Peter J. Fuller

Mechanisms of Mineralocorticoid Action

Sodium transport in epithelial tissues is regulated by the physiological mineralocorticoid aldosterone. The response to aldosterone is mediated by the mineralocorticoid receptor (MR), for which the crystal structure of the ligand-binding domain has recently been established. The classical mode of action for this receptor involves the regulation of gene transcription. Several genes have now been shown to be regulated by aldosterone in epithelial tissues. Of these, the best characterized is serum- and glucocorticoid-regulated kinase, which increases sodium influx through the epithelial sodium channel. Turnover of these channels in the cell membrane is mediated by Nedd4–2, a ubiquitin protein ligase; serum- and glucocorticoid-regulated kinase interacts with and phosphorylates Nedd4–2, thereby rendering it unable to bind the sodium channels. In nonepithelial tissues, particularly the cardiovascular system, aldosterone also has direct effects, activating an inflammatory cascade, leading to cardiac fibrosis. A critical role for the MR in cardiovascular disease has now been demonstrated by the beneficial response to MR blockade in 2 large clinical trials in patients with cardiac failure. It is these nonepithelial actions of MR activation that need to be exploited for the development of antagonists that target the cardiovascular system while avoiding the undesirable side effects of renal MR blockade.

Deletion of Mineralocorticoid Receptors From Macrophages Protects Against Deoxycorticosterone/Salt-Induced Cardiac Fibrosis and Increased Blood Pressure

Increased mineralocorticoid levels plus high salt promote vascular inflammation and cardiac tissue remodeling. Mineralocorticoid receptors are expressed in many cell types of the cardiovascular system, including monocytes/macrophages and other inflammatory cell types. Although mineralocorticoid receptors are expressed in monocytes/macrophages, their role in regulating macrophage function to date has not been investigated. We, thus, used the Cre/LoxP-recombination system to selectively delete mineralocorticoid receptors from monocytes/macrophages with the lysozyme M promoter used to drive Cre expression (MRflox/flox/LysMCre/− mice). Male mice from each genotype (MRflox/flox or wild-type and MRflox/flox/LysMCre/− mice) were uninephrectomized, given 0.9% NaCl solution to drink, and treated for 8 days or 8 weeks with either vehicle (n=10) or deoxycorticosterone (n=10). Equivalent tissue macrophage numbers were seen for deoxycorticosterone treatment of each genotype at 8 days; in contrast, plasminogen activator inhibitor type 1 and NAD(P)H oxidase subunit 2 levels were increased in wild-type but not in MRflox/flox/LysMCre/− mice given deoxycorticosterone. Baseline expression of other inflammatory genes was reduced in MRflox/flox/LysMCre/− mice compared with wild-type mice. At 8 weeks, deoxycorticosterone-induced macrophage recruitment and connective tissue growth factor and plasminogen activator inhibitor type 1 mRNA levels were similar for each genotype; in contrast, MRflox/flox/LysMCre/− mice showed no increase in cardiac fibrosis or blood pressure, as was seen in wild-type mice at 8 weeks. These data demonstrate the following points: (1) mineralocorticoid receptor signaling regulates basal monocyte/macrophage function; (2) macrophage recruitment is not altered by loss of mineralocorticoid receptor signaling in these cells; and (3) a novel and significant role is seen for macrophage signaling in the regulation of cardiac remodeling and systolic blood pressure in the deoxycorticosterone/salt model.

The steroid receptor superfamily: mechanisms of diversity.

The steroid receptor superfamily of ligand‐dependent transcription factors is characterized by marked conservation of both structure and function between the various receptors. Despite their well‐documented extensive similarities, these receptors respond to a diverse range of ligands, which results in an even more impressive diversity of function. A variety of strategies is used at each point in the pathway from ligand binding to gene expression to achieve this diversity. The nature of the ligand is important as are the tissue‐specific patterns of receptor gene expression, the presence of binding proteins, and the effects of cell‐ or tissue‐specific ligand‐modifying enzymes. Once bound to the receptor, the nature of which may vary as a result of either differential splicing or gene duplication yielding multiple isoforms, the activated receptor may form hetero‐ or homodimers. A complex interplay then occurs between the receptor dimer, other nuclear proteins, the response element, and the promoter complex to regulate gene expression. These elements may vary as a function of the cell type, other stimuli, and the context and sequence of the response element (or elements) in a given gene. By these mechanisms diversity may even be achieved for a given ligand, receptor subtype, gene, or cell. The observations may help to explain certain phenomena in hormone biology that are difficult to reconcile with the previous, simple, univariant model of steroid hormone action.—Fuller, P. J. The steroid receptor superfamily: mechanisms of diversity. FASEB J. 5: 3092‐3099; 1991.

