Prognostic value of total, free and lipoprotein fraction-bound plasma mitotane levels in advanced adrenocortical carcinoma: a prospective study of the ENDOCAN-COMETE-Cancer network.

PURPOSE: Mitotane is the only approved treatment for metastatic adrenocortical carcinoma (ACC). Monitoring plasma levels is recommended, but its predictive value is insufficient. METHODS: This prospective study of the French ENDOCAN-COMETE network aimed to investigate the prognostic role of plasma mitotane levels pharmacokinetics and free or bound to lipoprotein fraction measurements during six consecutive months. Lipoprotein fractions were isolated by ultracentrifugation, and mitotane level was determined by HPLC-UV. Total, free, and lipoprotein fraction bound plasma mitotane were monitored every two months for six months with morphological assessment. The primary endpoint was overall survival (OS). RESULTS: 21 patients with metastatic ACC were included. Median overall survival was 23 months. The median free mitotane level per patient was 12% (± 7%), and the majority (88%) was bound to lipoprotein fractions. Several pharmacokinetics measures of total mitotane were related to OS: first level at one month (p = 0.026), mean level (p = 0.055), and area under the curve (AUC) (p = 0.048), with higher exposure associated to longer OS. Free mitotane (not bounded) and mitotane bounded to lipoprotein subfraction added no prognostic values. The relationship between the mitotane level and OS suggested a minimum "effective" threshold of 10-15 mg/L or an area under the curve above 100 mg/L/month with no individualized maximum value. CONCLUSION: This prospective study did not identify any added prognostic value of free mitotane level over the total level. Early total mitotane level measurements (before 3-6 months) were related to OS with a higher and faster exposure related to more prolonged survival.

Expanding the phenotype of mitochondrial disease: Novel pathogenic variant in ISCA1 leading to instability of the iron-sulfur cluster in the protein.

We report a patient carrying a novel pathogenic variant p.(Tyr101Cys) in ISCA1 leading to MMDS type 5. He initially presented a psychomotor regression with loss of gait and language skills and a tetrapyramidal spastic syndrome. Biochemical analysis of patient fibroblasts revealed impaired lipoic acid synthesis and decreased activities of complex I and II of respiratory chain. While ISCA1 is involved in the mitochondrial machinery for iron-sulfur cluster biogenesis, these dysfunctions are secondary to impaired maturation of mitochondrial proteins containing the [4Fe-4S] clusters. Expression and purification of the human ISCA1 showed a decreased stability of the [2Fe-2S] cluster in the mutated protein.

[SF-1, a key player in adrenal and gonadal differentiation: implications in gonadal dysgenesis and primary ovarian insufficiency]. / SF-1, un acteur majeur de la différenciation surrénalienne et sexuelle : implications dans les dysgénésies gonadiques et l'insuffisance ovarienne prématurée.

Steroidogenic factor 1 (SF-1) gene, identified by Keith Parker in 1992, encodes for an orphan nuclear receptor, NR5A1, whose expression is detected during fetal life in adrenal and gonadal steroidogenic tissues, but also in the developing hypothalamus and in pituitary gonadotropic cells. SF-1 knock-out mouse models exhibit complete adrenal and gonadal agenesis. Human mutations of this transcription factor, were initially associated with primary adrenal failure and male gonadal dysgenesis with various degrees of under androgenization. More recently, identification of novel SF-1 mutations responsible for isolated 46, XY gonadal dysgenesis or 46, XX primary ovarian insufficiency, underscores its central role in the control and maintenance of adrenal and reproductive functions. A better understanding in the regulatory mechanisms of SF-1 signaling pathway, will open new avenues for diagnostic and therapeutic managements of sex differentiation disorders and infertilities.

Hibernoma development in transgenic mice identifies brown adipose tissue as a novel target of aldosterone action.

