Myeloid mineralocorticoid receptors contribute to skeletal muscle repair in muscular dystrophy and acute muscle injury.

Suppressing mineralocorticoid receptor (MR) activity with MR antagonists is therapeutic for chronic skeletal muscle pathology in Duchenne muscular dystrophy (DMD) mouse models. Although mechanisms underlying clinical MR antagonist efficacy for DMD cardiomyopathy and other cardiac diseases are defined, mechanisms in skeletal muscles are not fully elucidated. Myofiber MR knockout improves skeletal muscle force and a subset of dystrophic pathology. However, MR signaling in myeloid cells is known to be a major contributor to cardiac efficacy. To define contributions of myeloid MR in skeletal muscle function and disease, we performed parallel assessments of muscle pathology, cytokine levels, and myeloid cell populations resulting from myeloid MR genetic knockout in muscular dystrophy and acute muscle injury. Myeloid MR knockout led to lower levels of C-C motif chemokine receptor 2 (CCR2)-expressing macrophages, resulting in sustained myofiber damage after acute injury of normal muscle. In acute injury, myeloid MR knockout also led to increased local muscle levels of the enzyme that produces the endogenous MR agonist aldosterone, further supporting important contributions of MR signaling in normal muscle repair. In muscular dystrophy, myeloid MR knockout altered cytokine levels differentially between quadriceps and diaphragm muscles, which contain different myeloid populations. Myeloid MR knockout led to higher levels of fibrosis in dystrophic diaphragm. These results support important contributions of myeloid MR signaling to skeletal muscle repair in acute and chronic injuries and highlight the useful information gained from cell-specific genetic knockouts to delineate mechanisms of pharmacological efficacy.

ATP1A1 Mutant in Aldosterone-Producing Adenoma Leads to Cell Proliferation.

The molecular mechanisms by which ATP1A1 mutation-mediated cell proliferation or tumorigenesis in aldosterone-producing adenomas (APAs) have not been elucidated. First, we investigated whether the APA-associated ATP1A1 L104R mutation stimulated cell proliferation. Second, we aimed to clarify the molecular mechanisms by which the ATP1A1 mutation-mediated cell proliferated. We performed transcriptome analysis in APAs with ATP1A1 mutation. ATP1A1 L104R mutation were modulated in human adrenocortical carcinoma (HAC15) cells (ATP1A1-mutant cells), and we evaluated cell proliferation and molecular signaling events. Transcriptome and immunohistochemical analysis showed that Na/K-ATPase (NKA) expressions in ATP1A1 mutated APA were more abundant than those in non-functioning adrenocortical adenoma or KCNJ5 mutated APAs. The significant increase of number of cells, amount of DNA and S-phase population were shown in ATP1A1-mutant cells. Fluo-4 in ATP1A1-mutant cells were significantly increased. Low concentration of ouabain stimulated cell proliferation in ATP1A1-mutant cells. ATP1A1-mutant cells induced Src phosphorylation, and low concentration of ouabain supplementation showed further Src phosphorylation. We demonstrated that NKAs were highly expressed in ATP1A1 mutant APA, and the mutant stimulated cell proliferation and Src phosphorylation in ATP1A1-mutant cells. NKA stimulations would be a risk factor for the progression and development to an ATP1A1 mutant APA.

Immunohistochemistry of the Human Adrenal CYP11B2 in Normal Individuals and in Patients with Primary Aldosteronism.

