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Biomedical subjects

C M Bamberger

Publications and source records attributed to C M Bamberger.

At least 19 recordsLinked to original sources
PubMed2006

Expression of urocortin in the extravillous human trophoblast at the implantation site.

Urocortin (UCN) is a 40 amino acid peptide which is closely related to corticotropin-releasing hormone and binds with high affinity to both CRH type 1 and type 2 receptors. UCN is expressed in human reproductive tissues including endometrium, ovary, and placenta. This study was designed to investigate the cellular localization of UCN at the implantation site of the human blastocyst, as well as the regulation of the UCN promoter by two major intracellular signaling pathways, the cAMP/PKA and diacylglycerol/PKC pathways, in cells of placental origin. For this reason, immunohistochemistry was performed on tissue sections from paraffin-embedded human first trimester placentas and freshly isolated human invasive extravillous trophoblast cells (EVT) were analyzed for UCN expression using RT-PCR and immunofluorescence. Finally, UCN promoter activity was analyzed in the JEG3 human choriocarcinoma cell line. Immunohistochemistry revealed expression of UCN in the cytotrophoblast, the EVT and decidual cells. Both UCN mRNA and peptide were detectable in freshly isolated EVT. Finally, a human UCN promoter luciferase reporter construct transfected into JEG3 cells was significantly inducible by phorbol ester plus ionomycin, but not by phorbol ester alone or by forskolin. Collectively, the present study reports the expression of UCN in EVT and the activation of the UCN gene promoter by the diacylglycerol/PKC pathway. The functional significance of urocortin for the physiology of EVT requires further investigation.

Corticotropin-Releasing Hormone↗
PubMed2006

Expression of the glucocorticoid receptor in the human adrenal cortex.

Glucocorticoids produced in the adrenal cortex act by binding to a specific intracellular protein, the glucocorticoid receptor (GR), which then modulates gene transcription in target tissues. Whether the adrenal cortex itself is a glucocorticoid target tissue has not been analyzed as yet. Since the presence of GR would be a prerequisite for such "intracortical" glucocorticoid action, this study was designed to analyze GR expression in the normal human adrenal gland using RT-PCR, Western blot, and immunohistochemistry. RT-PCR revealed the presence of GR mRNA in adrenal cortex as well as in NCIh295 cells. These results were confirmed at the protein level by Western blot employing a specific anti-human GR antibody. Immunohistochemically, weak GR staining was observed in the adrenal medulla. In contrast, GR was strongly expressed in the adrenal cortex with the zona reticularis showing the most intense staining. Transfection of a GR-responsive luciferase reporter gene into NCIh295 cells resulted in dexamethasone-dependent induction of luciferase activity, indicating that GR is functional in this tissue. In this study, we show for the first time that GR is expressed in the human adrenal cortex. Its preferential expression in the zona reticularis may indicate a functional role in the regulation of adrenal androgen biosynthesis.

Adrenal Cortex↗
PubMed2004

Vitamin B6 modulates glucocorticoid-dependent gene transcription in a promoter- and cell type-specific manner.

