Marked Cortisol Production by Intracrine ACTH in GIP-Treated Cultured Adrenal Cells in Which the GIP Receptor Was Exogenously Introduced

The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing’s syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (–) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3β2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.     

Differential actions of metyrapone on the fetal pituitary-adrenal axis in the sheep fetus in late gestation

It is not clear if an increase in intra-adrenal cortisol is required to mediate the actions of adrenocorticotropic hormone (ACTH) on adrenal growth and steroidogenesis during the prepartum stimulation of the fetal pituitary-adrenal axis. We infused metyrapone, a competitive inhibitor of cortisol biosynthesis, into fetal sheep between 125 and 140 days of gestation (term = 147 +/- 3 days) and measured fetal plasma cortisol, 11-desoxycortisol, and ACTH; pituitary pro-opiomelanocortin mRNA and adrenal expression of ACTH receptor (melanocortin type 2 receptor), steroidogenic acute regulatory protein (StAR), 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), cytochrome P450 17-hydroxylase (CYP17), 3beta-hydroxysteroid dehydrogenase, and cytochrome P450 21-hydroxylase mRNA; and StAR protein in the fetal adrenal gland. Plasma ACTH and 11-desoxycortisol concentrations were higher (P < 0.05), whereas plasma cortisol concentrations were not significantly different in metyrapone- compared with vehicle-infused fetuses. The ratio of plasma cortisol to ACTH concentrations was higher (P < 0.0001) between 136 and 140 days than between 120 and 135 days of gestation in both metyrapone- and vehicle-infused fetuses. The combined adrenal weight and adrenocortical thickness were greater (P < 0.001), and cell density was lower (P < 0.01), in the zona fasciculata of adrenals from the metyrapone-infused group. Adrenal StAR mRNA expression was lower (P < 0.05), whereas the levels of mature StAR protein (30 kDa) were higher (P < 0.05), in the metyrapone-infused fetuses. In addition, adrenal mRNA expression of 11betaHSD2, CYP11A1, and CYP17 were higher (P < 0.05) in the metyrapone-infused fetuses. Thus, metyrapone administration may represent a unique model that allows the investigation of dissociation of the relative actions of ACTH and cortisol on fetal adrenal steroidogenesis and growth during late gestation.     

Ca2+ signal stimulates the expression of steroidogenic acute regulatory protein and steroidogenesis in bovine adrenal fasciculata-reticularis cells

Adrenal glucocorticoid synthesis is stimulated by ACTH or its nitrophenylsulphenyl derivative, NPS-ACTH. Acute stimulation of steroid hormone biosynthesis is highly dependent on the expression of steroidogenic acute regulatory (StAR) protein. To determine the regulatory mechanism of StAR expression in bovine fasciculata/reticularis cells, we analyzed the second messenger systems involved in StAR protein expression using cultured cells activated by ACTH and NPS-ACTH. We concluded that cAMP is not the essential second messenger for StAR protein expression, since NPS-ACTH activated StAR protein expression more than ACTH without increase in cellular cAMP. A 15-lipoxygenase metabolite(s) of arachidonic acid stimulated steroidogenesis without increase in StAR protein expression, since AA-861, a lipoxygenase inhibitor, inhibited steroidogenesis without affecting StAR protein expression. Stimulation of StAR protein expression and the corresponding increase in the steroidogenesis were inhibited by nicardipine in cells treated with ACTH or NPS-ACTH. These data indicate that the dominant second messenger for the stimulation of StAR protein expression is Ca2+. Calmodulin-dependent kinase II inhibitors KN-93 and KN-62 suppressed steroidogenic activity without affecting StAR expression. The protein kinase C inhibitor Ro 31-8220 did not show any effects on StAR expression and steroidogenesis. Calmodulin-dependent kinase II and protein kinase C can therefore be concluded not to be involved in StAR protein expression in bovine cells.     

Congenital adrenal hyperplasia: biochemical and molecular perspectives

Congenital adrenal hyperplasia is a disorder occurring in both sexes and is the commonest cause of ambiguous genitalia. It is a group of autosomal recessive disorders in which, on the basis of an enzyme defect the bulk of steroid hormone production by adrenal cortex shifts from corticosteroids to androgens. Autosomal recessive mutations in the CYP21, CYP17, CYP11B1 and 3betaHSD genes that encode steroidogenic enzymes, in addition to mutations in the gene encoding the intracellular cholesterol transport protein steroidogenic acute regulatory protein StAR can cause CAH. Each of the defects causes different biochemical consequences and clinical features. Deficiencies in 21 hydroxylase (21-OH) and 11beta-Hydroxylase (11beta-OH) are the two most frequent causes of CAH. All the biochemical defects impair cortisol secretion, resulting into compensatory hypersecretion of ACTH and consequent hyperplasia of the adrenal cortex. Research in recent years has clarified clinical, biochemical and genetic problems in diagnosis and treatment of the disorders. Expanding knowledge of the gene mutations associated with each of these disorders is providing valuable diagnostic tools in addition to the biochemical profile and phenotype. Genotyping is useful in selecting instances to provide genetic counseling and to clarify ambiguous cases.     