The CYP11B2 enzyme is the terminal enzyme in the biosynthesis of aldosterone. Immunohistochemistry using antibodies against CYP11B2 defines cells of the adrenal ZG that synthesize aldosterone. CYP11B2 expression is normally stimulated by angiotensin II, but becomes autonomous in primary hyperaldosteronism, in most cases driven by recently discovered somatic mutations of ion channels or pumps. Cells expressing CYP11B2 in young normal humans form a continuous band beneath the adrenal capsule; in older individuals they form discrete clusters, aldosterone-producing cell clusters (APCC), surrounded by non-aldosterone producing cells in the outer layer of the adrenal gland. Aldosterone-producing adenomas may exhibit a uniform or heterogeneous expression of CYP11B2. APCC frequently persist in the adrenal with an aldosterone-producing adenoma suggesting autonomous CYP11B2 expression in these cells as well. This was confirmed by finding known mutations that drive aldosterone production in adenomas in the APCC of clinically normal people. Unilateral aldosteronism may also be due to multiple CYP11B2-expressing nodules of various sizes or a continuous band of hyperplastic ZG cells expressing CYP11B2. Use of CYP11B2 antibodies to identify areas for sequencing has greatly facilitated the detection of aldosterone-driving mutations.

Myeloid cells are capable of synthesizing aldosterone to exacerbate damage in muscular dystrophy.

FDA-approved mineralocorticoid receptor (MR) antagonists are used to treat heart failure. We have recently demonstrated efficacy of MR antagonists for skeletal muscles in addition to heart in Duchenne muscular dystrophy mouse models and that mineralocorticoid receptors are present and functional in skeletal muscles. The goal of this study was to elucidate the underlying mechanisms of MR antagonist efficacy on dystrophic skeletal muscles. We demonstrate for the first time that infiltrating myeloid cells clustered in damaged areas of dystrophic skeletal muscles have the capacity to produce the natural ligand of MR, aldosterone, which in excess is known to exacerbate tissue damage. Aldosterone synthase protein levels are increased in leukocytes isolated from dystrophic muscles compared with controls and local aldosterone levels in dystrophic skeletal muscles are increased, despite normal circulating levels. All genes encoding enzymes in the pathway for aldosterone synthesis are expressed in muscle-derived leukocytes. 11ß-HSD2, the enzyme that inactivates glucocorticoids to increase MR selectivity for aldosterone, is also increased in dystrophic muscle tissues. These results, together with the demonstrated preclinical efficacy of antagonists, suggest MR activation is in excess of physiological need and likely contributes to the pathology of muscular dystrophy. This study provides new mechanistic insight into the known contribution of myeloid cells to muscular dystrophy pathology. This first report of myeloid cells having the capacity to produce aldosterone may have implications for a wide variety of acute injuries and chronic diseases with inflammation where MR antagonists may be therapeutic.

Gene expression effects of glucocorticoid and mineralocorticoid receptor agonists and antagonists on normal human skeletal muscle.

Mineralocorticoid and glucocorticoid receptors are closely related steroid hormone receptors that regulate gene expression through many of the same hormone response elements. However, their transcriptional activities and effects in skeletal muscles are largely unknown. We recently identified mineralocorticoid receptors (MR) in skeletal muscles after finding that combined treatment with the angiotensin-converting enzyme inhibitor lisinopril and MR antagonist spironolactone was therapeutic in Duchenne muscular dystrophy mouse models. The glucocorticoid receptor (GR) agonist prednisolone is the current standard-of-care treatment for Duchenne muscular dystrophy because it prolongs ambulation, likely due to its anti-inflammatory effects. However, data on whether glucocorticoids have a beneficial or detrimental direct effect on skeletal muscle are controversial. Here, we begin to define the gene expression profiles in normal differentiated human skeletal muscle myotubes treated with MR and GR agonists and antagonists. The MR agonist aldosterone and GR agonist prednisolone had highly overlapping gene expression profiles, supporting the notion that prednisolone acts as both a GR and MR agonist that may have detrimental effects on skeletal muscles. Co-incubations with aldosterone plus either nonspecific or selective MR antagonists, spironolactone or eplerenone, resulted in similar numbers of gene expression changes, suggesting that both drugs can block MR activation to a similar extent. Eplerenone treatment alone decreased a number of important muscle-specific genes. This information may be used to develop biomarkers to monitor clinical efficacy of MR antagonists or GR agonists in muscular dystrophy, develop a temporally coordinated treatment with both drugs, or identify novel therapeutics with more specific downstream targets.

