Mutant WD-repeat protein in triple-A syndrome.

Triple-A syndrome (MIM 231550; also known as Allgrove syndrome) is an autosomal recessive disorder characterized by adrenocorticotropin hormone (ACTH)-resistant adrenal insufficiency, achalasia of the oesophageal cardia and alacrima. Whereas several lines of evidence indicate that triple-A syndrome results from the abnormal development of the autonomic nervous system, late-onset progressive neurological symptoms (including cerebellar ataxia, peripheral neuropathy and mild dementia) suggest that the central nervous system may be involved in the disease as well. Using fine-mapping based on linkage disequilibrium in North African inbred families, we identified a short ancestral haplotype on chromosome 12q13 (<1 cM), sequenced a BAC contig encompassing the triple-A minimal region and identified a novel gene (AAAS) encoding a protein of 547 amino acids that is mutant in affected individuals. We found five homozygous truncating mutations in unrelated patients and ascribed the founder effect in North African families to a single splice-donor site mutation that occurred more than 2,400 years ago. The predicted product of AAAS, ALADIN (for alacrima-achalasia-adrenal insufficiency neurologic disorder), belongs to the WD-repeat family of regulatory proteins, indicating a new disease mechanism involved in triple-A syndrome. The expression of the gene in both neuroendocrine and cerebral structures points to a role in the normal development of the peripheral and central nervous systems.

Hormonal regulation of the mouse adrenal melanocortinergic system.

Adrenocortical cells of several species have been reported to express significant levels of Agouti-related protein (Agrp) as well as melanocortin 4-receptor (MC4-R). In this study, we used the mouse tumoral adrenal cell line ATC7- L that secretes corticosterone in basal conditions with a 2- fold increase in response to ACTH treatment. We reported that these cells expressed functional MC4-R. They also expressed Agrp mRNA and secreted immunoreactive Agrp in the culture medium. Long-term treatment of ATC7-L with (Nle4,D-Phe7)-alpha MSH (NDP-alpha MSH) or forskolin as well as Agrp strongly reduced MC4-R level by more than 30%. On the contrary, leptin treatment did not modify this level although it significantly reduced MC2-R level. These results could be correlated to some data obtained in vivo on adrenal glands removed from diet-induced obese mice exhibiting a hyperleptinemia, where the level of both MC2-R and MC4-R appeared to be reduced as Agrp mRNA expression level was increased compared to Control mice. All these data would suggest the existence of a link between the metabolic status and the activation of the adrenal melanocortinergic system.

Regulation of corticotropin receptor number and messenger RNA in cultured human adrenocortical cells by corticotropin and angiotensin II.

The regulation of ACTH receptor binding sites and mRNA by ACTH and angiotensin II (A-II) was studied using cultured human adrenal fasciculata reticularis cells (HAC). These cells expressed two major ACTH receptor transcripts of 1.8 and 3.4 kb and three minor ones of 4, 7, and 11 kb. ACTH increased the levels of all these transcripts in a time- and dose-dependent manner. At a maximal concentration of 10(-8) M, ACTH enhanced 21- and 4-fold the level of ACTH receptor mRNA and the number of receptors per cell, respectively. Pretreatment of HAC with A-II produced a dose-dependent enhancement of ACTH receptor mRNA that was associated with an increase of both ACTH receptor number and responsiveness to this hormone. The effects of A-II were completely blocked by an AT1 receptor subtype antagonist but not by an AT2 antagonist. The effects of ACTH together with A-II on ACTH receptor mRNA were greater than those induced by each hormone alone. These results show that ACTH receptor number and mRNA are positively regulated by the two main hormones (ACTH and A-II) which, in vivo, regulate adrenocortical functions. In addition, they also show that HAC are a target for A-II. Thus, regulation of ACTH receptors may be one mechanism by which ACTH and A-II regulate adrenocortical functions under both normal and pathological conditions.

Identification of Urop11, a novel leptin-modulated gene that is upregulated in the hypothalamus of mice with virus-induced obesity.

Obesity results from disturbances of tightly regulated interactions between the nervous, endocrine, and metabolic systems that can be caused by external factors, such as viral infections. A mouse model of obesity induced by brain infection with a morbillivirus, canine distemper virus, allowed us to identify obesity-related genes. Using a subtractive library for the hypothalamus, the main brain structure regulating energy homeostasis, we identified a new gene on mouse chromosome 19 which we named upregulated obese product (Urop) 11 and, which has no homology with any known mRNA. A step-by-step molecular approach allowed us to isolate the full-length mRNA, predict the protein sequence, and identify consensus sites. Urop11 was mainly detected in the hypothalamus and adipocytes, and was dramatically upregulated in these central and peripheral structures in obese mice. Urop11 was also expressed in human neural and lymphoid samples and its expression seemed to be regulated by the state of lymphocyte activation. Interestingly, Urop11 expression was strongly upregulated both in vivo in mouse hypothalamus and in vitro in mouse neural cell lines, after leptin treatment. Taken together, our data show that Urop11 is a target of leptin, the satiety factor produced by adipocytes, in physiological and pathological conditions, including obesity. This new gene can be considered a key molecule in the hypothalamic integration pathway and demonstrates the importance of Urop11 as a target of leptin action.

