Cell cycle-dependent regulation of extra-adrenal glucocorticoid synthesis in murine intestinal epithelial cells

Glucocorticoids are anti-inflammatory steroids with important applications in the treatment of inflammatory diseases. Endogenous glucocorticoids are mainly produced by the adrenal glands, although there is increasing evidence for extra-adrenal sources. Recent findings show that intestinal crypt cells produce glucocorticoids, which contribute to the maintenance of intestinal immune homeostasis. Intestinal glucocorticoid synthesis is critically regulated by the transcription factor liver receptor homologue-1 (LRH-1). As expression of steroidogenic enzymes and LRH-1 is restricted to the proliferating cells of the crypts, we aimed to investigate the role of the cell cycle in the regulation of LRH-1 activity and intestinal glucocorticoid synthesis. We here show that either pharmacological or molecular modulation of cell cycle progression significantly inhibited expression of steroidogenic enzymes and synthesis of glucocorticoids in intestinal epithelial cells. Synchronization of intestinal epithelial cells in the cell cycle revealed that expression of steroidogenic enzymes is preferentially induced at the G(1)/S stage. Differentiation of immature intestinal epithelial cells to mature nonproliferating cells also resulted in reduced expression of steroidogenic enzymes. This cell cycle-related effect on intestinal steroidogenesis was found to be mediated through the regulation of LRH-1 transcriptional activity. This mechanism may restrict intestinal glucocorticoid synthesis to the proliferating cells of the crypts.

P450 oxidoreductase deficiency - a new form of congenital adrenal hyperplasia

Patients with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley- Bixler syndrome (a craniosynostosis syndrome), are likely to have P450 oxidoreductase (POR) deficiency. Since our first report in 2004, about 26 recessive POR mutations have been identified in 50 patients. POR is the obligate electron donor to all microsomal (type II) P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46,XY newborns as well as overvirilization in those who are 46,XX. This may be explained by impaired aromatization of fetal androgens which may also lead to maternal virilization and low urinary estriol levels during pregnancy. A role for the alternate 'backdoor' pathway of androgen biosynthesis, leading to dihydrotestosterone production bypassing androstenedione and testosterone, has been suggested in POR deficiency but remains unclear. POR variants may play an important role in drug metabolism, as most drugs are metabolized by hepatic microsomal P450 enzymes. However, functional assays studying the effects of specific POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. Thus, the impact of POR mutations on drug metabolism by hepatic P450s requires further investigation.

Adrenal gland development and defects

The network regulating human adrenal development is complex. Studies of patients with adrenal insufficiency due to gene mutations established a central role for transcription factors GLI3, SF1 and DAX1 in the initial steps of adrenal formation. Adrenal differentiation seems to depend on adrenocorticotropic hormone (ACTH) stimulation and signalling, including biosynthesis and action of POMC, PC1, TPIT, MC2R, MRAP and ALADIN, all of which cause adrenocortical hypoplasia when mutated in humans. Studies of knockout mice revealed many more factors involved in adrenal development; however, in contrast to rodents, in humans several of those factors had no adrenal phenotype when mutated (e.g. WT1, WNT4) or, alternatively, human mutations have not (yet) been identified. Tissue profiling of fetal and adult adrenals suggested 69 genes involved in adrenal development. Among them were genes coding for steroidogenic enzymes, transcription and growth factors, signalling molecules, regulators of cell cycle and angiogenesis, and extracellular matrix proteins; however, the exact role of most of them remains to be elucidated.

The role of adrenal steroidogenesis in arterial hypertension

Adrenal aldosterone production, the major regulator of salt and water retention, is discussed with respect to hypertensive diseases. Physiological aldosterone production is tightly regulated, either stimulated or inhibited, in the adrenal zona glomerulosa by both circulating factors and/or by locally derived endothelial factors. Arterial hypertension caused by volume overload is the leading clinical symptom indicating increased mineralocorticoid hormones. Excessive aldosterone production is seen in adenomatous disease of the adrenals. The balance between stimulatory/proliferative and antagonistic signaling is disturbed by expression of altered receptor subtypes in the adenomas. Increased aldosterone production without a detectable adenoma is the most frequent form of primary aldosteronism. Both increased sensitivity to agonistic signals and activating polymorphisms within the aldosterone synthase gene (CYP11B2) have been associated with excessive aldosterone production. 17alpha-Hydroxylase deficiency and glucocorticoidremediable aldosteronism can also cause excessive mineralocorticoid synthesis. In contrast, the severe form of pregnancy-induced hypertension, preeclampsia, is characterized by a compromised volume expansion in the presence of inappropriately low aldosterone levels. Initial evidence suggests that compromised CYP11B2 is causative, and that administration of NaCl lowered blood pressure in pregnant patients with low aldosterone availability due to a loss of function.

Extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean?

Glucocorticoids (GC) are lipophilic hormones commonly used as therapeutics in acute and chronic inflammatory disorders such as inflammatory bowel disease due to their attributed anti-inflammatory and immunosuppressive actions. Although the adrenal glands are the major source of endogenous GC, there is increasing evidence for the production of extra-adrenal GC in the brain, thymus, skin, vasculature, and the intestine. However, the physiological relevance of extra-adrenal-produced GC remains still ambiguous. Therefore, this review attracts attention to discuss possible biological benefits of extra-adrenal-synthesized GC, especially focusing on the impact of locally synthesized GC in the regulation of intestinal immune responses.

Effects of thermal environment on hypothalamic-pituitary-adrenal axis hormones, oxytocin, and behavioral activity in periparturient sows

Provision of additional floor heating (33 to 34 degrees C) at birth and during the early postnatal hours is favorable for newborn piglets of domestic sows (Sus scrofa). We investigated whether this relatively high temperature influenced sow behavior and physiology around farrowing. One-half of 28 second-parity pregnant sows were randomly chosen to be exposed to floor heating 12 h after onset of nest building and until 48 h after birth of the first piglet (heat treatment), whereas the rest of the sows entered the control group (control treatment) with no floor heating. Hourly blood sampling from 8 h before and until 24 h after the birth of the first piglet was used for investigation of temporal changes in plasma concentrations of oxytocin, cortisol, and ACTH. In addition, occurrence and duration of sow postures were recorded -8 to +48 h relative to the birth of the first piglet. There was a clear temporal development in sow behavior and hormone concentrations (ACTH, cortisol, and oxytocin) across parturition (P < 0.001), independent of treatment. In general, hormone concentrations increased from the start to the end of farrowing. The observed oxytocin increase and peak late in farrowing coincided with the passive phase where sows lie laterally with an overall reduced activity. Floor heating increased the mean concentration of cortisol (P = 0.02; estimated as 29% greater than in controls) and tended to increase the mean concentration of ACTH (P = 0.08; estimated as 17% greater than in controls), but we did not find any treatment effect on mean oxytocin concentrations, the course of parturition, or the behavior of sows. Behavioral thermoregulation may, however, have lost some function for the sows because the floor was fully heated in our study. In addition, exposure to heat decreased the between-sow variation of plasma oxytocin (approximately 31% less relative to control) and ACTH (approximately 46% less relative to control). Whether this decreased variation may be indicative of acute stress or linked to other biological events is unclear. In conclusion, inescapable floor heating (around 33.5 degrees C) may be considered a stressor for sows around farrowing, giving rise to elevated plasma concentrations of cortisol, but without concurrent changes in oxytocin or behavioral activity.

STAR splicing mutations cause the severe phenotype of lipoid congenital adrenal hyperplasia: insights from a novel splice mutation and review of reported cases