Due to their immunosuppressive effects, glucocorticoids (GC) are widely used in the treatment of inflammatory and autoimmune states. However, long-term GC treatment is associated with severe side effects. To increase the ratio of wanted and unwanted GC effects, is, therefore, a desirable goal, which could be achieved by either developing new "dissociating" GC or by combining conventional GC therapy with substances that selectively interfere with glucocorticoid receptor (GR) function. Vitamin B6 was previously shown to inhibit GR transactivation in non-immune cells. In the present study, we tested whether vitamin B6 would also interfere with GR function in immune cells and/or with transrepression in non-immune cells. Normal human lymphocytes and Jurkat T lymphoma cells were transfected with luciferase reporter constructs under the control of the interleukin-2 (IL-2) and the leukemia inhibitory factor (LIF) promoter, respectively. Cells were stimulated with phorbol ester, ionomycin, and different concentrations of dexamethasone, either in the absence (a vitamin B6-free medium was especially prepared for this study) or presence of vitamin B6. Both promoters were strongly induced in response to phorbol ester and ionomycin. Dexamethasone inhibited this effect in a dose-dependent manner both in the presence and absence of vitamin B6. Similar results were obtained at the protein level (IL-2- and LIF-specific ELISAs). Induction of a glucocorticoid response element (GRE)-driven promoter construct by dexamethasone in lymphoid cells was only marginally reduced by vitamin B6. In contrast, GR-mediated transactivation was strongly inhibited by vitamin B6 in HeLa cells, while GR-mediated transrepression of a matrix metalloproteinase 9 (MMP9) promoter construct was not affected. Our data indicate that vitamin B6 does not interfere with GC action in immune cells (wanted GC effects) while selectively inhibiting GR-dependent transactivation in non-immune cells (unwanted GC effects). Combination of GC treatment with supraphysiological doses of vitamin B6 may, thus, reduce the side effects of this type of immunosuppressive therapy, provided that the observed effects can be reproduced at subtoxic vitamin B6 concentrations in vivo.

Cytokines↗
PubMed2001

The glucocorticoid receptor is specifically expressed in the stromal compartment of the human endometrium.

The human endometrium is a classical target tissue for steroid hormones. While the expression pattern and functional roles of both the estrogen receptor (ER) and the progesterone receptor (PR) are well defined, expression of the glucocorticoid receptor (GR) in this tissue has not been described so far. In the present study, we used immunohistochemistry to analyze the expression of GR in the normal human endometrium throughout the menstrual cycle. The expression of GR was compared to that of ER and PR, which were analyzed in parallel. We show that GR is expressed in the human endometrium with a pattern that markedly differs from the expression patterns of ER and PR. ER and PR are expressed in the nuclei of endometrial glands, whereas GR is completely absent from these structures. However, GR is strongly expressed in the stromal compartment of the endometrium throughout the cycle. Both stromal fibroblasts and lymphocytes are GR-positive. In addition GR expression is also observed in the endothelium of small endometrial blood vessels, which are ER- and PR-negative. Western blot analysis performed on endometrial tumor cell lines of glandular (HEC-1B) and mesodermal (SKUT-1B) origin, respectively, showed GR expression only in the latter. In summary, we demonstrate that GR is expressed in fibroblasts, lymphocytes and endothelial cells of the human endometrial stroma, while it is absent from the glandular compartment. The specific expression pattern of GR within the human endometrium points to a possible functional role of glucocorticoids in the process of decidualization which occurs primarily in the stromal compartment.

Adult↗
PubMed2000

Expression of leukemia inhibitory factor (LIF) and LIF receptor (LIF-R) in the human adrenal cortex: implications for steroidogenesis.

It is well established that steroidogenesis in the adrenal cortex is regulated by extraadrenal factors, such as ACTH and angiotensin II. However, over the last years, it has become increasingly clear that paracrine and autocrine mechanisms are also important for steroid synthesis in the adrenal gland. The current study was designed to analyze whether the pleiotropic cytokine leukemia inhibitory factor (LIF) and/or its receptor (LIF-R) are expressed in the normal human adrenal cortex, and whether they may play a role in regulating steroidogenesis. Using LIF- and LIF-R-specific primers, we show by RT-PCR that both mRNAs are expressed in this tissue, as well as in the NCI-H295 adrenal carcinoma cell line. The correct sequences of the PCR products were verified by restriction enzyme analysis and DNA sequencing. Immunohistochemistry, employing specific antibodies against LIF and LIF-R, reveals expression of both proteins in the normal human adrenal cortex. Finally, we show that LIF can significantly enhance basal and ACTH-induced production of cortisol and aldosterone in NCI-H295 cells. In summary, we show for the first time that LIF and its receptor are expressed in the normal human adrenal cortex. Our stimulation experiments indicate that the intraadrenal LIF/LIF-R system may participate in regulating adrenal steroidogenesis.

