NNT is a key regulator of adrenal redox homeostasis and steroidogenesis in male mice.
ABSTRACT: Nicotinamide nucleotide transhydrogenase, NNT, is a ubiquitous protein of the inner mitochondrial membrane with a key role in mitochondrial redox balance. NNT produces high concentrations of NADPH for detoxification of reactive oxygen species by glutathione and thioredoxin pathways. In humans, NNT dysfunction leads to an adrenal-specific disorder, glucocorticoid deficiency. Certain substrains of C57BL/6 mice contain a spontaneously occurring inactivating Nnt mutation and display glucocorticoid deficiency along with glucose intolerance and reduced insulin secretion. To understand the underlying mechanism(s) behind the glucocorticoid deficiency, we performed comprehensive RNA-seq on adrenals from wild-type (C57BL/6N), mutant (C57BL/6J) and BAC transgenic mice overexpressing Nnt (C57BL/6JBAC). The following results were obtained. Our data suggest that Nnt deletion (or overexpression) reduces adrenal steroidogenic output by decreasing the expression of crucial, mitochondrial antioxidant (Prdx3 and Txnrd2) and steroidogenic (Cyp11a1) enzymes. Pathway analysis also revealed upregulation of heat shock protein machinery and haemoglobins possibly in response to the oxidative stress initiated by NNT ablation. In conclusion, using transcriptomic profiling in adrenals from three mouse models, we showed that disturbances in adrenal redox homeostasis are mediated not only by under expression of NNT but also by its overexpression. Further, we demonstrated that both under expression or overexpression of NNT reduced corticosterone output implying a central role for it in the control of steroidogenesis. This is likely due to a reduction in the expression of a key steroidogenic enzyme, Cyp11a1, which mirrored the reduction in corticosterone output.
Project description:Synthetic glucocorticoids are widely prescribed for the treatment of numerous inflammatory and autoimmune diseases and they can also affect the way the adrenal gland produces endogenous glucocorticoids. Indeed, patients undergoing synthetic glucocorticoid treatment can develop adrenal insufficiency, a condition characterised by reduced responsiveness of the adrenal to ACTH stimulation or stressors (e.g. surgical or inflammatory stress). To better elucidate the long-term effect of synthetic glucocorticoids treatment and withdrawal on adrenal function, we have investigated the long-term effects of prolonged treatment with methylprednisolone on HPA axis dynamics and on the adrenal steroidogenic pathway, both in basal conditions and in response to an inflammatory stress (lipopolysaccharide, LPS). We have found that 5-days treatment with methylprednisolone suppresses basal ACTH and corticosterone secretion, as well as corticosterone secretion in response to a high dose of ACTH, and down-regulates key genes in the adrenal steroidogenic pathway, including StAR, MRAP, CYP11a1 and CYP11b1. These effects were paralleled by changes in the adrenal expression of transcription factors regulating steroidogenic gene expression, as well as changes in the expression of adrenal clock genes. Importantly, 5 days after withdrawal of the treatment, ACTH levels are restored, yet basal levels of corticosterone, as well as most of the key steroidogenic genes and their regulators, remain down regulated. We also show that, although 5-days treatment with methylprednisolone reduces the corticosterone response to LPS, an increase in intra-adrenal pro-inflammatory cytokine gene expression was observed. Our data suggests that the steroidogenic pathway is directly affected by synthetic glucocorticoid treatment in the long-term, presumably via a mechanism involving activation of the glucocorticoid receptor. Furthermore, our data suggests a pro-inflammatory effect of synthetic glucocorticoids treatment in the adrenal gland.
Project description:During pregnancy, fetal glucocorticoid is derived from both maternal supply and fetal secretion. We have created mice with a disruption of the Cyp11a1 gene resulting in loss of fetal steroid secretion but preserving the maternal supply. Cyp11a1null embryos have appreciable although lower amounts of circulating corticosterone, the major mouse glucocorticoid, suggesting that transplacental corticosterone is a major source of corticosterone in fetal circulation. These embryos thus provide a means to examine the effect of fetal glucocorticoids. The adrenal in Cyp11a1 null embryos was disorganized with abnormal mitochondria and oil accumulation. The adrenal medullary cells did not express phenylethanolamine N-methyltransferase and synthesized no epinephrine. Cyp11a1 null embryos had decreased diencephalon Hsd11b1, increased diencephalon Crh, and increased pituitary Pomc expression, leading to higher adrenocorticotropin level in the plasma. These data indicate blunted feedback suppression despite reasonable amounts of circulating corticosterone. Thus, the corticosterone synthesized in situ by the fetus is required for negative feedback suppression of the hypothalamus-pituitary-adrenal axis and for catecholamine synthesis in adrenal medulla.
