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International audience; The coding sequence of the human ACTH receptor, cloned in 1992, contains no intron, but the presence of one intron (of about 18 kb) separating the coding exon from an upstream exon has been demonstrated. One major transcription start site was located in this first exon. Northern blot analysis of cultured human adrenocortical cells revealed several transcripts that can be partly explained by the use of different polyadenylation sites. We have isolated a 1 kb fragment of genomic DNA upstream of exon 1 and studied its basal promoter activity. The sequence of this region shows several putative CREs that could be responsible for the stimulation by ACTH of its own receptors as demonstrated on human adrenocortical cells. To functionally characterize the human ACTH receptor, we have prepared cells stably transfected with either the normal receptor or a mutant receptor. This model allows the study of both binding to ACTH and coupling to adenylate cyclase. Two naturally mutated receptors, described in patients with Familial Glucocorticoid Deficiency, have been studied. Both mutations (C251F and D107N) strongly impaired the binding of ACTH to its receptors and are then responsible for the absence of biological response to ACTH.

The entire ACTH receptor (ACTH-R) cDNA was amplified by RT/PCR from mouse Y-1 adrenocortical cells, subcloned into the pMOSBlue T vector, sequenced and inserted into the pSVK3 mammalian vector to obtain pSVACTHR. Balb 3T3 fibroblasts were co-transfected with pSVACTHR plus pSV2-neo and the transfectants were selected with G418 and cloned. Genomic integration of pSVACTHR and transcription of ACTH-R cDNA were checked by Southern blot and RT/PCR respectively. Expression of active ACTH-R protein was tested by measuring cAMP production in response to ACTH. Two ACTH-R expressing transfectants (clones 03 and 07) increased cAMP accumulation in response to ACTH. They were morphologically identical to parental 3T3 cells, but required 10-20% FCS to grow. In these transfectants, ACTH induced c-FOS protein expression, but did not activate the ERK isoforms of MAP Kinase and did not stimulate DNA synthesis. Apparently, the ACTH-R in Balb 3T3 cells induces the c-fos gene by a pathway independent of cAMP/protein kinase A and ERK/MAP Kinase.

Two mutant clones (Y6 and OS3) derived from the ACTH-responsive Y1 mouse adrenocortical tumor cell line fail to respond to ACTH with increased adenylyl cyclase activity and, as a consequence, are resistant to the steroidogenic effects of the hormone. As determined from Northern blot and RNase protection assays, ACTH resistance in these mutants results from the failure to accumulate ACTH receptor transcripts. The ACTH receptor gene appears to be present in these mutants as determined by Southern blot hybridization analysis and can be activated following the growth of the mutant cells as tumors in mice, suggesting that the ACTH receptor gene is modified in a reversible manner. When mutant cells are transformed with a gene encoding the mouse beta 2-adrenergic receptor they respond to beta-adrenergic agonists with increased adenylyl cyclase activity in a manner that is indistinguishable from a similarly transformed parent Y1 cell line. These results suggest that the adenylyl cyclase system in the mutants is otherwise intact and that the failure to express ACTH receptor transcripts limits the responsiveness of these clones to the hormone.

The recent cloning of a family of melanocortin receptors (MC-R) has identified five distinct G protein- and adenylate cyclase-coupled receptors. The MC2-receptor (MC2-R) preferentially binds ACTH. It is expressed in the adrenal cortex and is hence considered to be the ACTH receptor. The MC5-receptor (MC5-R) binds ACTH and alpha-MSH and is more widely expressed. The aim of this work was to study the sites of MC5-R expression in the bovine adrenal cortex and to compare the regulation of the expression of MC2-R and MC5-R in bovine adrenocortical cells in primary culture. Analysis of the expression of MC5-R was obtained by RT-PCR, using total RNA purified from glomerulosa and fasciculata zones of bovine adrenocortical tissue. MC5-R expression could be detected in RNA from the glomerulosa zone but was undetectable in the fasciculata zone. In bovine adrenocortical cells in culture, ACTH stimulates MC5-R expression in the glomerulosa and fasciculata cells. A DNA fragment, was obtained using primers based on the bovine ACTH receptor (MC2-R) sequence. This fragment was detected in RNA from the two zones. The probe was used to quantify MC2-R by Ribonuclease Protection assay and we observed that MC2-R mRNA is 3.6-fold more abundant in glomerulosa than in fasciculata-reticularis cells.