OBJECTIVE The steroidogenic acute regulatory protein (StAR) transports cholesterol to the mitochondria for steroidogenesis. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH) which is characterized by impaired synthesis of adrenal and gonadal steroids causing adrenal insufficiency, 46,XY disorder of sex development (DSD) and failure of pubertal development. Partial loss of StAR activity may cause adrenal insufficiency only. PATIENT A newborn girl was admitted for mild dehydration, hyponatremia, hyperkalemia and hypoglycaemia and had normal external female genitalia without hyperpigmentation. Plasma cortisol, 17OH-progesterone, DHEA-S, androstendione and aldosterone were low, while ACTH and plasma renin activity were elevated, consistent with the diagnosis of primary adrenal insufficiency. Imaging showed normal adrenals, and cytogenetics revealed a 46,XX karyotype. She was treated with fluids, hydrocortisone and fludrocortisone. DESIGN, METHODS AND RESULTS Genetic studies revealed a novel homozygous STAR mutation in the 3' acceptor splice site of intron 4, c.466-1G>A (IVS4-1G>A). To test whether this mutation would affect splicing, we performed a minigene experiment with a plasmid construct containing wild-type or mutant StAR gDNA of exons-introns 4-6 in COS-1 cells. The splicing was assessed on total RNA using RT-PCR for STAR cDNAs. The mutant STAR minigene skipped exon 5 completely and changed the reading frame. Thus, it is predicted to produce an aberrant and shorter protein (p.V156GfsX19). Computational analysis revealed that this mutant protein lacks wild-type exons 5-7 which are essential for StAR-cholesterol interaction. CONCLUSIONS STAR c.466-1A skips exon 5 and causes a dramatic change in the C-terminal sequence of the protein, which is essential for StAR-cholesterol interaction. This splicing mutation is a loss-of-function mutation explaining the severe phenotype of our patient. Thus far, all reported splicing mutations of STAR cause a severe impairment of protein function and phenotype.

Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

A novel mutation L260P of the steroidogenic acute regulatory protein gene in three unrelated patients of Swiss ancestry with congenital lipoid adrenal hyperplasia.

CONTEXT Lipoid congenital adrenal hyperplasia (CAH) is the most severe form of CAH leading to impaired production of all adrenal and gonadal steroids. Mutations in the gene encoding steroidogenic acute regulatory protein (StAR) cause lipoid CAH. OBJECTIVE We investigated three unrelated patients of Swiss ancestry who all carried novel mutations in the StAR gene. All three subjects were phenotypic females with absent Müllerian derivatives, 46,XY karyotype, and presented with adrenal failure. METHODS AND RESULTS StAR gene analysis showed that one patient was homozygous and the other two were heterozygous for the novel missense mutation L260P. Of the heterozygote patients, one carried the novel missense mutation L157P and one had a novel frameshift mutation (629-630delCT) on the second allele. The functional ability of all three StAR mutations to promote pregnenolone production was severely attenuated in COS-1 cells transfected with the cholesterol side-chain cleavage system and mutant vs. wild-type StAR expression vectors. CONCLUSIONS These cases highlight the importance of StAR-dependent steroidogenesis during fetal development and early infancy; expand the geographic distribution of this condition; and finally establish a new, prevalent StAR mutation (L260P) for the Swiss population.

A retroperitoneal bronchogenic cyst mimicking a pancreatic or adrenal mass

Retroperitoneal location of bronchogenic cysts is extremely rare. Most commonly they are encountered in the posterior mediastinum. Bronchogenic cysts arise from developmental aberrations of the tracheobronchial tree in the early embryologic period. We report a 42-year-old female patient with a retroperitoneal bronchogenic cyst in the left adrenal region. She was admitted to our hospital with epigastric pain and subsequently underwent CT of the abdomen. The examination revealed a mass related to the left adrenal gland. Endocrine tests for adrenal hypersecretion were negative. Because of the uncertain entity, laparoscopic adrenalectomy was performed. Pathological examination revealed a bronchogenic cyst in proximity to an inconspicuous left adrenal gland. Although very rare, bronchogenic cysts should be considered in the differential diagnosis of retroperitoneal cystic lesions and surgical resection pursued for symptom resolution and to establish a definitive histology.

Novel insight into etiology, diagnosis and management of primary adrenal insufficiency

