several mitochondrially encoded OXPHOS genes. Applying α-amanitin, the specific inhibitor of DNA-dependent RNA polymerase II, the dexamethasone-induced transcription of the mitochondrial genes can still proceeds, whereas the DEX effect on transcription of the mitochondrial transcription factors is suppressed. Moreover, HepG2 cells overexpressing mitochondrial targeted GR showed increased RNA synthesis, cytrochrome oxidase subunit I protein expression, and mitochondrial ATP production. We conclude that glucocorticoids can stimulate directly mitochondrial transcription by the mitochondrially localized GR, affecting OXPHOS enzyme biosynthesis. This takes place in addition to their action on mitochondrial genes by way of induction of the nuclearly encoded mitochondrial transcription factors.

Diet and expression of estrogen alpha and progesterone receptors in malignant mammary tissue.

Estrogen receptor (ER) and/or progesterone receptor (PR) expression has been associated with more favorable breast cancer prognosis. Results on the differential association of diet with ER and/or PR positive and negative tumors have been inconclusive. In a large case-control study conducted in Athens, Greece, we investigated whether diet is associated with the expression of ER-alpha or PR in mammary tumors of 421 women with histologically confirmed breast cancer. Diet was assessed through an extensive food frequency questionnaire and results were analyzed using multiple logistic regression. After controlling for non-nutritional variables and mutually adjusting for energy-generating nutrients and ethanol, carbohydrate intake was inversely associated with ER-alpha (P = 0.04) and PR (P = 0.10) expression. The odds ratios (OR) per one standard deviation increment were 0.69 with 95% confidence interval (95% CI) 0.48-0.98 for ER-alpha and 0.72 (95% CI 0.49-1.07) for PR expression. No consistent or statistically significant associations were noted for any of the other energy-generating nutrients or food groups examined. Although in these data no strong relations of qualitative aspects of diet with hormone receptor expression in breast cancer tumors were evident, the inverse association of carbohydrate intake with ER-alpha, and perhaps PR, expression merits further study in future investigations.

Glucocorticoid receptors and other nuclear transcription factors in mitochondria and possible functions.

The central role of mitochondria in basic physiological processes has rendered this organelle a receiver and integrator of multiple regulatory signals. Steroid and thyroid hormones are major modulators of mitochondrial functions and the question arises as to how these molecules act at the molecular level. The detection in mitochondria of steroid and thyroid hormone receptors suggested their direct action on mitochondrial functions within the context of the organelle. The interaction of the receptors with regulatory elements of the mitochondrial genome and the activation of gene transcription underlies the hormonal stimulation of energy yield. Glucocorticoid activation of hepatocyte RNA synthesis is one of the experimental models exploited in this respect. Furthermore, the interaction of the receptors with apoptotic/antiapoptotic factors is possibly associated with the survival-death effects of the hormones. In addition to the steroid/thyroid hormone receptors, several other receptors belonging to the superfamily of nuclear receptors, as well as transcription factors with well defined nuclear actions, have been found in mitochondria. How these molecules act and interact and how they can affect the broad spectrum of mitochondrial functions is an emerging exciting field.

Diet and expression of estrogen alpha and progesterone receptors in the normal mammary gland.

OBJECTIVE: It has been recently reported that expression of estrogen alpha (ER-alpha) and progesterone (PR) receptors in the normal mammary gland is inversely associated with breast cancer risk among postmenopausal women. We investigated whether dietary intakes are associated with the expression of ER-alpha and PR receptors in the apparently normal, as opposed to pathological, mammary tissue. METHODS: In a study in Greece, we examined associations of dietary intakes with ER-alpha and PR expression in the adjacent-to-pathological apparently normal mammary tissue of 562 women with either breast cancer (267 women) or BBD (299 women). Diet was assessed through an extensive food frequency questionnaire and results were analyzed using multiple logistic regression. RESULTS: Monounsaturated (p = 0.03) and, to a lesser extent, polyunsaturated lipids (p = 0.08) were positively associated with ER-alpha expression. Cereals and starchy roots were inversely associated with ER-alpha (p = 0.01), whereas milk and dairy products were inversely associated with PR expression (p = 0.02). Ethanol intake was non-significantly inversely associated with ER-alpha expression (p = 0.07). CONCLUSIONS: Our findings suggest that the weak associations of diet with breast cancer risk could be explained, to some extent, by effects of diet on receptor expression in the normal mammary gland.