Adrenal Cortex↗
PubMed2000

The adhesion molecule CEACAM1 (CD66a, C-CAM, BGP) is specifically expressed by the extravillous intermediate trophoblast.

CEACAM1 (CD66a, C-CAM, BGP) is an adhesion molecule of the carcinoembryonic antigen family which has been shown to be normally expressed at the apical pole of epithelial cells, including the apical pole of endometrial surface and glandular epithelia. The purpose of the present study was to investigate its expression pattern at the maternal-fetal interface, and thus to determine whether CEACAM1 could be implicated in the human implantation process. For this purpose, we performed immunohistochemistry using the 4D1/C2 monoclonal antibody (mAb) as well as flow cytometry and Western blot on isolated trophoblast populations. On the maternal side of the maternal-fetal interface, CEACAM1 was present in epithelial cells of pregnancy endometrium as well as in small endometrial vessels, whereas it was absent from decidual cells. On the fetal side, CEACAM1 was strongly expressed by the extravillous (intermediate) trophoblast at the implantation site, as well as by extravillous trophoblast cells with invasive phenotype in primary culture, as shown by flow cytometry and Western blot. Expression was also observed in placental villous core vessels but was absent from both villous cyto- and syncytiotrophoblasts throughout the pregnancy. We conclude that, given its specific expression pattern, CEACAM1 can be a useful marker for extravillous intermediate trophoblast and might be functionally implicated in mediating trophoblast/endometrial and/or trophoblast/endothelial interactions during the trophoblastic invasion of the endometrium.

Antigens, CD↗
PubMed2000

Molecular mechanisms of dissociative glucocorticoid activity.

BACKGROUND: Glucocorticoids mediate their effects on target cells via transactivation and transrepression of certain target genes. While conventional glucocorticoids do not distinguish between transactivation and transrepression, new glucocoticoids should be able to dissociate these effects, thus lowering the potential of unwanted side-effects of glucocorticoids in clinical use. In this study, we developed a new experimental system to test potentially selective glucocorticoids in normal lymphocytes. MATERIALS AND METHODS: Following pretreatment with phytohaemagglutinin, normal lymphocytes were transfected, using electroporation, with pGL3 luciferase reporter vectors under the control: (1) of the human IL-2 promoter; and (2) of a glucocorticoid response element (GRE). Luciferase activity was measured in response to various steroid compounds, including the potentially dissociative glucocorticoid medroxyprogesterone acetate (MPA). RESULTS: The IL-2 promoter was induced 267.2 +/- 27.5-fold (mean +/- SD) by phorbol ester and ionomycin. In these cells, hydrocortisone and dexamethasone caused a 22.9 +/- 3.6% and a 38.4 +/- 10% reduction in luciferase activity, respectively. Under GRE control, hydrocortisone stimulated luciferase activity 6.4 +/- 0.50-fold and dexamethasone 8.2 +/- 0.4-fold. MPA-induced transrepression was 73.3 +/- 7.2% for the IL-2 promoter, and transactivation was 2.4 +/- 0.4-fold with the GRE-driven construct. The natural progestin progesterone did not have significant effects on either construct. CONCLUSIONS: This is the first system that allows efficient analysis of glucocorticoid-dependent transactivation and transrepression in normal human lymphocytes. Compared to conventional glucocorticoids, MPA can be referred to as a dissociative glucocorticoid, its transrepression/transactivation ratio being 6.6 (transrepression 1.91/transactivation 0.29), with dexamethasone being the standard (transrepression 1/transactivation 1). We conclude that MPA is a highly promising substance for the treatment of autoimmune/inflammatory diseases.

Glucocorticoids↗
PubMed2000

The peripheral CRH/urocortin system.