Mineralocorticoid receptors are present in skeletal muscle and represent a potential therapeutic target.

Early treatment with heart failure drugs lisinopril and spironolactone improves skeletal muscle pathology in Duchenne muscular dystrophy (DMD) mouse models. The angiotensin converting enzyme inhibitor lisinopril and mineralocorticoid receptor (MR) antagonist spironolactone indirectly and directly target MR. The presence and function of MR in skeletal muscle have not been explored. MR mRNA and protein are present in all tested skeletal muscles from both wild-type mice and DMD mouse models. MR expression is cell autonomous in both undifferentiated myoblasts and differentiated myotubes from mouse and human skeletal muscle cultures. To test for MR function in skeletal muscle, global gene expression analysis was conducted on human myotubes treated with MR agonist (aldosterone; EC50 1.3 nM) or antagonist (spironolactone; IC50 1.6 nM), and 53 gene expression differences were identified. Five differences were conserved in quadriceps muscles from dystrophic mice treated with spironolactone plus lisinopril (IC50 0.1 nM) compared with untreated controls. Genes down-regulated more than 2-fold by MR antagonism included FOS, ANKRD1, and GADD45B, with known roles in skeletal muscle, in addition to NPR3 and SERPINA3, bona fide targets of MR in other tissues. MR is a novel drug target in skeletal muscle and use of clinically safe antagonists may be beneficial for muscle diseases.

Third-generation Mineralocorticoid Receptor Antagonists: Why Do We Need a Fourth?

The first mineralocorticoid receptor (MR) antagonist, spironolactone, was developed almost 60 years ago to treat primary aldosteronism and pathological edema. Its use waned in part because of its lack of selectivity. Subsequently, knowledge of the scope of MR function was expanded along with clinical evidence of the therapeutic importance of MR antagonists to prevent the ravages of inappropriate MR activation. Forty-two years elapsed between the first and MR-selective second generation of MR antagonists. Fifteen years later, despite serious shortcomings of the existing antagonists, a third-generation antagonist has yet to be marketed. Progress has been slowed by the lack of appreciation of the large variety of cell types that express the MR and its diverse cell-type-specific actions, and also its unique complex interaction actions at the molecular level. New MR antagonists should preferentially target the inflammatory and fibrotic effects of MR and perhaps its excitatory effects on sympathetic nervous system, but not the renal tubular epithelium or neurons of the cortex and hippocampus. This review briefly describes efforts to develop a third-generation MR antagonist and why fourth generation antagonists and selective agonists based on structural determinants of tissue and ligand-specific MR activation should be contemplated.

mTOR Regulates Mineralocorticoid Receptor Transcriptional Activity by ULK1-Dependent and -Independent Mechanisms.

The mineralocorticoid receptor (MR) is a transcription factor for genes mediating diverse, cell-specific functions, including trophic effects as well as promoting fluid/electrolyte homeostasis. It was reported that in intercalated cells, phosphorylation of the MR at serine 843 (S843) by Unc-51-like kinase (ULK1) inhibits MR activation and that phosphorylation of ULK1 by mechanistic target of rapamycin (mTOR) inactivates ULK1, and thereby prevents MR inactivation. We extended these findings with studies in M1 mouse cortical collecting duct cells stably expressing the rat MR and a reporter gene. Pharmacological inhibition of ULK1 dose-dependently increased ligand-induced MR transactivation, while ULK1 activation had no effect. Pharmacological inhibition of mTOR and CRISPR/gRNA gene knockdown of rapamycin-sensitive adapter protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) decreased phosphorylated ULK1 and ligand-induced activation of the MR reporter gene, as well as transcription of endogenous MR-target genes. As predicted, ULK1 inhibition had no effect on aldosterone-mediated transcription in M1 cells with the mutated MR-S843A (alanine cannot be phosphorylated). In contrast, mTOR inhibition dose-dependently decreased transcription in the MR-S843A cells, though not as completely as in cells with the wild-type MR-S843. mTOR, Raptor, and Rictor coprecipitated with the MR and addition of aldosterone increased their phosphorylated, active state. These results suggest that mTOR significantly regulates MR activity in at least 2 ways: by suppressing MR inactivation by ULK1, and by a yet ill-defined mechanism that involves direct association with MR. They also provide new insights into the diverse functions of ULK1 and mTOR, 2 key enzymes that monitor the cell's energy status.