Aldosterone is a major regulator of salt balance and blood pressure, exerting its effects via the mineralocorticoid receptor (MR). To analyze the regulatory mechanisms controlling tissue-specific expression of the human MR (hMR) in vivo, we have developed transgenic mouse models expressing the SV40 large T antigen (TAg) under the control of each of the two promoters of the hMR gene (P1 or P2). Unexpectedly, all five P1-TAg founder animals died prematurely from voluminous malignant liposarcomas originating from brown adipose tissue, as evidenced by the expression of the mitochondrial uncoupling protein ucp1, indicating that the proximal P1 promoter was transcriptionally active in brown adipocytes. No such hibernoma occurred in P2-TAg transgenic mice. Appropriate tissue-specific usage of P1 promoter sequences was confirmed by demonstrating the presence of endogenous MR in both neoplastic and normal brown adipose tissue. Several cell lines were derived from hibernomas; among them, the T37i cells can undergo terminal differentiation into brown adipocytes, which remain capable of expressing ucp1 upon adrenergic or retinoic acid stimulation. These cells possess endogenous functional MR, thus providing a new model to explore molecular mechanisms of mineralocorticoid action. Our data broaden the known functions of aldosterone and suggest a potential role for MR in adipocyte differentiation and regulation of thermogenesis.

Nouveautés dans l'hyperplasie congénitale des surrénales: New insights in congenital adrenal hyperplasia.

Congenital adrenal hyperplasia is an autosomal recessive disease due to functional abnormalities of adrenal steroid enzymes. The most common form of the disease is due to a 21-hydroxylase deficiency. The classical forms (most severe) are characterized by a deficiency in cortisol and sometimes in aldosterone, which may compromise the vital prognosis of neonates, and by an increase in androgen synthesis, leading to the virilization of girls' external genitalia at birth, followed by clinical signs of hyperandrogenism during childhood and adolescence. Neonatal screening has improved management and reduced morbidity and mortality in the neonatal period, but its performance could be broadly optimised by adjusting the assay techniques or the biomarkers used. The genetic diagnosis is difficult owing to the large genetic heterogeneity of the 6p21.3 region, which contains the CYP21A2 gene, especially with respect to the use of new-generation techniques of sequencing. Prenatal diagnosis is now possible as early as 6 weeks of gestation, but prenatal treatment remains controversial, awaiting results from prospective cohorts evaluating its long-term impact. Since conventional therapies have limitations, new therapies are currently being developed to allow better control of androgen synthesis and a substitutive treatment that respects the physiological rhythm of cortisol secretion, which would limit the development of long-term complications.

Résistances aux hormones stéroïdes : physiologie et pathologie: Pathophysiology of Steroid Resistance Syndrome.

Steroid resistance syndrome (mineralocorticoids, glucocorticoids, estrogens, androgens) is a rare clinical disorder, androgen insensitivity syndrome being the most commonly described. Resistance syndromes are characterized by elevated steroid hormone levels, secondary to an impaired signal transduction and a lack of negative feedback, without any specific clinical signs of steroid excess. In most cases, steroid hormone resistance is generally caused by steroid receptor mutations. Several nonsense and missense mutations or deletions have already been described for all steroid receptors, except for the progesterone receptor. The number of reported mutations is extremely variable, depending on the steroid receptor, ranging from 3 for the estrogen receptor to more than 500 for the androgen receptor. Loss of function of others factors implicated on the steroid hormone signaling pathway, such as molecular chaperones or coactivators, might also be involved in the pathogenesis of steroid resistance. Therapy is essentially symptomatic, there is currently no pharmacological strategy enabling restoration of the impaired hormone signaling pathway. Identification of steroid resistance syndrome and their characterization should allow a better understanding of underlying molecular defects, opening new avenues for the development of novel alternative options for the disease management.

Characterization of the human mineralocorticoid receptor gene 5'-regulatory region: evidence for differential hormonal regulation of two alternative promoters via nonclassical mechanisms.

