Specificity in mineralocorticoid versus glucocorticoid action.

The mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) share considerable structural and functional homology. Overlapping effects on epithelial sodium transport are observed in vivo; in vitro, both are able to bind and transactivate through a common hormone response element. This has led several investigators to suggest that specificity is conferred primarily by prereceptor mechanisms, and we have addressed this question using both in vitro and in vivo approaches. Although the MR has been regarded as less transcriptionally active than the GR in vitro, significant differences are observed when epithelial rather than fibroblast cell lines are used. These differences are mediated by the N-termini of the receptors. Activation of intracellular signaling pathways differentially modulates MR- versus GR-mediated transactivation. Although these studies identify mechanisms by which specificity may be achieved, they do not prove that this occurs in vivo. Such studies have been limited by an absence of MR-regulated genes. Known candidate aldosterone-responsive genes have been examined in the rat distal colon; the time course and the specificity of the response to a single parenteral dose of corticosteroid has been characterized. The epithelial sodium channel beta and gamma subunit genes are both up-regulated within 60 minutes by either MR or GR activation. Similar responses are observed for the serum and glucocorticoid-regulated kinase and channel-inducing factor genes. All four genes show clear and rapid up-regulation of their mRNA levels by aldosterone, which is paralleled by GR-mediated up-regulation of expression. While they are indeed aldosterone-responsive genes, genes that are uniquely aldosterone-regulated remain to be identified.

Intracellular signaling pathways confer specificity of transactivation by mineralocorticoid and glucocorticoid receptors.

The glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) bind similar ligands and target genes in vitro yet have distinct roles in vivo. With a single exception, known mechanisms conferring specificity have been limited to prereceptor mechanisms. These alone cannot account for specificity, particularly at a transcriptional level. These studies aimed to determine whether receptor-specific transcriptional regulation via physiological modulators of cellular signaling pathways, and MR-, as well as GR-specific interactions, could be demonstrated. By comparing modulation of GR- and MR-mediated transactivation in renal LLC-PK1 cells, we have identified several activators of intracellular signaling pathways that discriminate between the GR and the MR and demonstrate that differential regulation occurs at relatively specific points in the signaling pathway. The phosphatase inhibitor, okadaic acid, and the protein kinase G activator, sodium nitroprusside, stimulate only GR-mediated transactivation, in contrast to modulators of other protein kinase pathways that act in parallel on both receptors. The GR-specific effect of okadaic acid is observed only at doses where both phosphatases 1 and 2A are inhibited. MR-specific modulators include a centrally active alpha-2 adrenergic agonist and the thyroid receptor. Comparison of the interaction between the thyroid receptor and the GR, or the MR, distinguish two types of repression, only one of which is receptor-specific. These studies identify several signal transduction pathways that can differentially activate either the MR or the GR at a transcriptional level and might play physiological roles in conferring MR- or GR-specific regulation.

Differential expression of nuclear 11beta-hydroxysteroid dehydrogenase type 2 in mineralocorticoid receptor positive and negative tissues.

Corticosteroid hormone action is controlled at a pre-receptor level by the activity of two isoforms of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), catalyzing the interconversion of hormonally active cortisol to inactive cortisone. In particular 11beta-HSD2 protects the mineralocorticoid receptor (MR) from glucocorticoid excess, enabling aldosterone to interact with the MR. We have analyzed the subcellular localization of 11beta-HSD2 in relation to the expression of the MR in human colon and placenta. 3H-aldosterone binding studies confirmed expression of the MR in human colon but not term placental trophoblast. Enzyme activity studies and Western blot analyses carried out on subcellular fractions confirmed the presence of 11beta-HSD2 in microsomes. In colon, but not placenta, 11beta-HSD2 was also localized to the microsome-free, nuclear fraction. Protection upon the MR by 11beta-HSD2 in "classical" mineralocorticoid target tissues such as colon can be subserved at both a nuclear and extra-nuclear level. Tissue specific factors are responsible for the subcellular localization of 11beta-HSD2 and we postulate that one such factor may be the MR itself.

Determinants of specificity of transactivation by the mineralocorticoid or glucocorticoid receptor.

Glucocorticoids and mineralocorticoids have distinct in vivo roles despite close structural homology and similarities in vitro. Known mechanisms of specificity focus on factors extrinsic to the receptor; interactions that directly regulate the receptor to confer specificity are less well understood, particularly for the mineralocorticoid receptor (MR). To examine relative MR vs. glucocorticoid receptor (GR) function in a more physiological context, we compared transactivation by GR and MR in the standard experimental fibroblast CV-1 cell line, the renal epithelial LLC-PK1 line, and neuronal medullary raphe RN33B cells. Maximal transactivational activity mediated by MR, relative to that mediated by GR, is enhanced in both of these cell lines and is primarily conferred by an N-terminal-mediated enhancement of the MR response. In addition, the ligand concentration required for maximal transcriptional activity of the GR varies significantly between cell lines. This is independent of binding affinity or 11beta-hydroxysteroid dehydrogenase-mediated inactivation and may contribute to in vivo tissue-specific differences in responses to the GR. Although ligand binding affinity is clearly conferred by the LBD, receptor-specific variations between cell lines in transcriptional sensitivity to ligand appear, rather, to be associated with the N-terminus. These studies demonstrate that the specificity of the MR vs. the GR response may be mediated via unique cellular factors, as well as suggesting a novel means of expanding the cellular response to cortisol.

The molecular basis of RU486 resistance in the Tammar Wallaby, Macropus eugenii.

RU486 acts as a potent anti-progestin in humans but does not antagonise progesterone action in the chicken or hamster reflecting a substitution in the ligand binding domain (LBD) of cysteine for glycine in both the chicken and the hamster progesterone receptor (PR), at the position corresponding to codon 722 of the human PR. The tammar wallaby, Macropus eugenii, is also resistant to the effects of RU486. Cloning of a partial cDNA of the PR in the tammar wallaby reveals a glycine to alanine substitution (gly 722 in the human PR), as well as a glutamine to histidine substitution two amino acids upstream of this alanine residue. Both the glycine and glutamine residues are substituted in all three resistant species. These substitutions are also found in the mineralocorticoid receptor, which also does not bind RU486, and suggest an important role for these residues in the formation of the 11-beta pocket of the receptor, which accommodates the bulky side-chains of 11-beta substituted steroids.

Detection of discrete androgen receptor epitopes in prostate cancer by immunostaining: measurement by color video image analysis.

To determine whether multiple features of immunohistochemical staining of the androgen receptor (AR) in prostate cancer could reliably predict androgen dependence, tumor biopsy specimens from 30 patients (stages A-D2) were stained using anti-peptide antibodies to the amino- and carboxyl-terminal of the AR. Measurements were made of the mean area and total amount (i.e., integrated optical density) of AR staining in at least 20 fields per section using a color video image analysis system, and the mean intensity of AR staining per cell and the percentage of AR positive tumor cells were derived. Video image analysis measurement identified quantitative differences in AR staining between the two antibodies, suggesting that this approach may provide a means of identifying receptor variants in prostate tumors. The AR staining measurements were analyzed by discriminant function analysis to assign individual cases to good and poor clinical outcome groups. AR staining features measured with a single antibody (e.g., amino-terminal) were sufficient to predict outcome following hormonal therapy in stage D2 patients (predictive value, 1.0), whereas all features of AR staining measured with both antibodies were required for the entire patient group (predictive value, 0.97). The principal discriminant in both patient groups contributing to the correct assignment of outcome was the mean intensity of AR staining per cell. These findings suggest that AR staining features measured by video image analysis have the potential to predict outcome in prostate cancer.