Differential distribution of glucocorticoid and estrogen receptor isoforms: localization of GRbeta and ERalpha in nucleoli and GRalpha and ERbeta in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines.

The localization of glucocorticoid and estrogen receptors alpha (GRalpha, ERalpha) and beta (GRbeta, ERbeta) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In HepG2 and SaOS-2 cells GRbeta and ERalpha were localized mainly in the nucleus, particularly concentrated in nuclear structures, which on the basis of their staining with antibody against C23-nucleolin, were characterized as nucleoli. A faint, diffuse GRbeta and ERalpha staining was also observed in the cytoplasm. GRalpha and ERbeta were specifically enriched at the site of cell mitochondria, which were visualized by labelling with the vital dye CMX. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of glucocorticoid and estrogen receptor isoforms in the cell lines studied. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of GRbeta and ERalpha in nucleolar-related processes in HepG2 and SaOS-2 cells.

The mitochondrion as a primary site of action of regulatory agents involved in neuroimmunomodulation.

A major system of neuroimmunomodulation is the hypothalamic-pituitary-adrenocortical (HPA) axis, acting through glucocorticoids and their intracellular signaling components, exerting both stimulatory and inhibitory effects on the immune reaction. Glucocorticoids inhibit the production of proinflammatory cytokines by interacting with nuclear transcription factors (nuclear factor [NF]-kappaB, activated protein [AP]-1) and induce the production of several anti-inflammatory cytokines by gene activation. In some cells and/or in extreme stress conditions, apoptosis is evoked. In most processes related to neuroimmunomodulation a prominent role is emerging for mitochondria. These organelles generate more than 90% of the cell's energy requirements through oxidative phosphorylation (OXPHOS), which is regulated by several agents, including steroid and thyroid hormones. These hormones are inducers of nuclear and mitochondrial OXPHOS gene transcription and they exert a primary action not only on nuclear but also on mitochondrial genes by way of cognate receptors. Recently, additional nuclear transcription factors involved in neuroimmunomodulation have been detected in mitochondria (NF-kappaB, AP-1, p53, calcium/cAMP response element binding protein [CREB]), and binding sites of these and putative binding sites of other nuclear transcription factors have been identified in the mitochondrial genome. The interaction of these factors with mitochondrial regulatory proteins, with receptors and with the genome has been shown and, in some cases, modulation of mitochondrial transcription was observed with possible effects on energy yield. The mitochondria store a host of critical apoptotic activators and inhibitors in their intermembrane space and the release of these factors could be another possible mode of action of the mitochondrially translocated regulatory agents and receptors.

The mitochondrion as a primary site of action of steroid and thyroid hormones: presence and action of steroid and thyroid hormone receptors in mitochondria of animal cells.

Mitochondria are key cellular organelles that regulate events related to energy production and apoptosis. These processes are modulated, in turn, by steroid and thyroid hormones in the course of their actions on metabolism, growth and development. In this context, a direct effect of these hormones on the mitochondrial-linked processes, possibly by way of cognate mitochondrial receptors, has been proposed. In this paper we review data from the literature and present new findings supporting this concept. Receptors for steroid hormones, glucocorticoids and estrogens, and for T(3), have been detected in mitochondria by immunofluorescence labeling and confocal laser microscopy, by Western blotting of mitochondrial proteins and by immunogold electron microscopy. Furthermore, the mitochondrial genome contains nucleotide sequences with high similarity to known hormone-responsive elements, which interact with the appropriate receptors to confer hormone-dependent activation of reporter genes in transfection experiments. Thus, thyroid hormone stimulates mitochondrial transcription mediated by the cognate receptor when added to an in organello mitochondrial system, capable of faithful transcription.

Estrogen receptors alpha and beta (ERalpha and ERbeta) and androgen receptor (AR) in human sperm: localization of ERbeta and AR in mitochondria of the midpiece.