Regulation of aldosterone biosynthesis by the Kir3.4 (KCNJ5) potassium channel.

The G-protein-activated inwardly rectifying potassium channel Kir3.4 is expressed in the zona glomerulosa cell membrane and transports potassium out of the cell. Angiotensin II stimulation of aldosterone secretion is mediated, in part, by suppression of the transcription of KCNJ5, the gene coding for Kir3.4, and blocking channel activity. This results in membrane depolarization, mobilization of intracellular calcium, activation of the calcium-calmodulin pathway and increasing gene transcription of steroidogenic enzymes required for aldosterone secretion. In 40-60% of aldosterone-producing adenomas there is a somatic mutation in the region of the KCNJ5 gene that codes for the selectivity filter that decreases potassium selectivity, allowing sodium to leak into the cells, thus depolarizing the membrane and initiating events that result in increased aldosterone synthesis. The mechanism by which mutated KCNJ5 induces cell proliferation and adenoma formation remains unclear.

An Abbreviated History of Aldosterone Metabolism, Current and Future Challenges.

The initial isolation of adrenal steroids from large quantities of animal adrenals resulted in an amorphous fraction resistant to crystallization and identification and had potent effects on electrolyte transport. Aldosterone was eventually isolated and identified in the fraction and was soon shown to cause hypertension when in excess. The autonomous and excessive production of aldosterone, primary aldosteronism, is the most common cause of secondary hypertension. Aldosterone is metabolized in the liver and kidney, and its metabolites are conjugated with glucuronic acid for excretion. The most common liver metabolite is 3α,5ß-tetrahydroaldosterone-3-glucuronide, while that of the kidney is aldosterone-18-oxo-glucuronide. In terms of their value, especially the aldosterone-18-oxo-glucuronide, is commonly used for the diagnosis of primary aldosteronism because they provide an integrated value of the total daily production of aldosterone. Conversion of aldosterone to 18-oxo-glucuronide is impeded by drugs, like some common non-steroidal anti-inflammatory drugs that compete for UDP-glucuronosyltransferase-2B7, the most important glucuronosyltransferase for aldosterone metabolism. Tetrahydroaldosterone is the most abundant metabolite and the most reliable for the diagnosis of primary aldosteronism, but it is not commonly measured.

18-Oxocortisol: A journey.

The search for mineralocorticoids to explain some cases of low renin hypertension with suppressed aldosterone levels led to the isolation of the abundant steroid 18-hydroxycortisol in human urine. 18-Hydroxycortisol proved to be inactive, but because of its similarity to precursors for the synthesis of aldosterone, bullfrog adrenals were incubated with cortisol, resulting in the discovery of 18-oxocortisol which is structurally similar to aldosterone, but with a 17α-hydroxy group like cortisol. 18-Oxocortisol is a weak mineralocorticoid. Its synthesis occurs primarily in the zona glomerulosa where co-expression of the CYP11B2 (aldosterone synthase) and the CYP17A1 (17α-hydroxylase) occurs in a variable number of cells. The clinical value of the measurement of 18-oxocortisol is that it serves to distinguish subtypes of primary aldosteronism. It is significantly elevated in patients with aldosterone-producing adenomas in comparison to those with idiopathic bilateral hyperaldosteronism and helps predict the type of somatic mutation in the aldosterone-producing adenomas, as it is higher in those with KCNJ5 mutations compared to other gene mutations.

The ubiquitous mineralocorticoid receptor: clinical implications.