Vitamin D3 enhances ATRA-mediated neurosteroid biosynthesis in human glioma GI-1 cells

Emerging evidence indicates that vitamin D (VD) is an important modulator of brain development and function. To investigate whether VD modulates neurosteroid biosynthesis in neural cells, we investigated the effect of VD(3) on steroidogenic gene expression in human glioma GI-1 cells. We found that VD(3) enhanced CYP11A1 and 3β-hydroxysteroid dehydrogenase gene expression. The induction of CYP11A1 gene expression by VD(3) was dose- and incubation time-dependent. Calcipotriol, a VD(3) receptor (VDR) agonist, also induced CYP11A1 gene expression in GI-1 cells, indicating that VDR is involved in this induction. The induction of progesterone (PROG) de novo synthesis was observed along with the induction of steroidogenic genes by VD(3). Furthermore, VD(3) enhanced all-trans retinoic acid (ATRA)-induced CYP11A1 gene expression and PROG production. This suggests cooperative regulation of steroidogenic gene expression by the two fat-soluble vitamins, A and D. In addition, a mixed culture of neuronal IMR-32 cells and GI-1 cells treated with ATRA and VD(3) resulted in the induction of PROG-responsive gene expression in the IMR-32 cells. This result shows a paracrine action of PROG that is induced in and released by the GI-1 cells. The relationship between neurological dysfunction associated with VD deficiency and neurosteroid induction by VD is discussed.     

Prolyl oligopeptidase regulates progesterone secretion via the ERK signaling pathway in murine luteal cells

Prolyl oligopeptidase (POP), one of the most widely distributed serine endopeptidases, is highly expressed in the ovaries. However, the physiological role of POP in the ovaries is not clear. In this study, we investigated the significance of POP in the corpus luteum. Murine luteal cells were cultured in vitro and treated with a POP selective inhibitor, (2S)‐1[[(2 S)‐1‐(1‐oxo‐4‐phenylbutyl)‐2‐pyrrolidinyl carbonyl]‐2‐pyrrolidinecarbonitrile (KYP‐2047). We found that KYP‐2047 treatment decreased progesterone secretion. In contrast, POP overexpression increased progesterone secretion. Three essential steroidogenic enzymes, including p450 cholesterol side‐chain cleavage enzyme (CYP11A), 3β‐hydroxysteroid dehydrogenase (3β‐HSD), and the steroidogenic acute regulatory protein (StAR), were regulated by POP. Further studies showed that POP overexpression increased ERK1/2 phosphorylation and increased the expression of steroidogenic factor 1 (SF1), while KYP‐2047 treatment decreased ERK1/2 phosphorylation and SF1 expression. To clarify the role of ERK1/2 signaling in POP‐regulated progesterone synthesis, U0126‐EtOH, an inhibitor of the ERK signaling pathway, was used to treat luteal cells. We found that U0126‐EtOH decreased progesterone production and the expression of steroidogenic enzymes and SF1. POP overexpression did not reverse the effects of U0126‐EtOH. Overall, POP regulates progesterone secretion by stimulating the expression of CYP11A, 3β‐HSD, and StAR in luteal cells. ERK signaling and downstream SF1 expression contribute to this process.     

Mechanisms of inhibition of dehydroepiandrosterone upon corticosterone release from rat zona fasciculata-reticularis cells

We have demonstrated that dehydroepiandrosterone (DHEA) acts directly on rat zona fasciculata-reticularis (ZFR) cells to diminish corticosterone secretion by an inhibition of post-cAMP pathway, and decreases functions of steroidogenic enzymes after P450(scc) as well as steroidogenic acute regulatory (StAR) protein expression. However, the mechanisms by which DHEA engages with environmental messenger signals which translate into interfering StAR protein expression are still unclear. This study explored the effects of DHEA on the phosphorylation/activation of extracellular signal-regulated kinases (ERKs). ERK activation resulted in enhancing phosphorylation of steroidogenic factor-1 (SF-1) and increased StAR protein expression. ZFR cells were incubated in the presence or absence of adrenocorticotropin (ACTH), forskolin (FSK), 25-OH-cholesterol, U0126, and H89 at 37 degrees C. The concentration of corticosterone released into the media was measured by radioimmunoassay (RIA). The cells were used to extract protein for Western blot analysis of ERKs or StAR protein expression or immunoprecipitation of SF-1 analysis. The results suggested that (1) ERK pathway of rat ZFR cells might be PKA dependent, (2) ERK activity was required for SF-1 phosphorylation to upregulate steroidogenesis in rat ZFR cells, and (3) DHEA did not affect ERK phosphorylation, however, it attenuated forskolin-stimulated SF-1 phosphorylation to affect StAR protein expression.