The pro-opiomelanocortin (POMC)-derived peptides, pro-gamma-MSH (16K fragment), and Lys-gamma3-MSH, have been shown to potentiate the steroidogenic action of corticotrophin (ACTH) on the adrenal cortex. Using a continuously perfused adrenal cell column system, we have tested the hypothesis that gamma-MSH peptides exert their effect through the Melanocortin 3 Receptor (MC3-R), since this is the only known receptor to have high affinity for gamma-MSH peptides and has been suggested to be expressed in the rat adrenal. To investigate this hypothesis we tested whether the MC3-R agonist MTII and antagonist SHU9119 could mimic or block the actions of pro-gamma-MSH. We found that MTII could not mimic, and SHU9119 could not block pro-gamma-MSH mediated potentiation of ACTH-induced steroidogenesis. These results suggest that the MC3-R is not involved in mediating the potentiation effect, adding further evidence to the argument that another melanocortin receptor exists.

Sphingolipids are a diverse family of phospholipids and glycolipids that mediate cell-cell interactions, participate in signal transduction pathways and modulate the activity of various cellular proteins and receptors. The objective of the present studies was to characterize the role of the sphingolipid biosynthetic pathway in adrenocorticotropin (ACTH)-dependent steroidogenic gene expression and cortisol production. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt2cAMP) for various time periods and the content of sphingolipids was quantified by mass spectrometry. Treatment of H295R cells with ACTH and Bt2cAMP activated sphingolipid metabolism within five minutes. Decreases were found in the cellular levels of several sphingolipids, including sphingomyelin (SM) and glucosylceramide. ACTH/cAMP rapidly decreased levels of the signaling molecules ceramide, sphingosine and sphingosine-1-phosphate (S1P). The effect of these bioactive sphingolipids on steroidogenic gene expression was also examined. Both sphingosine and S1P were found to increase endogenous CYP17 mRNA and activate the transcriptional activity of CYP17-luciferase reporter constructs. Further, sphingosine and S1P rapidly increase cortisol biosynthesis in H295R cells. In summary, our studies establish a link between ACTH/cAMP-dependent steroidogenesis and sphingolipid metabolism in the human adrenal cortex. Finally, these findings suggest that sphingolipids may serve as signaling mediators in ACTH-stimulated cortisol biosynthesis.

The physiological effects of the pituitary hormone, adrenocorticotropic hormone (ACTH) on the adrenal are mediated by the melanocortin 2 receptor (MC2R), a G protein coupled receptor (GPCR) that signals via adenylate cyclase to elevate intracellular cyclic AMP (cAMP) levels. The function and expression of the receptor is likely to be a major determinant of the response to ACTH. Following repeated stimulation, the cAMP signal is diminished or desensitized. Prolonged desensitization may involve internalization of the receptor. Internalization may occur by at least two mechanisms--receptor mediated endocytosis via clathrin-coated pits and by caveolae mediated internalization. The mode of internalization for the endogenous MC2R in Y1 cells was determined using radiolabelled ACTH. Treatment of Y1 cells with hypertonic sucrose or with concanavalin A, which inhibit clathrin-mediated endocytosis, blocked internalization. Filipin and nystatin, which inhibit caveolae formation, did not influence internalization. A dominant negative GRK2 inhibited internalization whilst the protein kinase A (PKA) consensus site mutant MC2R (S208A) internalized normally. However, dominant negative V53D beta-arrestin-1 did not inhibit ACTH internalization in Y1 cells. In conclusion, it appears that the MC2R in Y1 cells internalizes by a G protein coupled receptor kinase (GRK) dependent clathrin-coated pit mechanism.