Mineralocorticoid receptors (MR) exist in many tissues, in which they mediate diverse functions crucial to normal physiology, including tissue repair and electrolyte and fluid homeostasis. However, inappropriate activation of MR within these tissues, and especially in the brain, causes hypertension and pathological vascular, cardiac, and renal remodeling. MR binds aldosterone, cortisol and corticosterone with equal affinity. In aldosterone-target cells, co-expression with the 11ß-hydroxysteroid dehydrogenase 2 (HSD2) allows aldosterone specifically to activate MR. Aldosterone levels are excessive in primary aldosteronism, but in conditions with increased oxidative stress, like CHF, obesity and diabetes, MR may also be inappropriately activated by glucocorticoids. Unlike thiazide diuretics, MR antagonists are diuretics that do not cause insulin resistance. Addition of MR antagonists to standard treatment for hypertension and cardiac or renal disease decreases end-organ pathology and sympathetic nerve activation (SNA), and increases quality of life indices.

Role of mineralocorticoid action in the brain in salt-sensitive hypertension.

1. The mechanisms by which excessive salt causes hypertension involve more than retention of sodium and water by the kidneys and are far from clear. Mineralocorticoids act centrally to increase salt appetite, sympathetic drive and vasopressin release, resulting in hypertension that is prevented by the central infusion of mineralocorticoid receptor (MR) antagonists. The MR has similar affinity for aldosterone and the glucocorticoids corticosterone or cortisol. Specificity is conferred in transport epithelia by the colocalization of the MR with 11ß-hydroxysteroid dehydrogenase Type 2. Coexpression also occurs in some neurons, notably those of the nucleus tractus solitarius that are activated by sodium depletion and aldosterone and mediate salt-seeking behaviour. 2. The salt-induced hypertension of the Dahl salt-sensitive rat is mitigated by the central infusion of a mineralocorticoid antagonist even though circulating aldosterone is normal or reduced in salt-sensitive (SS). Contrary to reports that salt appetite in the Dahl salt-sensitive rat is depressed, we found that it is increased compared with that in Spraque-Dawley rats. 3. Extra-adrenal aldosterone synthesis in the brain occurs in minute amounts that could only be relevant locally. Expression of aldosterone synthase mRNA and aldosterone concentrations in the brain of Dahl salt-sensitive rats are increased compared with Spraque-Dawley rats. The central infusion of inhibitors of aldosterone synthesis lowers salt-induced hypertension in the Dahl salt-sensitive rat, suggesting a role for excessive Dahl salt-sensitive synthesis in the brain. Brain MR, particularly those in the paraventricular nuclei, regulate inflammatory processes that are exacerbated by sodium and lead to cardiovascular dysfunction.

Mammalian Target of Rapamycin Inhibition Decreases Angiotensin II-Induced Steroidogenesis in HAC15 Human Adrenocortical Carcinoma Cells.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors suppress adrenal cortical carcinoma cell proliferation and cortisol production; the relationship between mTOR and aldosterone production has not been examined. METHODS: HAC15 cells were incubated with an mTOR activator and several inhibitors including AZD8055 (AZD) in the presence and absence of angiotensin II (AngII). The expression of rapamycin-sensitive adapter protein of mTOR (Raptor) and rapamycin-insensitive companion of mTOR (Rictor), adaptor proteins of mTOR complex 1 and 2, respectively, were studied in the HAC15 cells and deleted by CRISPR/gRNA. RESULTS: The mTOR inhibitors decreased aldosterone induced by AngII. Inhibition of mTOR by AZD significantly suppressed AngII-induced aldosterone and cortisol formation in a dose-dependent manner, whereas the mTOR activator MHY had no effect. AZD did not alter forskolin-induced aldosterone production showing that it is specific to the AngII signaling pathway. AngII-mediated ERK and mTOR activation were suppressed by AZD, along with a concomitant dose-dependent reduction of AngII-induced steroidogenic enzymes including steroidogenic acute regulatory protein, 3ß-hydroxysteroid dehydrogenase-type 2, CYP17A1, and aldosterone synthase protein. Furthermore, mTOR components ribosomal protein S6 kinase (P70S6K) and protein kinase B phosphorylation levels were decreased by AZD. As mTOR exerts its main effects by forming complexes with adaptor proteins Raptor and Rictor, the roles of these individual complexes were studied. We found an increase in the phosphorylation of Raptor and Rictor by AngII and that their CRISPR/gRNA-mediated knockdown significantly attenuated AngII-induced aldosterone and cortisol production. CONCLUSION: mTOR signaling has a critical role in transducing the AngII signal initiating aldosterone and cortisol synthesis in HAC15 cells and that inhibition of mTOR could be a therapeutic option for conditions associated with excessive renin-angiotensin system-mediated steroid synthesis.