The mineralocorticoid receptor (MR) is a ligand-dependent transcription factor involved in the regulation of sodium homeostasis. Two distinct mRNA isoforms of the human MR (hMR) differing in their untranslated 5'-ends have recently been identified, suggesting the existence of alternative promoters. To eludicate the regulatory mechanisms controlling hMR gene expression, we have isolated and characterized approximately 15 kb of hMR 5'-flanking region. Various deletion mutants of regions located immediately upstream of the untranslated exons 1 alpha and 1 beta (P1: 1 kb and P2: 1.7 kb, respectively) were inserted into a luciferase reporter gene and used in transient transfection experiments in CV-1 and human differentiated renal H5 cells. Both regions were shown to possess significant functional promoter activity, more pronounced in renal cells, although P1 directed higher levels of basal transcription. Cotransfection experiments with hMR or human glucocorticoid receptor (hGR) revealed that, while both promoters were glucocorticoid inducible, only the distal P2 promoter was stimulated by aldosterone in a dose- and hMR-dependent manner. Furthermore, we demonstrate that hMR and hGR are able to synergistically activate the P2 promoter, consistent with cooperativity between the two transduction pathways. Mineralocorticoid induction was localized to a region between -318 and +123 bp of P2. This region does not contain any consensus hormone responsive element, and direct binding of hMR to this DNA sequence was not observed, indicating that mineralocorticoid-induced transcriptional enhancement is mediated by nonclassical mechanisms. On the other hand, Sp1 and AP-2 bind to definite sequences on both promoters, suggesting that they represent important regulators of hMR promoter activity. Our results indicate that hMR gene expression is under the control of complex regulatory mechanisms involving alternative promoters and differential hormonal control, which might allow tissue-specific modulation of aldosterone action.

Modulation of human mineralocorticoid receptor function by protein kinase A.

The mineralocorticoid receptor (MR) acts as a ligand-dependent transcription factor modulating specific gene expression in sodium-transporting epithelia. Physiological evidence suggest a cross-talk between the cAMP- and aldosterone-signaling pathways. We provide evidence that protein kinase A (PKA), a major mediator of signal transduction pathways, modulates transcriptional activity of the human MR (hMR). Using transient transfection assays in HepG2 cells, we show that 8-bromo-cAMP, a protein kinase A activator, stimulates glucocorticoid response element (GRE)-containing promoters in a ligand-independent manner. This effect was strictly MR dependent since no activation of the reporter gene was observed in the absence of cotransfected hMR expression plasmid. Furthermore, a synergistic activation was achieved when cells were treated with both aldosterone and cAMP. This synergistic effect was also observed in the CV1 and the stable hMR-expressing M cells but was dependent on the promoter used. In particular, synergism was less pronounced in promoters containing several GREs. We show that (protein kinase-inhibiting peptide (PKI), the peptide inhibitor of PKA, prevented both cAMP and aldosterone induction, which indicates that a functional cAMP pathway is required for stimulation of transcription by aldosterone. Using MR-enriched baculovirus extracts in gel shift assays, we have shown that the binding of the MR to a GRE-containing oligonucleotide was enhanced by PKA. Increased DNA binding of hMR is likely to reflect an increase in the number of active receptors, as measured by Scatchard analysis. Using a truncated MR, we show that the N-terminal domain is required for the effect. Finally, the N-terminal truncated MR was not directly phosphorylated by PKA in vitro. We conclude that PKA acts indirectly, probably by relieving the effect of an MR repressor.

A new human MR splice variant is a ligand-independent transactivator modulating corticosteroid action.

Aldosterone effects are mediated by the MR, which possesses the same affinity for mineralocorticoids and glucocorticoids. In addition to the existence of mechanisms regulating intracellular hormone availability, we searched for human MR splice variants involved in tissue-specific corticosteroid function. We have identified a new human MR isoform, hMRDelta5,6, resulting from an alternative splicing event skipping exons 5 and 6 of the human MR gene. hMRDelta5,6 mRNAs are expressed in several human tissues at different levels compared with wild-type human MR, as shown by real time PCR. Introduction of a premature stop codon results in a 75-kDa protein lacking the entire hinge region and ligand binding domain. Interestingly, hMRDelta5,6 is still capable of binding to DNA and acts as a ligand-independent transactivator, with maximal transcriptional induction corresponding to approximately 30-40% of aldosterone-activated wild-type human MR. Coexpression of hMRDelta5,6 with human MR or human GR increases their transactivation potential at high doses of hormone. Finally, hMRDelta5,6 is able to recruit the coactivators, steroid receptor coactivator 1, receptor interacting protein 140, and transcription intermediary factor 1alpha, which enhance its transcriptional activity. Ligand-independent transactivation and enhancement of both wild-type MR and GR activities by hMRDelta5,6 suggests that this new variant might play a role in modulating corticosteroid effects in target tissues.