Identification of a splice variant of the rat estrogen receptor β gene

Recently a second estrogen receptor termed estrogen receptor β (ERβ) has been cloned and characterized, and shown to be expressed at the highest levels in ovarian granulosa cells and prostatic epithelium. In the course of amplifying a region of the ligand-binding domain of the rat ERβ cDNA we identified a second, larger transcript which appears to arise through differential splicing. The second isoform has 54 nucleotides inserted after position 1372 encoding 18 additional amino acids. Both isoforms are expressed at similar relative abundance in a range of tissues.

Tissue distribution of rat glucagon receptor and GLP-1 receptor gene expression.

The regulation of glucose metabolism by glucagon and GLP-1 is well established, but novel functions for these and other proglucagon-derived peptides are less well defined. This paper highlights the diversity of both GLP-1 and glucagon activity by studying the tissue distribution of glucagon and GLP-1 receptor gene expression by both Southern blot analysis of RT-PCR products and nuclease protection assays. By Southern blot analysis of RT-PCR products, GLP-1 receptor mRNA was detected in lung, hypothalamus, hippocampus, cerebral cortex, kidney, pancreas, and throughout the gastrointestinal tract. Glucagon receptor expression was detected in liver, kidney, spleen, thymus, adrenal glands, pancreas, cerebral cortex, lung, and throughout the gastrointestinal tract. Nuclease protection assay revealed glucagon receptor expression to be highest in liver and kidney, whereas GLP-1 receptor expression was only detected by protection assay in lung, stomach, and large bowel. Despite previous evidence that other receptors for proglucagon-derived peptides may exist, no evidence of novel receptors or multiple isoforms of the glucagon and GLP-1 receptors was found, indicating that the two cloned receptors may mediate all the effects of proglucagon-derived peptides, or that novel receptors may share less homology with the glucagon and GLP-1 receptors than previously anticipated.

6 Mineralocorticoid resistance

Summary Mineralocorticoid resistance, or pseudohypoaldosteronism (PHA), is a rare cause of salt wasting in young children. It may be inherited as an autosomal dominant or recessive trait, it may occur sporadically or, rarely, it may develop secondary to other conditions. It is characterized by episodes of dehydration and hyponatraemia in the face of high aldosterone levels. In most cases, after a short period of salt supplementation no further ill effects are experienced. The condition is of great interest because it provides insights into both the mechanisms by which salt and water balance are controlled and the actions of aldosterone. This article reviews the normal physiology of aldosterone, with particular reference to its biosynthesis and its actions in specific target tissues. Current knowledge regarding the molecular mechanisms involved in aldosterone action is discussed in some detail. The clinical features of PHA are reviewed and diagnostic issues and clinical management considered. Finally, current views regarding the pathophysiology of the condition are presented. Here, considerable uncertainty remains. Whilst in many cases of PHA there is greatly reduced binding of aldosterone to its receptor, the underlying abnormality is yet to be identified; in particular, in spite of strong reasons for suspecting a defect or defects in the mineralocorticoid receptor, there is so far no direct evidence to support this hypothesis. The article concludes with a discussion of other possible explanations for the underlying abnormality in PHA.

Rapid upregulation of serum and glucocorticoid-regulated kinase (sgk) gene expression by corticosteroids in vivo

The molecular mechanisms by which corticosteroids regulate epithelial sodium transport remain to be fully elucidated. Expression of the serum and glucocorticoid-regulated kinase (sgk) has recently been reported to be regulated acutely by corticosteroids in the amphibian A6 cell line and in cortical collecting tubule cells in vitro. In order to extend this observation to a mammalian system in vivo, the acute response of the sgk gene to a single parenteral dose of aldosterone or dexamethasone was examined in the rat kidney and distal colon. The sgk mRNA levels were significantly elevated by both steroids by 30 min in the distal colon, reaching a peak at 2 h. A more modest increase in sgk mRNA levels was also seen in the kidney in response to both steroids. In both tissues, sgk mRNA has a very short half-life. As for other corticosteroid-regulated genes, the response appears to be mediated by both the mineralocorticoid and glucocorticoid receptors. The response to aldosterone in the distal colon in the presence of cycloheximide was superinduced, strongly suggesting that this is a primary response. The responses to both adrenalectomy and carbenoxolone sodium treatment suggest that the observed responses to corticosteroids can occur in the physiological range of endogenous circulating corticosteroids. These studies provide strong evidence that sgk is an aldosterone-induced gene in vivo in a mammalian system.

The FOXL2 C134W mutation is characteristic of adult granulosa cell tumors of the ovary.