Origin of circulating 18-oxocortisol in the normal human adrenal.

18-Oxocortisol is the product of the metabolism of 11-deoxycortisol by the mitochondrial enzyme aldosterone synthase (CYP11B2). The traditional concept is that the CYP11B2 is exclusively expressed in zona glomerulosa cells and the 17α-hydroxylase (CYP17A1) enzyme, required to synthesize 11-deoxycortisol, is in the zona fasciculata of the human adrenal. It has been postulated that the substrate for 18-oxocortisol is either cortisol from the circulation or from zona fasciculata cells adjacent to the zona glomerulosa. P-glycoprotein, which is highly expressed in steroidogenic cells of the adrenal gland, efficiently expels cortisol from the cell. Double immunofluorescence staining for the CYP11B2 and CYP17A1 enzymes in 7 human adrenals demonstrated that a highly variable number of cells in different areas of the zona glomerulosa co-expressed both enzymes. In addition, there were a variable number of cells that exclusively expressed the CYP17A1 embedded within the zona glomerulosa surrounded by CYP11B2-expressing cells. 18-Oxocortisol in the media of human adrenocortical HAC15 cells was measured by ELISA after incubation with and without 10 nM of angiotensin II to stimulate CYP11B2 activity, with and without the 3ß-hydroxysteroid dehydrogenase (HSD3B) inhibitor trilostane, and with variable amounts of cortisol or 11-deoxycortisol. Cortisol was a poor substrate, while 11-deoxycortisol was a significant substrate for the synthesis of 18-oxocortisol. These data suggest that the biosynthesis of 18-oxocortisol in the human adrenal is likely catalyzed by co-expression of the two crucial enzymes CYP17A1 and CYP11B2 in a small proportion of cells within the zona glomerulosa. It is also possible that 11-deoxycortisol diffusing from cells expressing only CYP17A1 interspersed with cells expressing the CYP11B2 enzyme may be a paracrine substrate in the synthesis of 18-oxocortisol.

11ß-hydroxysteroid dehydrogenases: A growing multi-tasking family.

This review briefly addresses the history of the discovery and elucidation of the three cloned 11ß-hydroxysteroid dehydrogenase (11ßHSD) enzymes in the human, 11ßHSD1, 11ßHSD2 and 11ßHSD3, an NADP+-dependent dehydrogenase also called the 11ßHSD1-like dehydrogenase (11ßHSD1L), as well as evidence for yet identified 11ßHSDs. Attention is devoted to more recently described aspects of this multi-functional family. The importance of 11ßHSD substrates other than glucocorticoids including bile acids, 7-keto sterols, neurosteroids, and xenobiotics is discussed, along with examples of pathology when functions of these multi-tasking enzymes are disrupted. 11ßHSDs modulate the intracellular concentration of glucocorticoids, thereby regulating the activation of the glucocorticoid and mineralocorticoid receptors, and 7ß-27-hydroxycholesterol, an agonist of the retinoid-related orphan receptor gamma (RORγ). Key functions of this nuclear transcription factor include regulation of immune cell differentiation, cytokine production and inflammation at the cell level. 11ßHSD1 expression and/or glucocorticoid reductase activity are inappropriately increased with age and in obesity and metabolic syndrome (MetS). Potential causes for disappointing results of the clinical trials of selective inhibitors of 11ßHSD1 in the treatment of these disorders are discussed, as well as the potential for more targeted use of inhibitors of 11ßHSD1 and 11ßHSD2.