Estrogen alpha and progesterone receptor expression in the normal mammary epithelium in relation to breast cancer risk.

Estrogens play a central role in the etiology of breast cancer, and results from observational studies and randomized trials have also implicated progestins. The effects of these hormones in the mammary tissue are exerted through binding with specific receptor proteins in the cell nucleus. It has been proposed that higher estrogen receptor alpha expression in the normal breast epithelium may increase breast cancer risk. In a study in Greece, we determined estrogen alpha and progesterone receptor expression in normal mammary tissue adjacent to the pathological tissue from 267 women with breast cancer and 299 women with benign breast disease. Mouse monoclonal antibodies specific for estrogen receptor alpha and progesterone receptor were applied. The H-index, which incorporates frequency and intensity of staining of the cells, and can range from 0 to 300, was deemed positive when it exceeded 9. Among premenopausal women, there was no evidence for an association with breast cancer risk for expression of either type of receptors. Among postmenopausal women, breast cancer risk was inversely associated with expression of both estrogen alpha (odds ratio (OR)=0.39; p=0.015) and progesterone (OR=0.40; p=0.008) receptors. The hypothesis that overexpression of estrogen receptors alpha or progesterone receptors in normal breast epithelium may increase the risk of breast cancer was not supported by our data. Instead, we found evidence that overexpression of these receptors may be associated with reduced risk for breast cancer in line with the well-known association of expression of these receptors in the malignant tissue and better breast cancer prognosis.

Steroid and thyroid hormone receptors in mitochondria.

Receptors for glucocorticoids, estrogens, androgens, and thyroid hormones have been detected in mitochondria of various cell types by Western blotting, immunofluorescence labeling, confocal microscopy, and immunogold electron microscopy. A role of these receptors in mitochondrial transcription, OXPHOS biosynthesis, and apoptosis is now being revealed. Steroid and thyroid hormones regulate energy production, inducing nuclear and mitochondrial OXPHOS genes by way of cognate receptors. In addition to the action of the nuclearly localized receptors on nuclear OXPHOS gene transcription, a parallel direct action of the mitochondrially localized receptors on mitochondrial transcription has been demonstrated. The coordination of transcription activation in nuclei and mitochondria by the respective receptors is in part realized by their binding to common trans acting elements in the two genomes. Recent evidence points to a role of the mitochondrial receptors in cell survival and apoptosis, exerted by genomic and nongenomic mechanisms. The identification of additional receptors of the superfamily of nuclear receptors and of other nuclear transcription factors in mitochondria increases their arsenal of regulatory molecules and further underlines the central role of these organelles in the integration of growth, metabolic, and cell survival signals.

Nuclear receptors and other nuclear transcription factors in mitochondria: regulatory molecules in a new environment.

The mitochondrion is the major energy generating organelle of the cell and the site of other basic processes, including apoptosis. The mitochondrial functions are performed in concert with other cell compartments and are regulated by various extracellular and intracellular signals. Several nuclear receptors and other nuclear transcription factors, such as NF-kappa B, AP-1, CREB and p53, involved in growth, metabolic and developmental processes, have been detected in mitochondria. This finding raises the question as to the role of these regulatory molecules in their "new" environment. Experimental evidence supports the action of the mitochondrially localized transcription factors on mitochondrial transcription, energy yield and apoptosis, extending the known nuclear role of these molecules outside the nucleus. A principle of coordination of nuclear and mitochondrial gene transcription has been ascertained as regards the regulatory action of steroid and thyroid hormones on energy yield. Accordingly, the same nuclear receptors, localized in the two compartments-nuclei and mitochondria-regulate transcription of genes serving a common function by way of interaction with common binding sites in the two genomes. This principle is now expanding to encompass other nuclearly and mitochondrially localized transcription factors.