Granulosa cell tumors of the ovary represent ∼5% of malignant ovarian cancers. It has recently been reported that 95–97% of adult granulosa cell tumors carry a unique somatic mutation in the FOXL2 gene. We undertook this study to verify the presence of the FOXL2 Cys134Trp mutation in two geographically independent cohorts of granulosa cell tumors and to examine the expression pattern of FOXL2 in these tumors. A total of 56 tumors with the histological diagnosis of adult granulosa cell tumor from two centers, Melbourne and Helsinki, were examined for the presence of the mutation using direct sequence analysis. Two granulosa cell tumor-derived cell lines, COV434 and KGN, three juvenile granulosa cell tumors and control tissues were also examined. The expression of the FOXL2 gene was determined using quantitative RT-PCR and/or immunohistochemistry. We found that 52 of the 56 adult granulosa cell tumors harbor the mutation, of which three were hemi/homozygous. Of the four cases with wild-type FOXL2 sequence, reappraisal suggests that three may have been misclassified at primary diagnosis. The KGN cells were heterozygous for the mutation, whereas the COV434 cells had a wild-type FOXL2 genotype. The expression levels of FOXL2 were similar across the adult granulosa cell tumors and the normal ovary controls; one mutation-negative granulosa cell tumor had high FOXL2 mRNA levels, whereas the COV434 cells and two of the three juvenile granulosa cell tumors lacked the expression of FOXL2. Our data provide confirmation of the frequent presence of the FOXL2 C134W mutation in adult granulosa cell tumors and demonstrate that the mutation is not associated with altered FOXL2 expression. The mutation analysis may be a useful tool to differentiate particularly between cell-rich diffuse granulosa cell tumors and mitotically active sex cord-stromal tumors. This unique FOXL2 mutation appears to be characteristic of adult granulosa cell tumors.

Molecular Pathogenesis of Granulosa Cell Tumors of the Ovary

Granulosa cell tumors of the ovary (GCT) comprise a distinct subset of ovarian cancers that account for approximately 5% of all ovarian malignancies. They are thought to arise from normal proliferating granulosa cells of the late preovulatory follicle and exhibit many morphological and biochemical features of these cells. GCT are distinct from other ovarian carcinomas in their hormonal activity; their ability to secrete estrogen, inhibin, and Müllerian inhibiting substance accounts for some of the clinical manifestations of the disease and also provides useful tumor markers for disease surveillance. Although considered to be of low malignant potential, GCT are commonly associated with slow, indolent disease progression, and frequent yet long delays to tumor recurrence are characteristic of this disease. Unlike the more intensely investigated epithelial ovarian tumors, relatively little is known about the molecular and genetic changes that give rise to GCT. To date, many investigations have centered around pathways known to be involved in normal granulosa cell proliferation, including those activated by FSH receptor stimulation. Most recently, the finding that approximately 97% of adult GCT harbor a somatic missense mutation in the FOXL2 gene (c.402C→G; p.C134W) represents an exciting advancement in the field of GCT research. The high frequency with which the mutation occurs in adult GCT, along with its absence from juvenile GCT and other human malignancies is suggestive of an oncogenic or gain-of-function mutation and, indeed, that the mutation is pathognomonic for adult GCT. In this review, we explore the implications of this finding and the most recent work characterizing molecular pathways of potential pathogenetic significance in GCT.

Interdomain interactions in the mineralocorticoid receptor

The potential for interaction between the N-terminal domain and the C-terminal region (hinge and ligand-binding domain) of the mineralocorticoid receptor (MR) was examined using the mammalian-2-hybrid assay. The MR C-terminal region was fused to the GAL4 DNA-binding domain (GAL4-MRC). To examine if the AF-2 is involved in the interaction, as has been reported for other steroid hormone receptors, it was inactivated by point mutation (E962A). The N-terminal domain was fused to the VP16 transactivation domain (VP16-MRNT). In the mammalian-2-hybrid assay both GAL4-MRC and GAL4-MRC(E962A) interact with VP16-MRNT in an aldosterone-dependent manner. The GAL4-MRC(E962A) construct was used in subsequent experiments to examine the AF-2-independent N/C-interaction. The MR antagonist spironolactone inhibits the aldosterone-mediated association of the two domains. GAL4-MRC(E962A) interacts weakly with the GR or AR N-terminal domains in the presence of aldosterone. No dimerization between GAL4-MRC(E962A) and VP16-MRC is observed. Interestingly, cortisol produces a much weaker N/C-interaction than aldosterone, and it is possible that the N/C-interaction may contribute to observed functional differences in the MR bound to the two ligands.