[Genetics of congenital lipodystrophies]. / Génétique des lipodystrophies congénitales.

Congenital lipodystrophies are heterogeneous genetic diseases, leading to the loss of adipose tissue. This loss of adipose tissue can be generalized or partial, thus defining different phenotypes. These lipodystrophies have a major metabolic impact, secondary to lipotoxicity. This lipotoxicity is responsible for insulin resistance, dyslipidemia and hepatic steatosis. The severity of the metabolic impact correlates with the severity of the loss of adipose tissue. Mutations in 15 predisposition genes are currently described; BSCL2 and AGPT2 genes are the major genes in the generalized forms. On the contrary, LMNA and PPARG gene mutations are recovered in partial lipodystrophies forms. These different genes encode for proteins involved in adipocyte physiology, altering adipocyte differentiation, triglycerides synthesis and lysis or playing a major role in the lipid droplet formation. Congenital lipodystrophies treatment is based on the management of metabolic comorbidities but recombinant leptin therapy appears to have promising results. These different points have been recently discussed during the 2015 Endocrine Society Congress, notably by S. O'Rahilly and are highlighted in this review.

The mineralocorticoid receptor discriminates aldosterone from glucocorticoids independently of the 11 beta-hydroxysteroid dehydrogenase.

To investigate the mechanisms involved in the in vivo aldosterone selectivity of the mineralocorticoid receptor (MR), we studied the respective contribution of the receptor and the enzyme 11 beta-hydroxysteroid dehydrogenase (11HSD), which converts glucocorticoids into inactive metabolites. Using a cotransfection assay in CV-1 cells, aldosterone activated mouse mammary tumor virus promoter through human MR (hMR) with an ED50 of 0.01 nM. An at least 100-fold higher concentration of cortisol (F), corticosterone (B), or dexamethasone was required to obtain half-maximum transactivation, indicating a functional preference of hMR for aldosterone over glucocorticoids. The catalytic activity of 11HSD was analyzed using HPLC by measuring the tritiated metabolites produced in CV-1 and COS cells. Both cell types displayed a significant dehydrogenase activity (20 fmol/10 min.10(3) cells) inhibitable by carbenoxolone, but no detectable reductase activity. In this model, B was more rapidly metabolized than F. Carbenoxolone treatment of hMR-transfected CV-1 cells did not result in a shift of the dose-response transactivation curves of B and F toward lower concentrations, ruling out the implication of 11HSD in the aldosterone MR selectivity of these conditions. Despite similar affinity constants of aldosterone and glucocorticoids for the hMR, kinetic experiments showed that the off-rate of aldosterone from hMR was 5 times lower than that of glucocorticoids, pointing to an intrinsic discriminating property of the receptor. Therefore, we propose that in addition to 11HSD, MR plays an active role in the mechanism of aldosterone selectivity.

Tissue-specific expression of alpha and beta messenger ribonucleic acid isoforms of the human mineralocorticoid receptor in normal and pathological states.

Expression of the mineralocorticoid receptor (MR) is restricted to some sodium-transporting epithelia and a few nonepithelial target tissues. Determination of the genomic structure of the human MR (hMR) revealed two different untranslated exons (1alpha and 1beta), which splice alternatively into the common exon 2, giving rise to two hMR mRNA isoforms (hMR alpha and hMR beta). We have investigated expression of hMR transcripts in renal, cardiac, skin, and colonic tissue samples by in situ hybridization with exon 1alpha and 1beta specific riboprobes, using an exon 2 probe as internal control. Specific signals for either exon 1alpha- and 1beta-containing mRNAs were detected in typically hMR-expressing cells in all tissues analyzed. hMR alpha and hMR beta were present in distal tubules of the kidney, in cardiomyocytes, in enterocytes of the colonic mucosa, and in keratinocytes and sweat glands. Interestingly, although both isoforms appear to be expressed at approximately the same level, the relative abundance of each message compared with that of exon 2-containing mRNA strikingly differs among aldosterone target tissues, suggesting the possibility of other tissue-specific transcripts originating from alternative splicing. Finally, functional hypermineralocorticism was associated with reduced expression of hMR beta in sweat glands of two patients affected by Conn's and Liddle's syndrome, whereas normal levels of hMR isoforms were found in one case of pseudohypoaldosteronism. Altogether, our results indicate a differential, tissue-specific expression of hMR mRNA isoforms, hMR beta being down-regulated in situations of positive sodium balance, independently of aldosterone levels.