Project description:Nicotinamide nucleotide transhydrogenase (NNT) is a proton pump in the inner mitochondrial membrane that generates reducing equivalents in the form of NAPDH, which can be used for anabolic pathways or to remove reactive oxygen species (ROS). A number of studies have linked NNT dysfunction to cardiomyopathies and increased risk of atherosclerosis; however, biallelic mutations in humans commonly cause a phenotype of adrenal insufficiency, with rare occurrences of cardiac dysfunction and testicular tumours. Here, we compare the transcriptomes of the hearts, adrenals and testes from three mouse models: the C57BL/6N, which expresses NNT; the C57BL/6J, which lacks NNT; and a third mouse, expressing the wild-type NNT sequence on the C57BL/6J background. We saw enrichment of oxidative phosphorylation genes in the C57BL/B6J in the heart and adrenal, possibly indicative of an evolved response in this substrain to loss of <i>Nnt.</i> However, differential gene expression was mainly driven by mouse background with some changes seen in all three tissues, perhaps reflecting underlying genetic differences between the C57BL/B6J and -6N substrains.
Project description:Wnt/?-catenin (?cat) signaling is critical for adrenal homeostasis. To elucidate how Wnt/?cat signaling elicits homeostatic maintenance of the adrenal cortex, we characterized the identity of the adrenocortical Wnt-responsive population. We find that Wnt-responsive cells consist of sonic hedgehog (Shh)-producing adrenocortical progenitors and differentiated, steroidogenic cells of the zona glomerulosa, but not the zona fasciculata and rarely cells that are actively proliferating. To determine potential direct inhibitory effects of ?cat signaling on zona fasciculata-associated steroidogenesis, we used the mouse ATCL7 adrenocortical cell line that serves as a model system of glucocorticoid-producing fasciculata cells. Stimulation of ?cat signaling caused decreased corticosterone release consistent with the observed reduced transcription of steroidogenic genes Cyp11a1, Cyp11b1, Star, and Mc2r. Decreased steroidogenic gene expression was correlated with diminished steroidogenic factor 1 (Sf1; Nr5a1) expression and occupancy on steroidogenic promoters. Additionally, ?cat signaling suppressed the ability of Sf1 to transactivate steroidogenic promoters independent of changes in Sf1 expression level. To investigate Sf1-independent effects of ?cat on steroidogenesis, we used Affymetrix gene expression profiling of Wnt-responsive cells in vivo and in vitro. One candidate gene identified, Ccdc80, encodes a secreted protein with unknown signaling mechanisms. We report that Ccdc80 is a novel ?cat-regulated gene in adrenocortical cells. Treatment of adrenocortical cells with media containing secreted Ccdc80 partially phenocopies ?cat-induced suppression of steroidogenesis, albeit through an Sf1-independent mechanism. This study reveals multiple mechanisms of ?cat-mediated suppression of steroidogenesis and suggests that Wnt/?cat signaling may regulate adrenal homeostasis by inhibiting fasciculata differentiation and promoting the undifferentiated state of progenitor cells.
Project description:The adrenal glands are able to modulate immune responses through neuroimmunoendocrine interactions and cortisol secretion that could suppress exacerbated inflammation such as in inflammatory bowel disease (IBD). Therefore, here we evaluated the role of these glands in experimental colitis induced by 3% dextran sulfate sodium (DSS) in C57BL/6 mice subjected to adrenalectomy, with or without glucocorticoid (GC) replacement. Mice succumbed to colitis without adrenals with a higher clinical score and augmented systemic levels of IL-6 and lower LPS. Furthermore, adrenalectomy negatively modulated systemic regulatory markers. The absence of adrenals resulted in augmented tolerogenic lamina propria dendritic cells but no compensatory local production of corticosterone and decreased mucosal inflammation associated with increased IFN-? and FasL in the intestine. To clarify the importance of GC in this scenario, GC replacement in adrenalectomized mice restored different markers to the same degree of that observed in DSS group. Finally, this is the first time that adrenal-derived hormones, especially GC, were associated with the differential local modulation of the gut infiltrate, also pointing to a relationship between adrenalectomy and the modulation of systemic regulatory markers. These findings may elucidate some neuroimmunoendocrine mechanisms that dictate colitis outcome.
Project description:Hypoxia, a common stressor in prematurity, leads to sexually dimorphic, short- and long-term effects on the adult hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes. We hypothesized that these effects are due to stress-induced increases in testosterone during early postnatal life. We evaluated this phenomenon by systematically assessing the short-term effects of normoxic or hypoxic separation on male and female pups at birth, postnatal hours (H) 2, 4, and 8, and postnatal days (PD) 2 to 7. Our findings were (a) hypoxic separation led to a large increase in plasma corticosterone from 4H-PD4, (b) neither normoxic nor hypoxic separation affected critical adrenal steroidogenic pathway genes; however, a significant decrease in baseline Cyp11a1, Mc2r, Mrap, and Star adrenal expression during the first week of neonatal life confirmed the start of the adrenal stress hyporesponsive period, (c) a luteinizing hormone/follicle-stimulating hormone-independent increase in plasma testosterone occurred in normoxic and hypoxic separated male pups at birth, (d) testicular Cyp11a1, Lhcgr, and Star expression was high at birth and decreased thereafter suggesting a hyporesponsive period in the testes, and (e) elevated estrogen in the early neonatal period occurred independently of gonadotropin stimulation. We conclude that a large corticosterone response to hypoxia during the first 5 days of life occurs as an adaptation to neonatal stress, that the testosterone surge during the first hours after birth occurs independently of gonadotropins but is associated with upregulation of the steroidogenic pathway genes in the testes, and that high postnatal estrogen production also occurs independently of gonadotropins.