Although cAMP has long been regarded as the primary intracellular messenger for ACTH-stimulated cortisol secretion, a requirement for Ca2+ is well established. However, a specific mechanism which couples ACTH receptor activation to increased intracellular calcium concentration in the adrenal cortical cell has not been elucidated. Here, we present evidence for a specific model in which ACTH at picomolar concentrations induces cAMP which acts through kinase-dependent and independent pathways to stimulate cortisol secretion. Along one of these pathways, cAMP acts directly to depolarize cells by inhibition of a specific non-inactivating K+ channel (I(AC)). This model provides a specific mechanism whereby cAMP-mediated inhibition of I(AC) is tightly coupled to depolarization-dependent Ca2+ entry and cortisol secretion. Ca2+ and cAMP are dual second messengers in the ACTH signalling pathway that are linked through I(AC) K+ channels.

Cultured bovine adrenal fasciculata cells were used to characterize angiotensin II (A-II) and corticotropin (ACTH) receptors and to study their homologous and heterologous regulation. These cells contain one type of high affinity binding sites for A-II (KD congruent to 2.4 +/- 0.3 10(-9) M) and about 100000 sites/cell. Photoaffinity labeling followed by SDS-PAGE under reducing conditions revealed a single macromolecule of apparent MR 65,000. Treatment of cells with increasing concentrations of A-II produced down-regulation of its own receptors and marked homologous and heterologous (ACTH) steroidogenic desensitization. However, the desensitization was not correlated with receptor loss and was mainly due to alterations of the steroidogenic pathway. Pretreatment of cells with ACTH also reduced A-II receptors, but this was not associated with steroidogenic desensitization. Bovine fasciculata cells contain two binding sites for ACTH: one of high affinity (KD congruent to 2.6 +/- 0.4 10(-10) M) and low capacity (2030 +/- 390 sites/cell) and the other of low affinity and high capacity. Affinity cross-linking of ACTH to plasma membranes prepared from adrenal cells revealed a labeled macromolecule of apparent MR 43000. However, cross-linking experiments to intact cells revealed, both under reducing and non-reducing conditions, two labeled macromolecules of apparent MR of 123000 and 43000. Pretreatment of cells with ACTH enhanced its receptor and the cAMP and cortisol responses to further ACTH stimulation. These effects were time- and dose-dependent. The maximal effects were observed at 10(-10) to 10(-9) M. A-II alone had no effect but it blocked partially the stimulatory action of ACTH.

Rat adrenocortical cells utilize both low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol for steroid hormone production. In addition to exogenous lipoprotein-derived cholesterol, cells produce cholesterol de novo. Adrenocorticotropin (ACTH) increases both steroid hormone secretion and uptake of LDL and HDL. We studied the expression of LDL receptor mRNA and CLA- I (a putative HDL receptor) mRNA in cultured rat adrenocortical cells. ACTH increased the amounts of LDL receptor mRNA during 2 to 48 h of stimulation, the highest levels being detected after 2–4 h. Similar results were obtained with cyclic AMP (cAMP) derivatives, 8-bromo cAMP (8-Br cAMP) or dibutyryl cAMP. ACTH increased CLA-1 mRNA during 2 to 24 h of stimulation, the highest levels being detected after 4 h. In conclusion, ACTH up regulates both LDL and HDL receptor mRNA in rat adrenocortical cells.

The rapid, cAMP mediated increase produced by ACTH in adrenal corticosteroidogenesis depends also on the rapid synthesis of protein. This obligatory involvement of protein synthesis has been established in several laboratories mainly on the basis of studies demonstrating a parallelism, under a variety of experimental conditions, of the capacity of adrenal cells to synthesize protein with their ability to increase steroid production in response to ACTH or cAMP. More recent correlative studies have disclosed the existence of two proteins, denoted as p and i. Protein i appears only in ACTH or cAMP stimulated cells and with the same time course and ACTH dose response as the increase in corticosteroid synthesis. Protein p, which closely resembles i in molecular weight and primary structure but differs in pI, is synthesized only in unstimulated cells. Neither the function of these two proteins nor the exact nature of the interplay of their synthesis on regulation has been established, although it seems reasonably certain that i is stimulatory rather than p inhibitory. The protein glycosylation inhibitor tunicamycin was found to inhibit the ACTH produced increase in steroidogenesis and also to inhibit the synthesis of protein i under conditions which did not inhibit overall protein synthesis. Therefore it seems probable that i is an N-glycosylated form of protein p and that adrenal cells are unresponsive to acute ACTH action if they are incapable of synthesizing glycosylated proteins specifically.