Glucocorticoid receptor isoforms in human hepatocarcinoma HepG2 and SaOS-2 osteosarcoma cells: presence of glucocorticoid receptor alpha in mitochondria and of glucocorticoid receptor beta in nucleoli.

In the context of a possible direct action of glucocorticosteroids on mitochondrial transcription and/or apoptosis by way of cognate mitochondrial receptors, the possible localization of glucocorticoid receptors alpha and beta (GRalpha and GRbeta) in mitochondria was explored in human hepatocarcinoma HepG2 and osteosarcoma SaOS-2 cells, in which glucocorticoids exert an anabolic and apoptotic effect, respectively. In both cell types, GRalpha was detected in mitochondria, in nuclei and in cytosol by immunofluorescence labeling and confocal scanning microscopy, by immunogold electron microscopy and by Western blotting. GRbeta was shown to be almost exclusively restricted to the nucleus of the two cell types, being particularly concentrated in nucleoli, pointing to a solely nuclear role of this receptor isoform and to a possible function in nucleoli related processes. Computer analysis identified a putative internal mitochondrial targeting sequence within the glucocorticoid receptor. The demonstration of mitochondrially localized GRalpha in HepG2 and SaOS-2 cells corroborates previous findings in other cell types and further supports a direct role of this receptor in mitochondrial functions.

Tumour-associated trypsin inhibitor, carcinoembryonic antigen and acute-phase reactant proteins CRP and alpha1-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.

The dynamic localization of the glucocorticoid 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.

Oligonucleotide sequences similar to transcription factor consensi of nuclear genes are present in the human mitochondrial genome.

Screening the mitochondrial genome for binding sites for known nuclear transcription factors revealed oligonucleotide sequences identical or with over 85% similarity to consensi sequences for twenty-one transcription factors, modulating nuclear genes involved, among others, in cell proliferation, inflammation and synthesis of ribosomal and mitochondrial proteins. Two of these sequences were found in the D-loop, the others dispersed among structural genes for respiratory enzyme subunits, for t-RNAs and for rRNAs. We hypothesize that the transcription factors corresponding to the detected mitochondrial binding sites and the agents controlling the availability of these factors could play a regulatory role in the diverse functions of mitochondria, such as energy production, differentiation and apoptosis.

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

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 hGRalpha. 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 hGRalpha. Wild type hGRalpha 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 hGRalpha. 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.

Effects of dexamethasone on K(+)-evoked glutamate release from rat hippocampal slices.

Dexamethasone (DEX) at physiologically elevated (stress) concentration (1 microM) decreased K(+)-evoked glutamate release from rat hippocampal slices under superfusion in the presence of Ca2+. On the contrary 10 microM DEX increased this K(+)-evoked glutamate release while 0.1 microM DEX had no effect. The glucocorticoid antagonist for the "classic" receptor, RU 486, completely reversed the effect of 1 microM DEX. Actinomycin D had no effect. Dexamethasone at 1 microM had no effect on the Ca2(+)-independent (10 mM Mg2+ replacing 1 mM Ca2+) K(+)-evoked glutamate release. Dexamethasone at 1 microM or 10 microM had no effect on the phosphate-activated glutaminase--the key enzyme for the biosynthesis of neurotransmitter glutamate. These results suggest that the effect of DEX on K(+)-evoked glutamate release: (i) depends on its concentration; (ii) is exerted on the Ca2(+)-dependent (neurotransmitter release), at least at physiological stress concentrations; and (iii) is exerted via the classical receptor but is nongenomic.