11ßHSD2 Efficacy in Preventing Transcriptional Activation of the Mineralocorticoid Receptor by Corticosterone.

Affinity of the mineralocorticoid receptor (MR) is similar for aldosterone and the glucocorticoids (GC) cortisol and corticosterone, which circulate at concentrations far exceeding those of aldosterone. 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) inactivation of GC within the immediate vicinity of the MR is credited with prereceptor specificity for aldosterone in cells coexpressing MR and 11ßHSD2. 11ßHSD2 efficacy is also critical to other recently described 11ßHSD2 substrates. The aim of this work was to address doubts that low levels of expression of 11ßHSD2 in aldosterone target tissues suffice to prevent the initiation of gene transcription by the MR activated by physiological concentrations of corticosterone. Cell models stably expressing an MR/Gaussia luciferase reporter and various levels of constitutive or induced 11ßHSD2 at concentrations lower than those in rat kidney homogenates and microsomes were produced. Aldosterone and corticosterone were equipotent transactivators of the MR reporter gene in cells without 11ßHSD2. Rate of conversion of tritiated corticosterone to 11-dehydrocorticosterone increased and corticosterone-induced nuclear translocation of MR decreased, as 11ßHSD2 expression increased. The 50% maximal MR activation for the reporter gene stimulation by corticosterone rose with increasing 11ßHSD2 expression, shifting the steroid dose-response curve for corticosterone-induced MR transactivation to the right. Several stable cell lines expressing an easily and reproducibly measured MR reporter system and consistent incremental amounts of 11ßHSD2 protein were produced and used to document that 11ßHSD2 within low physiological levels inactivates relevant concentrations of GC and decreases MR transactivation by GC in a dose-dependent fashion, laying to rest doubts of the efficacy of this enzyme.

Angiotensin II-regulated transcription regulatory genes in adrenal steroidogenesis.

Transcription regulatory genes are crucial modulators of cell physiology and metabolism whose intracellular levels are tightly controlled in response to extracellular stimuli. We previously reported a set of 29 transcription regulatory genes modulated by angiotensin II in H295R human adrenocortical cells and their roles in regulating the expression of the last and unique enzymes of the glucocorticoid and mineralocorticoid biosynthetic pathways, 11ß-hydroxylase and aldosterone synthase, respectively, using gene expression reporter assays. To study the effect of this set of transcription regulatory genes on adrenal steroidogenesis, H295R cells were transfected by high-efficiency nucleofection and aldosterone and cortisol were measured in cell culture supernatants under basal and angiotensin II-stimulated conditions. BCL11B, BHLHB2, CITED2, ELL2, HMGA1, MAFF, NFIL3, PER1, SERTAD1, and VDR significantly stimulated aldosterone secretion, while EGR1, FOSB, and ZFP295 decreased aldosterone secretion. BTG2, HMGA1, MITF, NR4A1, and ZFP295 significantly increased cortisol secretion, while BCL11B, NFIL3, PER1, and SIX2 decreased cortisol secretion. We also report the effect of some of these regulators on the expression of endogenous aldosterone synthase and 11ß-hydroxylase under basal and angiotensin II-stimulated conditions. In summary, this study reports for the first time the effects of a set of angiotensin II-modulated transcription regulatory genes on aldosterone and cortisol secretion and the expression levels of the last and unique enzymes of the mineralocorticoid and glucocorticoid biosynthetic pathways. Abnormal regulation of mineralocorticoid or glucocorticoid secretion is involved in several pathophysiological conditions. These transcription regulatory genes may be involved in adrenal steroidogenesis pathologies; thus they merit additional study as potential candidates for therapeutic intervention.