The hypothalmus-pituitary-adrenal (HPA) axis and the immune system communicate at multiple levels: On the one hand, immune system-derived substances, such as interleukin-1, interleukin-6, tumor necrosis factor alpha, and leukemia inhibitory factor can stimulate the HPA axis. On the other hand, HPA axis-derived substances, most importantly glucocorticoids, can modulate the immune response. Furthermore, factors that were originally thought to be restricted to the HPA axis have been found to be expressed by immune cells. Proteins belonging to the CRH (corticotropin-releasing hormone) family represent important examples of such hormones. In the early 1990s, it was shown that immunoreactive CRH was present at sites of chemically induced inflammation. Administration of anti-CRH antibodies reduced the degree of inflammation, pointing to a pro-inflammatory role of "peripheral" CRH. We and others could show that lymphocytes are one source of immunoreactive CRH; however, the antiserum used in our study as well as in previous reports crossreacted with urocortin, a newly discovered member of the CRH family. Using RT-PCR, we could clearly demonstrate that human lymphocytes expressed urocortin but not CRH mRNA. These results were confirmed by immunocytochemistry, employing urocortin- and CRH-specific antibodies, respectively. The possible functional roles of urocortin expression in the immune system are discussed.

Animals↗
PubMed1999

[Molecular and clinical endocrinology of the endometrium].

The ovarian steroid hormones estradiol and progesterone exert their effect by interacting with their intracellular receptors, which, after ligand binding translocate to the nucleus and bind to the promoter regions of target genes. The consequence is a change in the transcription rate of the target genes, followed by a change in production of the corresponding proteins. Target genes of the sexual steroid hormones include cytokines and growth factors, among them CSF-1, TGF-beta and LIF. The rhythm and activity of steroidogenesis, receptor modulation and transcription are reflected by cycle-specific proliferation and differentiation processes in the endometrium. Quantitative and/or qualitative molecular endocrinology is of increasing interest for better definition of morphological changes, although, as yet, the pathological laboratory test is of much less practical consequence than a suspicious vaginal sonography. In spite of the high standard of ultrasound techniques, however, most cases with slightly increased endometrial thickness show histologically benign changes of the endometrium rather than endometrial precancer or cancer. This is especially true for perimenopausal women with no other clinical findings. Yet, the cancer risk is increased in women under tamoxifen therapy. Hence, as a rule, these cases, when endometrial thickness exceeds 5 mm, need a diagnostic biopsy or abrasio.

Biopsy↗
PubMed1999

Strongly reduced expression of the cell cycle inhibitor p27 in endometrial neoplasia.

In the present study we investigated the expression of the cell cycle inhibitor p27 in endometrial neoplasia using immunohistochemistry with a p27-specific antibody. Expression of p27 in endometrial carcinomas was compared with expression in the normal endometrium throughout the cycle. Normal endometrial cells showed strong nuclear expression of p27. Expression was present throughout the cycle and was stronger during the secretory phase. We found strongly reduced or abolished expression of p27 in endometrial carcinoma (85.3% of cases). The 41 tumours analysed were classified according to p27 staining intensity and percentage of positive cells into the following categories of p27 expression: negative/very low (56.0%); low (29.3%); moderate (14.7%) and high (0.0%). All the p27-positive tumours were well-differentiated endometrioid carcinomas of malignancy grade G1. Comparison with the p53 status showed that all tumours with strong p53 expression had low/negative p27 staining, while those that were positive for p27 had negative/low p53 staining. Reduced or absent p27 levels were also observed by Western blot analysis both in tumour samples and in HEC-1B endometrial adenocarcinoma cells. It thus seems that p27 expression is essential for the control of normal endometrial proliferation, and reduced or absent p27 expression may be an important step in endometrial carcinogenesis.

Adenocarcinoma↗
PubMed1999

Telomerase activity in benign and malignant adrenal tumors.