Presence of multiple functional polyadenylation signals in the 3'-untranslated region of human corticotropin receptor cDNA.

We present 2.59 kb of the 3'-non-coding region of the ACTH receptor cDNA that contains seven potential polyadenylation signals. Among these signals, five are functional as detected by 3'-RACE and are consistent with the transcripts of 1.8, 3.4 and 4 kb visualized on Northern blots. We propose that the most likely molecular mechanism for the multiple ACTH-R mRNA transcripts is the alternative use of polyadenylation signals.

Expression of the human melanocortin-4 receptor gene is controlled by several members of the Sp transcription factor family.

The melanocortin-4 receptor (MC4-R) plays a key role in the hypothalamic control of food intake, lending importance to the understanding of the mechanisms that regulate its expression. To identify factors controlling the expression of the human (h) MC4-R gene, a fragment containing 1253 bp of the 5'-flanking region of the hMC4-R gene was isolated. A series of hMC4-R luciferase constructs were developed and used to transiently transfect HEK293 and GT1-7 cell lines, both expressing endogenous MC4-R mRNA. Deletion analysis of the 1253 bp fragment showed that the basal promoter activity is mainly restricted to the 179 bp upstream of the transcription start site in both cell types. Mutation of a putative Sp1-binding site located at position -76 bp resulted in a dramatic reduction of the luciferase activity in HEK293 and GT1-7 cells by 87 and 80% respectively. Both in vitro and in vivo studies (gel shift and chromatin immunoprecipitation analyses) revealed binding of both Sp1 and Sp3 to this site in HEK293 cells. Cotransfection with an Sp1 expression vector in Drosophila cells that do not express Sp1, in conjunction with treatment of HEK293 cells with mithramycin A, a specific inhibitor of Sp1, confirmed the role of Sp1. For the first time, we have demonstrated that the constitutive activity of the hMC4-R promoter is dependent upon Sp transcription factors.

Functional relationships between three novel homozygous mutations in the ACTH receptor gene and familial glucocorticoid deficiency.

Familial glucocorticoid deficiency (FGD) is an autosomal recessive disorder characterized by a glucocorticoid adrenal insufficiency without mineralocorticoid deficiency. Mutations of the ACTH receptor (MC2-R) gene have been reported in some FGD cases, but only a few of them have been functionally studied. We reported clinical features and MC2-R gene analysis in three families. For each proband, an homozygous mutation was identified after amplification and sequencing of the whole intronless MC2-R gene. One mutation converted Val-142 located in the second intracellular loop to Leu. Another mutation in the sixth transmembrane domain converted Ala-233 to Pro. The last mutation converted the negatively charged Asp-103 in the first extracellular loop to an uncharged Asn. Functional studies of these mutations as well as the S120R mutation were performed after stable transfection of M3 cells and measurement of ACTH-induced cAMP production. For the S120R, V142L, and A233P mutated MC2-R, cAMP production curves were similar to that obtained with M3 parental cells, confirming that these mutations are responsible for the FGD in the affected patients. The D103N-mutated MC2-R had an impaired cAMP response to physiological doses of ACTH, but the maximal response at very high concentrations of ACTH was similar to that obtained for the wild-type MC2-R. All these results demonstrated clear relationships based on functional studies between MC2-R homozygous mutations and FGD phenotype.

Regulation of expression of male-specific rat liver microsomal 3 beta-hydroxysteroid dehydrogenase.

In the steroidogenic pathways present in the gonads and adrenal cortex, 3 beta-hydroxysteroid dehydrogenase isomerase (3 beta HSD) is a key enzyme which controls the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids. Herein, we used an antibody against human placental 3 beta HSD and a rat testicular 3 beta HSD cDNA probe to study the expression of rat liver 3 beta HSD mRNA and protein. Rat liver microsomal 3 beta HSD activity has been previously reported to exhibit a significant sex difference, with much higher activity in the male. We have shown an age-dependent increase in levels of immunoreactive 3 beta HSD through the time of maturation of the male rat. The immunoreactive protein, of similar molecular size to the human placental and rat testicular 3 beta HSD, was localized to the microsomal fraction of liver and was concentrated in pericentral locations. Immunoreactive protein was not detected in liver of immature (before 25 days of age) rats of either sex or in adult female liver. Northern blot analysis of liver and testicular RNA with a rat testicular 3 beta HSD cDNA probe revealed the presence of a 1.6-kilobase mRNA species in addition to the major 2.1-kilobase mRNA species in adult male liver, neither of which was detected in immature or adult female liver RNA. Hypophysectomy of female rats or treatment with testosterone implants caused induction of liver 3 beta HSD protein, while continuous infusion of GH to male rats decreased the level of 3 beta HSD protein. Similarly, the levels of the mRNA species were decreased after GH treatment. Using [3 alpha-3H]dehydroepiandrosterone as substrate for 3 beta HSD activity, we determined the apparent Km for liver microsomal NAD(+)-dependent 3 beta HSD activity to be 20 microM in both adult male and female liver and was much greater than the Km of rat Leydig tumor 3 beta HSD activity (0.2 microM). Liver 3 beta HSD activity was inhibited by trilostane, a proven inhibitor of gonadal and adrenal 3 beta HSD activity. A rat liver 3 beta HSD cDNA was isolated from a male liver cDNA library that was closely related to the type II 3 beta HSD form of rat ovary but different from type III liver 3 beta HSD. The enzyme obtained upon expression of this cDNA had properties characteristic of male-specific NAD(+)-dependent liver microsomal 3 beta HSD (i.e. high apparent Km for dehydroepiandrosterone) and distinct from those of the high affinity gonadal type I 3 beta HSD.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetics of ACTH insensitivity syndromes.