Human skin as target for aldosterone: coexpression of mineralocorticoid receptors and 11 beta-hydroxysteroid dehydrogenase.

The expression of mineralocorticoid receptors (MR) and 11 beta-hydroxysteroid dehydrogenase (11HSD) activity has been investigated in the epidermis and appendages of the human skin. Aldosterone binds to MR and regulates sodium transport in tight epithelia. Mineralocorticoid selectivity is achieved through coexpression of MR and 11HSD, which prevents permanent MR occupancy by glucocorticoids. Some forms of hypertension may involve abnormalities of MR and/or 11HSD. However, their direct assessment in humans remains difficult in the kidney or colon. This led us to explore this system in human skin easily accessible to biopsy. In situ hybridization with specific MR complementary ribonucleic acid probes and immunohistochemistry using three different anti-MR antibodies showed that MR was expressed at both the messenger ribonucleic acid and protein levels in the keratinocytes of the epidermis, in the sweat and sebaceous glands, and in the hair follicles. A significant 11HSD activity was found in isolated sweat gland ducts (5 fmol/3-mm length.10-min incubation with 10 nmol/L corticosterone as substrate) and was very low in the epidermis. In both structures, reductase activity was 10 times lower than that of dehydrogenase. Studies on the cofactor specificity of the enzyme showed a nicotinamide-adenine-dinucleotide preference in sweat glands, contrasting with a nicotinamide-adenine-dinucleotide phosphate dependence in epidermis. Human skin appears as a new target for aldosterone because it coexpresses MR and 11HSD. Our findings present the possibility to explore the functionality of the MR system in human tissue and its implications in various physiopathological situations.

Functional importance of Myc-associated zinc finger protein for the human parathyroid hormone (PTH)/PTH-related peptide receptor-1 P2 promoter constitutive activity.

The aim of the present study was to analyze the functional importance for the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) gene P2 promoter activity of the putative proximal Myc-associated zinc finger protein (MAZ) site localized at position bp -45 to -39 bp, taking advantage of a G/A mutation identified at position -40 in the human sequence. Wild-type 'full-length' (1285P2) and truncated (760P2) promoter sequences were inserted upstream to the luciferase basic (pLucB) and enhancer (pLucE) reporter gene expression vectors. Transient transfections in osteoblast-like SaOS-2 cells and renal cells (RC.SV3A2) showed that the -40 G/A mutation significantly impaired transcriptional activity of wild-type 1285P2-pLucB and 760P2-pLucE promoter constructs. Further truncation of the P2 sequence demonstrated that the sequence -109/-37 bp was essential for promoter activity. Co-transfection with a MAZ expression vector did not modify the wild-type 1285P2-pLucB construct reporter activity but significantly increased 2-fold the mutated construction activity (P<0.05). Electrophoretic mobility shift assays using SaOS-2 nuclear extracts and a double-stranded DNA fragment encompassing the -45 to -39 putative MAZ site (ds-MAZ-oligo) disclosed two specific DNA-protein complexes. Complex II (fast moving) had a lower affinity for the mutated MAZ motif than for the wild-type MAZ motif while complex I (slow moving) had the same affinity for both wild-type or mutated MAZ sequences. Competition studies with Sp1 consensus oligonucleotide (ds-Sp1-oligo) markedly reduced complex I intensity, with a concomitant increase in that of complex II. Finally, ribonuclease protection assays showed that P2-specific PTHR1 mRNA transcript expression was significantly decreased in SaOS-2 cells transfected with ds-MAZ-oligo as compared with that for control (P<0.001) and ds-Sp1-oligo (P<0.05). Taken together, our studies suggest that the putative -45 to -39 MAZ-binding site regulates the constitutive activity of human PTHR1 P2 promoter.