BACKGROUND: The central role of estrogens and androgens in the male reproductive system has focused attention on the presence and distribution of their cognate receptors [estrogen receptor (ER) alpha, ERbeta and androgen receptor (AR)] in male reproductive tissues and cells. Since the presence of steroid hormone receptors in mitochondria of mammalian cells has been well documented, we investigated the possibility of mitochondrial localization of sex steroid hormone receptors in sperm. METHODS AND RESULTS: Applying immunofluorescence labelling and confocal laser scanning microscopy we show that the estrogen receptor beta and the AR of human sperm are specifically enriched in the midpiece, at the site of the mitochondria, which were visualized by labelling with the vital dye CMX. Nuclear and mitochondrial localization of AR was also detected in LnCap human prostate cancer cells. Differentially, most of the ERalpha immunostaining is in the form of a compact zone at a region corresponding to the equatorial segment of the upper post-acrosomal region of the sperm head. Immunoblotting experiments using sperm extracts revealed the presence of a 66 and a 45 kDa protein reacting with the ERalpha antibody, one 64 kDa protein reacting with the ERbeta antibody and a 110 and a 90 kDa protein reacting with the antibody against AR. CONCLUSIONS: Our findings suggest that the differential localization of AR and ER isoforms in human sperm reveals distinct roles of these receptors in the physiology of sperm cells and, perhaps, also in the process of fertilization.

Differential subcellular distribution of estrogen receptor isoforms: localization of ERalpha in the nucleoli and ERbeta in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines.

The localization of estrogen receptors alpha (ERalpha) and beta (ERbeta) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In both cell types, ERalpha was localized mainly in the nucleus, particularly concentrated on nuclear structures, which on the basis of their staining with pyronin and with antibodies against the nucleoli-specific Ki67 antigen and C23-nucleolin, were characterized as nucleoli. A faint, diffuse ERalpha staining was also observed in the cytoplasm. ERbeta was specifically enriched at the site of the mitochondria, visualized by labelling with the vital dye CMX and antibody against the mitochondrial-specific cytochrome oxidase subunit I. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of ERalpha and ERbeta. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of ERalpha in nucleolar-related processes.

Co-expression of trypsin and tumour-associated trypsin inhibitor (TATI) in colorectal adenocarcinomas.

Trypsin and its specific inhibitor, TATI (tumour-associated trypsin inhibitor), are expressed in normal human pancreas and in a variety of tumours. The aim of the present study was to assess the parallel expression of trypsin and TATI in colorectal cancer, in comparison with their expression in normal epithelial tissue, since proteases and their inhibitors are thought to be co-expressed in malignant neoplasms. We also assessed the possible significance of their expression as a means of differentiation between normal and malignant tissue. We examined qualitatively and semi-quantitatively the immunohistochemical expression of trypsin and TATI on paraffin-embedded serial tissue sections from 91 colorectal adenocarcinomas. The reverse-transcriptase-polymerase-chain reaction (RT-PCR) was also performed on fresh malignant tissue from 55 of the above adenocarcinomas. Normal and non-malignant tissues adjacent to the tumours were also evaluated. Cytoplasmic expression of trypsin (more than 25% of the cancer cells positive) was found in 67 (73.6%) adenocarcinomas, whereas TATI was expressed in the cytoplasm of 59 (64.8%) cases studied. Statistical analysis using Spearman's test has demonstrated a significant correlation between trypsin and TATI immunohistochemical expression (p<0.01). RT-PCR showed co-expression of trypsin and TATI mRNA in all carcinomas studied. Distinct patterns of trypsin and TATI immunohistochemical expression were observed in adjacent, non-malignant tissues, where both trypsin and TATI mRNA were also detected. Normal tissues were negative by immunohistochemistry. Our results indicate co-expression of trypsin and TATI in colorectal tumours both at the mRNA and protein level. We conclude that in colorectal neoplasms, high levels of trypsin and TATI may be important for malignant tumour formation and/or metastatic process.