Project description:CONTEXT:Mitochondrial cytochrome P450scc converts cholesterol to pregnenolone in all steroidogenic tissues. Although progesterone production from the fetally-derived placenta is necessary to maintain pregnancy to term, four patients with mutations in the gene encoding P450scc (CYP11A1), have been described, one in a 46,XX female and three in underandrogenized 46,XY individuals, all with primary adrenal failure. OBJECTIVE:Our aim was to determine whether P450scc mutations might be found in other children and to explore genotype/phenotype correlations. METHODS AND PATIENTS:We performed mutational analysis of CYP11A1 in individuals with 46,XY disorders of sex development and primary adrenal failure, followed by functional studies of P450scc activity and of P450scc RNA splicing. RESULTS:Among nine 46,XY infants with adrenal failure and disordered sexual differentiation, two infants had compound heterozygous mutations in CYP11A1. One patient harbored the novel P450scc missense mutations L141W and V415E, which retained 38 and 0% activity, respectively. The other carried a CYP11A1 frameshift mutation c835delA (0% activity) and a splice site mutation [IVS3+(2-3)insT] that prevented correct splicing of P450scc mRNA. CONCLUSIONS:P450scc deficiency is a recently recognized disorder that may be more frequent than originally thought. The phenotypic spectrum ranges from severe loss-of-function mutations associated with prematurity, complete underandrogenization, and severe, early-onset adrenal failure, to partial deficiencies found in children born at term with clitoromegaly and later-onset adrenal failure. In contradistinction to congenital lipoid adrenal hyperplasia caused by steroidogenic acute regulatory protein mutations, adrenal hyperplasia has not been reported in any of the six patients with P450scc deficiency.
Project description:In the adrenal gland, adrenocorticotropin (ACTH) acting through the cAMP protein kinase (PKA) transduction pathway is the main regulator of genes involved in glucocorticoid synthesis. The prolactin (PRL) receptor is expressed in the adrenal cortex of most mammals, but experimental proof that PRL ensures direct control on glucocorticoid synthesis in rodents remains elusive. To unravel the physiological importance of PRL in adrenocortical functions, we measured steroidogenic capacity of Prlr-deficient mice (Prlr(-/-)) and explored the influence of JAK/STAT signaling, the major PRL transduction pathway, on the steroidogenic activity of adrenocortical cell cultures. We demonstrate that lack of Prlr does not affect basal (nor stress-induced) corticosterone levels in mice. PRL triggers JAK2/STAT5-dependent transcription in adrenal cells, but this does not influence corticosterone release. In contrast, pharmacological or siRNA-mediated inhibition of JAK2 reveals its essential role in both basal and ACTH/cAMP-induced steroidogenesis. We demonstrate that nuclear JAK2 regulates the amount of active transcription factor CREB (cAMP response element-binding protein) through tyrosine phosphorylation and prevention of proteasomal degradation, which in turn leads to transcriptional activation of the rate-limiting steroidogenic Star gene. Hence, we describe a novel link between PKA and JAK2 by which nuclear JAK2 signaling controls adrenal steroidogenesis by increasing the stability of CREB.
Project description:DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1; also known as NROB1, nuclear receptor subfamily 0, group B, member 1) encodes a nuclear receptor that is expressed in embryonic stem (ES) cells, steroidogenic tissues (gonads, adrenals), the ventromedial hypothalamus (VMH), and pituitary gonadotropes. Humans with DAX1 mutations develop an X-linked syndrome referred to as adrenal hypoplasia congenita (AHC). These boys typically present in infancy with adrenal failure but later fail to undergo puberty because of hypogonadotropic hypogonadism (HHG). The adrenal failure reflects a developmental abnormality in the transition of the fetal to adult zone, resulting in glucocorticoid and mineralocorticoid deficiency. The etiology of HHG involves a combined and variable deficiency of hypothalamic GnRH secretion and/or pituitary responsiveness to GnRH resulting in low LH, FSH and testosterone. Treatment with exogenous gonadotropins generally does not induce spermatogenesis. Animal models indicate that DAX1 also plays a critical role in testis development and function. As a nuclear receptor, DAX1 has been shown to function as a transcriptional repressor, particularly of pathways regulated by other nuclear receptors, such as steroidogenic factor 1 (SF1). In addition to reproductive tissues, DAX1 is also expressed at high levels in ES cells and plays a role in the maintenance of pluripotentiality. Here we review the clinical manifestations associated with DAX1 mutations as well as the evolving information about its function based on animal models and in vitro studies.
Project description:Using targeted exome sequencing, we identified mutations in NNT, an antioxidant defense gene, in individuals with familial glucocorticoid deficiency. In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human adrenocortical cell line resulted in impaired redox potential and increased reactive oxygen species (ROS) levels. Our results suggest that NNT may have a role in ROS detoxification in human adrenal glands.