The effects of steroid hormones on the transcription of genes encoding enzymes of oxidative phosphorylation.

Regulation of energy metabolism is one of the major functions of steroid hormones. In this process, mitochondria, by way of oxidative phosphorylation, play a central role. Depending on the energy needs of the cell, on the tissue, on the developmental stage and on the intensity of the hormonal stimulus, the response can be an activation of pre-existing respiratory chain components, an increased transcription of nuclear-encoded and/or mitochondrial-encoded respiratory chain enzyme (OXPHOS) genes and of biosynthesis of the respective enzyme subunits or, in extreme cases of high energy needs, an increase in the number of mitochondria and mitochondrial DNA content per cell. Some of the hormonally regulated systems involving effects on nuclear and mitochondrial OXPHOS genes are reviewed in this paper. The possible molecular mechanisms of steroid hormone action on nuclear and mitochondrial gene transcription and possible ways of coordination of transcription in these two separate cell compartments involving direct interaction of steroid receptors with hormone response elements in nuclear OXPHOS genes and in mitochondria and induction/activation of nuclear-encoded regulatory factors affecting mitochondrial gene transcription are presented.

Mitochondrial localization of glucocortocoid receptor in glial (Müller) cells in the salamander retina.

Glucocorticoid hormones regulate the transcription of nuclear genes by way of their receptors. In addition, these hormones modulate mitochondrial gene transcription by mechanisms that remain poorly understood. Using immunofluorescence labeling in isolated Müller and photoreceptor cells and in intact salamander retina, we found that the glucocorticoid receptor (GR) is localized in both cell types. Confocal laser scanning microscopy and double staining with cytochrome oxidase (COX) showed that GR is localized in the mitochondria of Müller cells, but not in the mitochondria of photoreceptors. GR also colocalizes with glutamine synthetase (GS) in the cytoplasm of Müller cells. GR is also localized in the microvilli of the distal process of Müller cells and in the synaptic terminal of photoreceptors. Pre-incubation of Müller cells with 1 microM dexamethasone (DEX) for 7 h led to greater than 50% inhibition of the glutamate-induced increase in mitochondrial NADH. This late effect of glucocorticoids on glutamate metabolism could be ascribed, in part, to a direct action of steroid hormones on mitochondrial metabolism.

Glucocorticoid and thyroid hormone receptors in mitochondria of animal cells.

This article concerns the localization of glucocorticoid and thyroid hormone receptors in mitochondria of animal cells. The receptors are discussed in terms of their potential role in the regulation of mitochondrial transcription and energy production by the oxidative phosphorylation pathway, realized both by nuclear-encoded and mitochondrially encoded enzymes. A brief survey of the role of glucocorticoid and thyroid hormones on energy metabolism is presented, followed by a description of the molecular mode of action of these hormones and of the central role of the receptors in regulation of transcription. Subsequently, the structure and characteristics of glucocorticoid and thyroid hormone receptors are described, followed by a section on the effects of glucocorticoid and thyroid hormones on the transcription of mitochondrial and nuclear genes encoding subunits of OXPHOS and by an introduction to the mitochondrial genome and its transcription. A comprehensive description of the data demonstrates the localization of glucocorticoid and thyroid hormone receptors in mitochondria as well as the detection of potential hormone response elements that bind to these receptors. This leads to the conclusion that the receptors potentially play a role in the regulation of transcription of mitochondrial genes. The in organello mitochondrial system, which is capable of sustaining transcription in the absence of nuclear participation, is presented, responding to T3 with increased transcription rates, and the central role of a thyroid receptor isoform in the transcription effect is emphasized. Lastly, possible ways of coordinating nuclear and mitochondrial gene transcription in response to glucocorticoid and thyroid hormones are discussed, the hormones acting directly on the genes of the two compartments by way of common hormone response elements and indirectly on mitochondrial genes by stimulation of nuclear-encoded transcription factors.