Prolactin potentiates insulin-stimulated leptin expression and release from differentiated brown adipocytes.

The pituitary hormone prolactin (PRL) exerts pleiotropic effects, which are mediated by a membrane receptor (PRLR) present in numerous cell types including adipocytes. Brown adipose tissue (BAT) expresses uncoupling proteins (UCPs), involved in thermogenesis, but also secretes leptin, a key hormone involved in the control of body weight. To investigate PRL effects on BAT, we used the T37i brown adipose cell line, and demonstrated that PRLRs are expressed as a function of cell differentiation. Addition of PRL leads to activation of the JAK/STAT and MAP kinase signaling pathways, demonstrating that PRLRs are functional in these cells. Basal and catecholamine-induced UCP1 expression were not affected by PRL. However, PRL combined with insulin significantly increases leptin expression and release, indicating that PRL potentiates the stimulatory effect of insulin as revealed by the recruitment of insulin receptor substrates and the activation of phosphatidylinositol 3-kinase. To explore the in vivo physiological relevance of PRL action in BAT, we showed that leptin content was significantly increased in BAT of PRLR-null mice compared with wild-type mice, highlighting the involvement of PRL in the leptin secretion process. This study provides the first evidence for a functional link between PRL and energy balance via a cross-talk between insulin and PRL signaling pathways in brown adipocytes.

Inactivating mutations of the mineralocorticoid receptor in Type I pseudohypoaldosteronism.

Type I pseudohypoaldosteronism (PHA1) is a rare form of mineralocorticoid resistance characterized by neonatal renal salt wasting and failure to thrive. Typical biochemical features include high levels of plasma aldosterone and renin, hyponatremia and hyperkalemia. Different mutations of the human mineralocorticoid receptor (hMR) gene have been identified in subjects affected by the autosomal dominant or sporadic form of the disease. Our laboratory has investigated a large number of subjects with familial and sporadic PHA1. Several different mutations have been detected, which are localized in different coding exons of the hMR gene. These mutations either create truncated proteins, either affect specific amino acids involved in receptor function. In this paper, we review hMR mutations described to date in PHA1 and their functional characterization. We discuss the absence of mutations in some kindreds and the role of precise phenotypic and biological examination of patients to allow for identification of other genes potentially involved in the disease.

A key enzyme in the biosynthesis of neurosteroids, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD), is expressed in rat brain.

In rat brain, the presence of pregnenolone and progesterone, not attributable to peripheral glandular sources, has been demonstrated and thus the two compounds can be classified as neurosteroids. In vitro experiments have shown the conversion of pregnenolone, a 3 beta-hydroxy-delta 5-ene steroid, into progesterone, a delta 4-oxo steroid, thus demonstrating a 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) enzymatic activity. The conversion of 3 beta-hydroxy-delta 5-derivatives into the corresponding delta 4-oxo steroids by 3 beta-HSD is an essential step in the biosynthesis of all steroid hormones in endocrine glands. To date, four isoforms of 3 beta-HSD have been characterized in the rat. We report here the selective expression of a 3 beta-HSD isoform in rat brain. An in situ hybridization study, using an oligonucleotide common to the 4 known isoforms, demonstrated 3 beta-HSD mRNA in neurons of the olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, habenula, hippocampus and cerebellum. The cerebellum showed the highest level of 3 beta-HSD mRNA corresponding to a transcript of 1.8 kb. Nucleotide sequencing of PCR-amplified cDNA fragments from cerebellar mRNA indicated the expression of an isoform of 3 beta-HSD cDNA very closely related to the isoform I expressed in the adrenals and gonads. Further evidence for the expression of 3 beta-HSD gene in the brain was demonstrated utilizing anti-peptide 3 beta-HSD antibodies which revealed an immunoreactive protein of approximately 45 kDa in the cerebellum. Our results demonstrate for the first time the expression of the enzyme 3 beta-HSD in the brain, at both the mRNA and protein levels. Since several neuroactive neurosteroids are substrates or products of the 3 beta-HSD enzymatic activity, our findings offer new possibilities to study the regulatory mechanisms governing their biosynthesis in the brain.