Primary adrenal insufficiency (PAI) is a rare condition in childhood which is either inherited (mostly) or acquired. It is characterized by glucocorticoid and maybe mineralocorticoid deficiency. The most common form in children is 21-hydroxylase deficiency, which belongs to the steroid biosynthetic defects causing PAI. Newer forms of complex defects of steroid biosynthesis are P450 oxidoreductase deficiency and (apparent) cortisone reductase deficiency. Other forms of PAI include metabolic disorders, autoimmune disorders and adrenal dysgenesis, e.g. the IMAGe syndrome, for which the underlying genetic defect has been recently identified. Newer work has also expanded the genetic causes underlying isolated, familial glucocorticoid deficiency (FGD). Mild mutations of CYP11A1 or StAR have been identified in patients with FGD. MCM4 mutations were found in a variant of FGD in an Irish travelling community manifesting with PAI, short stature, microcephaly and recurrent infections. Finally, mutations in genes involved in the detoxification of reactive oxygen species were identified in patients with unsolved FGD. Most mutations were found in the enzyme nicotinamide nucleotide transhydrogenase, which uses the mitochondrial proton pump gradient to produce NADPH. NADPH is essential in maintaining high levels of reduced forms of antioxidant enzymes for the reduction of hydrogen peroxide. Similarly, mutations in the gene for TXNRD2 involved in this system were found in FGD patients, suggesting that the adrenal cortex is particularly susceptible to oxidative stress.

Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla.

The endocannabinoid system (ECS) comprises the cannabinoid receptors CB1 and CB2 and their endogenous arachidonic acid-derived agonists 2-arachidonoyl glycerol and anandamide, which play important neuromodulatory roles. Recently, a novel class of negative allosteric CB1 receptor peptide ligands, hemopressin-like peptides derived from alpha hemoglobin, has been described, with yet unknown origin and function in the CNS. Using monoclonal antibodies we now identified the localization of RVD-hemopressin (pepcan-12) and N-terminally extended peptide endocannabinoids (pepcans) in the CNS and determined their neuronal origin. Immunohistochemical analyses in rodents revealed distinctive and specific staining in major groups of noradrenergic neurons, including the locus coeruleus (LC), A1, A5 and A7 neurons, which appear to be major sites of production/release in the CNS. No staining was detected in dopaminergic neurons. Peptidergic axons were seen throughout the brain (notably hippocampus and cerebral cortex) and spinal cord, indicative of anterograde axonal transport of pepcans. Intriguingly, the chromaffin cells in the adrenal medulla were also strongly stained for pepcans. We found specific co-expression of pepcans with galanin, both in the LC and adrenal gland. Using LC-MS/MS, pepcan-12 was only detected in non-perfused brain (∼40 pmol/g), suggesting that in the CNS it is secreted and present in extracellular compartments. In adrenal glands, significantly more pepcan-12 (400-700 pmol/g) was measured in both non-perfused and perfused tissue. Thus, chromaffin cells may be a major production site of pepcan-12 found in blood. These data uncover important areas of peptide endocannabinoid occurrence with exclusive noradrenergic immunohistochemical staining, opening new doors to investigate their potential physiological function in the ECS. This article is part of a Special Issue entitled 'Fluorescent Neuro-Ligands'.

Utility of 30 and 60 minute cortisol samples after high-dose synthetic ACTH-1-24 injection in the diagnosis of adrenal insufficiency

BACKGROUND: In clinical practise the high dose ACTH stimulation test (HDT) is frequently used in the assessment of adrenal insufficiency (AI). However, there is uncertainty regarding optimal time-points and number of blood samplings. The present study compared the utility of a single cortisol value taken either 30 or 60 minutes after ACTH stimulation with the traditional interpretation of the HDT. METHODS: Retrospective analysis of 73 HDT performed at a single tertiary endocrine centre. Serum cortisol was measured at baseline, 30 and 60 minutes after intravenous administration of 250 µg synthetic ACTH1-24. Adrenal insufficiency (AI) was defined as a stimulated cortisol level <550 nmol/l. RESULTS: There were twenty patients (27.4%) who showed an insufficient rise in serum cortisol using traditional HDT criteria and were diagnosed to suffer from AI. There were ten individuals who showed insufficient cortisol values after 30 minutes, rising to sufficient levels at 60 minutes. All patients revealing an insufficient cortisol response result after 60 minutes also had an insufficient result after 30 minutes. The cortisol value taken after 30 minutes did not add incremental diagnostic value in any of the cases under investigation compared with the 60 minutes' sample. CONCLUSIONS: Based on the findings of the present analysis the utility of a cortisol measurement 30 minutes after high dose ACTH injection was low and did not add incremental diagnostic value to a single measurement after 60 minutes.