The detection and characterization of the physiologically relevant receptors for corticotropin (ACTH) in rat adrenocortical cells is described. By the use of a radioligand with full biological potency and high specific radioactivity (1800 +/- 75 Ci/mmol), a single class of receptors with an apparent Kd of 1.41 +/- 0.21 nM was detected and the number of sites was estimated to be 3840 +/- 1045 per cell. The binding curve was superimposable on the concentration-response curve for cAMP but dissociated from that for steroidogenesis. These data are best explained by the receptor-reserve model in which occupancy of a small fraction of the receptors is sufficient for inducing maximal steroidogenesis.

ACTH has both short-term (acute) and long-term (chronic) effects to regulate steroid hormone biosynthesis in the adrenal cortex. The acute action of ACTH involves the mobilization of cholesterol and its binding to cytochrome P-450scc. The long-term action of ACTH involves the regulation of synthesis of the various enzymes involved in steroidogenesis. Evidence is presented that cholesterol may have a role to play in this regulatory process as it does in the short-term action of ACTH, consistent with the concept that substrates of specific forms of cytochrome P-450 are frequently able to regulate the synthesis of these specific forms.

Studies of ACTH functions in adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y1 cells. In order to obtain alternative cell lines with a more differentiated zona fasciculata (ZF) phenotype we used targeted tumorigenesis strategy. We have generated transgenic mice expressing the SV40 T antigen under the control of the ACTH-dependent promoter for the AKR1B7/MVDP gene (aldo-keto reductase 1B7/mouse vas deferens protein), which encodes an enzyme responsible for detoxifying isocaproaldehyde, the product of side-chain cleavage of cholesterol generated by steroidogenesis. Our previous data indicated that in the mouse adrenal, AKR1B7 expression was restricted to the ZF and that a 0.5-kb promoter region was able to target specific adrenal expression in transgenic mice. In situ hybridization analyses indicate that AKR1B7 expression during fetal and post-natal periods paralleled the onset of glucocorticoid synthesis and the development of ZF. In transgenic mice, ACTH control and developmental programming of the CAT gene driven by the 0.5-kb promoter followed endogenous gene regulation. Then transgenic mice harboring the 0.5-kb/SV40 T antigen construct were generated and two founders out of three developed adrenal tumors. Cells derived from the tumor of founder 1 (ATC1) were grown in presence of forskolin to maintain ACTH receptor expression and were tested for ACTH responsiveness by immunocychemistry and northern blot analyses. Even after several passages, the ACTH induced AKR1B7 and P450c11beta mRNAs accumulations were similar to that observed in mouse primary adrenocortical cell cultures. Our findings suggest that ATC1 cells have conserved essential features of ZF cells. In order to achieve complete characterization of these cells further analyses are currently performed to investigate their steroidogenic activity.

The mechanism of the altered adrenal responsiveness during chronic stress was studied by analysis of ACTH and Ang II responses and the expression and activity of steroidogenic enzymes in the adrenal cortex of rats subjected to repeated immobilization (2 hr/day for 14 days), or repeated i.p. injection of 1.5 M NaCl. Concomitant with increased pregnenolone production and reduced aldosterone secretion by isolated adrenal glomerulosa cells of chronically stressed rats, P-450scc mRNA were increased and P-450aldo mRNA levels were decreased in adrenal zona glomerulosa. Consistent with elevated plasma corticosterone levels, isolated adrenal fasciculata cells from stressed rats showed higher cAMP, pregnenolone and corticosterone responses to ACTH. Adrenal fasciculata area and levels of P-450scc, but not those of P-450(11s) hydroxylase were significantly increased. The effects of repeated stress on adrenal steroidogenesis were mimicked by repeated ACTH injections. The half life of corticosterone in plasma measured with [3H]corticosterone was increased in stressed rats but not in ACTH injected rats. This study shows that chronic stress leads to a) inhibition of mineralocorticoid secretion due to inhibition of the late biosynthetic pathway, and b) increased circulating glucocorticoids due to increased ACTH receptor activity, expression and activity of the early pathway, and decreased glucocorticoid clearance. Altered adrenal glomerulosa and fasciculata function, but not changes in glucocorticoid clearance, are probably mediated by increased ACTH secretion during chronic stress.