Histological analysis of surgically removed adrenal masses often fails to differentiate between benign and malignant tumors. In normal cells, the telomeric ends of the chromosomes are shortened with each cell division, leading to chromosome destabilization and cellular senescence after a critical number of cell cycles. In tumor cells, telomere shortening is prevented by a specific DNA polymerase, called telomerase. In an effort to clarify the role of telomerase in the pathogenesis of adrenal tumors, and to test whether its activity could serve as marker of malignancy, we measured telomerase activity in 41 human adrenal tissue samples that were classified both by the clinical course and by histological examination. Telomerase activity was determined by TRAP ELISA and expressed as high (>50% of positive control telomerase activity), medium (31-50%), low (11-30%), very low (< or = 10%), or absent (0%). The 8 normal adrenal tissue samples showed very low levels of telomerase activity. Mean telomerase activity also very low in 3/3 incidentalomas, 6/6 Cushing adenomas, 6/6 Conn adenomas, 7/7 adrenocortical carcinomas, 8/8 benign pheochromocytomas, and 2/3 malignant pheochromocytomas. In contrast, one malignant pheochromocytoma showed high telomerase activity. These data indicate that telomerase activity may not be a suitable marker for malignancy in the adrenal gland. Our results also challenge the current dogma of close correlation between cell dedifferentiation and telomerase activity.

Adenoma↗
PubMed1999

Dissociative glucocorticoid activity of medroxyprogesterone acetate in normal human lymphocytes.

The immunosuppressive effects of glucocorticoids (GC) have led to their wide application in the treatment of inflammatory and autoimmune states. However, long term GC treatment is associated with severe side-effects. The development of agents displaying a more favorable ratio of wanted and unwanted GC effects, is, therefore, a major goal of pharmacological and clinical research. In this study, the progesterone receptor agonist medroxyprogesterone acetate (MPA), which also binds to the glucocorticoid receptor (GR), was tested with regard to its immunosuppressive properties. Using a recently established electroporation protocol, we show that MPA (but not progesterone) can suppress a human interleukin-2 (IL-2) promoter-luciferase construct to the same extent as the synthetic GC dexamethasone in normal human lymphocytes. MPA also markedly suppressed IL-2 (as well as IL-1 and IL-6) release, as assessed by specific enzyme-linked immunosorbent assays. In contrast, a highly dexamethasone-inducible glucocorticoid response element-driven promoter construct was only marginally stimulated by MPA in both normal human lymphocytes and HeLa cells. RT-PCR and Western blot analysis of normal human lymphocytes revealed that they do not express progesterone receptor messenger ribonucleic acid and protein, respectively. In contrast, the GR protein was clearly detectable in all samples and was shown to mediate the effects of MPA in transfected Jurkat T lymphoma cells. Our data indicate that 1) MPA can transrepress the human IL-2 gene in normal human lymphocytes in the absence of significant trans-activation; and 2) this effect is mediated by GR. Because of its dissociative GC activity, MPA is a highly promising substance for the treatment of inflammatory/autoimmune states.

Blotting, Western↗
PubMed1999

Reduced expression levels of the cell-cycle inhibitor p27Kip1 in human pituitary adenomas.

The molecular mechanisms leading to increased cellular proliferation rates and, thus, tumor formation in the anterior pituitary gland are poorly understood. The cyclin-dependent kinase inhibitor p27Kip1 is a key molecule regulating the G1 phase of the cell cycle in many cell types. Furthermore, it was shown that p27 knock-out mice develop pro-opiomelanocortin-positive pituitary tumors. In an effort to clarify the role of p27 in the normal and tumorous human pituitary, we studied the expression of p27 by immunohistochemistry, using a highly specific mouse monoclonal anti-human p27 antibody. Normal pituitaries and 54 pituitary adenomas (twelve somatotrope adenomas, nine prolactinomas, twelve corticotrope adenomas, three TSH-producing tumors, six gonadotrope adenomas, six null cell adenomas, and six oncocytomas) were analyzed. p27 expression was determined semiquantitatively with regard to both the percentage of positive cells and the intensity of the staining. Normal human pituitaries showed strong expression of p27 in most nuclei. In contrast, the levels of p27 were reduced in the majority of the tumors analyzed. Twenty-two tumors (six somatotrope adenomas, five prolactinomas, four corticotrope adenomas, two TSH-producing tumors, two gonadotrope adenomas, and three null cell adenomas) were completely p27-negative. In 18 tumors, p27 expression was found in < or = 10% of the cells. In the other ten tumors, 11-80% of the cells were p27-positive. In summary, we were able to demonstrate reduced expression levels of the cell-cycle inhibitor p27 in tumors derived from all pituitary cell types. Our data indicate that p27 may be an important regulator of cellular proliferation in the anterior pituitary, the underexpression of which could play a role in pituitary tumorigenesis.