Differences between aldosterone and its antagonists in binding kinetics and ligand-induced hsp90 release from mineralocorticosteroid receptor.

We have previously reported that mineralocorticosteroid receptor (MR) is a 8-9 S heterooligomeric complex that includes the 90 kDa heat shock protein (hsp90). To elucidate how antagonist-receptor complexes are biologically inactive in terms of transcriptional regulation, we analyzed the binding of mineralocorticosteroid agonists and antagonists with MR and the ligand-induced transformation of its heterooligomeric structure. This study was performed in the cytosol of adrenalectomized rat kidney and of COS cells transiently transfected with human MR cDNA. Although aldosterone antagonists (SC9420 and RU26752) bind MR with the same affinity as aldosterone, they dissociate much more rapidly from the 8-9 S form of both rat and human MR than does aldosterone. Using sedimentation gradient analysis, we showed that the interaction between hsp90 and the steroid binding subunit of MR is highly dependent upon the nature of the steroid ligand since the binding of aldosterone antagonists results in an easy release of hsp90. We propose that both rapid dissociation of ligand and weakened hsp90-receptor interaction play a key role in the mechanism of mineralocorticosteroid antagonism. In the COS cell model, cortisol, described as a weak mineralocorticosteroid agonist, dissociates also more rapidly from human MR than does aldosterone. Our results suggest that ligand binding kinetics and ligand dependent modification in receptor structure are important modulators of MR function as a transcriptional regulatory factor.

A novel monoclonal anti-rabbit hsp90 antibody: usefulness for studies on hsp90-steroid receptor interaction.

The M(r) 90,000 protein associated with steroid receptors in their non-transformed state has been identified as a heat shock protein (hsp90) but the relationship between hsp90 binding and receptor function is still poorly understood. In this work, we have obtained and characterized one monoclonal anti-rabbit hsp90 antibody (7C10), among more than 2000 wells plated. This antibody was able to complex both free and rabbit uterine progesterone receptor-associated hsp90 as demonstrated by sedimentation analysis on sucrose gradients. As assessed by ELISA, 7C10 displayed a high binding affinity for hsp90 (approximately 4 nM). A standardized and specific competitive binding assay was developed for accurate quantification of hsp90 in rabbit tissues including reticulocyte lysate. 7C10 also permitted immunolocalization of hsp90 in various rabbit tissues. In Western blot, the monoclonal antibody recognized a single polypeptide band of M(r) approximately 90,000 in crude or purified rabbit preparations but failed to cross-react with any other mammalian or avian hsp90. These findings suggest that hsp90, a highly conserved protein, is a weak immunogen and elicits a strict species specific immunological response. Owing to its high affinity and specificity for rabbit hsp90, the monoclonal antibody 7C10 was used for purification and total depletion of hsp90 from the reticulocyte lysate, an efficient system for in vitro receptor translation and reconstitution studies. Thus, 7C10 represents a new powerful tool to further investigate the importance of hsp90 in steroid hormone receptor function.

A monoclonal anti-idiotypic 'internal image' antibody that recognizes the A1 adenosine receptor potentiates the alpha 1-adrenergic activation of phospholipase C in primary cultures of mouse striatal astrocytes.

To determine which subtype of adenosine receptor mediates the potentiating effect of 2-chloroadenosine on the noradrenaline-induced inositol-phosphate formation, we used the monoclonal anti-idiotypic antibody AA1 that acts as an 'internal image' of adenosine and specifically recognizes the A1 adenosine receptor. In cultured mouse striatal astrocytes, AA1 increased the noradrenaline-evoked inositol phosphate (IP) accumulation, thus demonstrating a biological activity of an anti-idiotypic antibody. This effect was inhibited by PACPX, a selective A1 antagonist. Inhibitors of phospholipase A2 activity prevented the potentiation. These results establish the involvement of A1 adenosine receptors in the modulation of phospholipase C activity.