Adenoma↗
PubMed1999

Relaxin-like factor (RLF): a new specific marker for Leydig cells in the ovary.

Relaxin-like factor (RLF), also known as the Leydig cell insulin-like factor (Ley-I-L), is a novel member of the insulin-IGF-relaxin family of hormones and growth factors that has recently been shown to be strongly expressed in testicular Leydig cells. Expression of the RLF peptide in the human ovary and in ovarian tumors has not been studied. In the present study, the expression of the RLF peptide in the human ovary was investigated by immunohistochemistry using a specific antibody raised against human RLF. By this method, RLF was found to be expressed in hilus (Leydig) cells and theca interna cells but absent in granulosa cells, ovarian stromal cells, and surface epithelium. RLF expression was also observed in the corpus luteum, although at a lower level than in theca cells. Thirty-seven sex cord-stromal tumors and five cases of hilar Leydig cell hyperplasia were also investigated for RLF expression. RLF was found to be strongly expressed in hilar Leydig cell hyperplasia and sex cord-stromal tumors with a component of Leydig or luteinized cells. Of the analyzed theca cell tumors, two displayed a diffuse staining pattern. As expected, RLF was not expressed in granulosa cell tumors. In conclusion, RLF appears to be a useful marker for Leydig cells in the human ovary and may be a diagnostic supplement in hyperplasias and tumors derived therefrom.

Biomarkers, Tumor↗
PubMed1998

Human lymphocytes produce urocortin, but not corticotropin-releasing hormone.

Hypothalamic corticotropin-releasing hormone (CRH) is the principal regulator of the hypothalamus-pituitary-adrenal axis in mammals. In addition, immunoreactive CRH is also present at peripheral sites, where it is thought to act as a proinflammatory peptide. However, the source of peripheral CRH has remained obscure. Human lymphocytes were shown to produce immunoreactive CRH, yet the data on CRH mRNA expression in these cells are equivocal. More recently, Vaughan et al. discovered a new member of the CRH family, termed urocortin. Urocortin was shown to act through the same receptors as CRH. The current study was designed to investigate both mRNA and protein expression of CRH and urocortin in human lymphocytes. Using a commercial CRH(1-41) radioimmunoassay, we demonstrate that normal human lymphocytes and Jurkat T lymphoma cells produce significant amounts of immunoreactive peptide. However, no CRH mRNA was detectable by RT-PCR in these cells. In contrast, a band of the correct size and sequence was amplified with urocortin-specific primers. Immunocytochemical analysis of human lymphocytes using antibodies that could distinguish between CRH and urocortin revealed significant expression of urocortin but not of CRH, consistent with our RT-PCR data. We conclude that human lymphocytes produce urocortin, but not CRH.

Cells, Cultured↗
PubMed1997

Regulation of the human interleukin-2 gene by the alpha and beta isoforms of the glucocorticoid receptor.

The immunosuppressive effects of glucocorticoids are largely due to transcriptional inhibition of immunologically relevant genes, such as the interleukin-2 (IL-2) gene. These effects are mediated by the intracellular glucocorticoid receptor (GR). In humans, alternative splicing of the GR precursor mRNA gives rise to two receptor isoforms, termed GRalpha and GRbeta. We previously demonstrated that GRbeta could antagonize GRalpha-mediated transactivation of a glucocorticoid-responsive element (GRE)-driven reporter gene in COS-7 cells. The present study was designed to analyze the roles of the two GR isoforms on glucocorticoid-mediated transrepression of the IL-2 gene. Using a recently developed transfection technique, we demonstrate that in primary human lymphocytes, stimulation of a 548 bp IL-2 promoter-luciferase reporter construct by phorbol ester and calcium ionophore is reversed by dexamethasone to a similar extent as in Jurkat T lymphoma cells transfected with a GRalpha expression vector. Transfection of a GRbeta expression vector alone did not result in IL-2 promoter repression in response to glucocorticoids. Furthermore, GRbeta did not antagonize the repressive effects of GRalpha on IL-2 promoter activity. Surprisingly, overexpression of GRbeta in Jurkat cells did not cause significant inhibition of GRalpha-induced transactivation of a GRE-dependent luciferase reporter gene either. We conclude that the transrepressive effect of glucocorticoids on IL-2 gene transcription is exclusively mediated by GRalpha. GRbeta can neither antagonize GRalpha-mediated transactivation nor transrepression in Jurkat cells, indicating a cell type-specific pattern of GRbeta-mediated antiglucocorticoid activity.

Calcium↗
PubMed1997

Inhibition of mineralocorticoid and glucocorticoid receptor function by the heat shock protein 90-binding agent geldanamycin.

The effects of mineralocorticoids and glucocorticoids are mediated by the intracellular mineralocorticoid glucocorticoid receptor (MR) and glucocorticoid receptor (GR), respectively. Several studies suggest that hormone binding and, thus, receptor activation depend on the association of both MR and GR with the 90-kDa heat shock protein (hsp 90). However, there are few reports analyzing the functional relevance of this association in vivo. The present study was designed to determine how the new hsp 90-binding agent geldanamycin, which was previously shown to disrupt the formation of steroid receptor/hsp complexes, interferes with MR- and GR-mediated transactivation in intact cells. We show that geldanamycin inhibits aldosterone-dependent transactivation of a mineralocorticoid-responsive reporter genes in a concentration-dependent manner. Similar effects were observed for the dexamethasone-activated GR. However, geldanamycin did not affect transcription from a retinoic acid-dependent reporter gene. Inhibition of GR-mediated transactivation was observed both in HeLa cells expressing endogenous GR and in COS-7 cells transfected with a GRa expression vector. Binding studies indicate that geldanamycin disrupts receptor function by reducing hormone binding affinity without lowering intracellular receptor protein levels. Our data support the current model of hsp 90-dependent steroid receptor activation. Furthermore, we show for the first time that MR function also depends on the interaction with hsp 90 in intact cells. Finally, we demonstrate that the function of endogenous is thought to keep the receptor protein in an inactive, yet ligand-activable state (9-17). Ligand binding induces a conformational change in the receptor molecule, which causes it to dissociate from the hsp complex, to translocate to the cell nucleus, and, finally, to interact with specific hormone response elements in the promoter regions of hormone-responsive genes (6-8). Both MR and GR bind as homodimers to identical palindromic sequences on the target DNA, termed glucocorticoid response elements (GREs) (18). The formation of GR/MR heterodimers has also been described (19,20) and may have profound functional consequences (21). The current model of MR and GR function holds that these receptors are unable to bind their respective hormones as long as they are not associated with the hsp complex (9-17). However, experimental support for this model is mainly based on in vitro work. There are few reports analyzing the functional relevance of GR/hsp interactions in mammalian cells. In the most recent study, Whitesell et al. showed that the hspE90-binding agent geldanamycin can specifically disrupt GR/hsp association, thus inhibiting glucocorticoid-mediated transcriptional activation (22). MR is even less well studied in this respect. To our knowledge, there have not been any data supporting a functional role for proper MR/hsp interaction in intact cells. In this study, we show for the first time that MR function depends on the interaction with hsp 90 in intact human cells. Furthermore, we demonstrate that geldanamycin inhibits GR-mediated transcriptional activation in two human cells lines, confirming the results by Whitesell et al. and extending them to transfected as opposed to endogenous GR.

Aldosterone↗