The last decade has seen remarkable progress in our understanding of the genetic causes of these potentially lethal conditions. Clearly, other genes that cause FGD remain to be discovered, and further work is required on the functions of MRAP and ALADIN in the expectation that they will provide insights into essential biological processes and perhaps identify key therapeutic strategies and targets for these diseases.

Prostaglandin E2 is a positive regulator of adrenocorticotropin receptors, 3 beta-hydroxysteroid dehydrogenase, and 17 alpha-hydroxylase expression in bovine adrenocortical cells.

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of cAMP. Herein we examine the effects of prostaglandin E2 (PGE2) on the differentiated functions of bovine adrenocortical (BAC) cells in primary culture. PGE2 (10 microM) treatment for 3 h stimulated steroidogenesis and cAMP production by over 100-fold. In addition, the cAMP antagonist Rp-cAMP (1 mM) inhibited PGE2 stimulation of steroidogenesis by 60%. This observation suggests that the cAMP second messenger system is responsible for much of the PGE2-activated steroid hormone synthesis. Chronic treatment of BAC cells with PGE2 caused induction of 3 beta-hydroxysteroid dehydrogenase and steroid 17 alpha-hydroxylase cytochrome P-450 expression as determined by the examination of enzyme activity, enzyme levels by immunoblotting, and specific messenger RNA (mRNA) levels by Northern analysis. The positive effects of PGE2 on expression of 3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase cytochrome P-450 were similar to the effects seen after ACTH treatment of BAC cells. In addition, treatment of BAC cells with PGE2 for 3 days caused a 3-fold induction of ACTH receptors as determined by increased cell binding of [125I]ACTH. Finally, we determined that BAC cells produced PGE2 and that the level of synthesis increased 10-fold after treatment with the hormone angiotensin II. Taken together these data indicate that PGE2 is a positive regulator of BAC cell differentiation acting on ACTH receptors, steroid metabolizing enzymes, and steroidogenesis. The ability of BAC cells to produce PGE2 leaves open the possibility for paracrine and autocrine regulation within the adrenal.

Regulation of 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase expression by adrenocorticotropin in bovine adrenocortical cells.

In the steroidogenic pathway, 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta HSD) catalyzes the formation of hormonally active delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids. In the present study the regulation of 3 beta HSD by ACTH action on bovine adrenocortical (BAC) cells in primary culture was evaluated. Western blot analysis was accomplished using an antibody against human placental 3 beta HSD. The relative molecular mass of 3 beta HSD in these cells was 45K, which was similar to that in human placenta. A significant effect of ACTH was not detected until day 6 of culture due to the high basal levels of the enzyme in BAC cells. Treatment of cells with ACTH on day 8 of culture resulted in a marked increase in the amount of 3 beta HSD protein, and this effect was correlated directly with enzymatic activity. The effects of ACTH were time and dose dependent, with an increase detectable only after 48 h of treatment; the maximal response was obtained with 10(-9) M ACTH. As demonstrated by Northern analysis, ACTH action was manifested by increasing the steady state level of 3 beta HSD mRNA. A human 3 beta HSD cDNA probe, which was used in this study, hybridized to a 1.7-kilobase species of BAC RNA. The effects of ACTH on 3 beta HSD activity and increases in 3 beta HSD protein and mRNA in BAC cells were mimicked by treatment with (Bu)2cAMP. The findings of this study suggest that ACTH controls 3 beta HSD gene expression in BAC cells by a cAMP-dependent mechanism similar to that involved in the expression of steroid hydroxylase genes. However, because the different stabilities of 3 beta HSD and hydroxylase proteins and/or mRNAs may play a critical role in determining the zone-specific steroids secreted from the adrenal cortex, other cAMP-dependent or independent regulatory mechanisms may also be important in regulating the expression of adrenal 3 beta HSD.

Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase in mouse tissues: male-specific isoforms are expressed in the gonads and liver.

Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta HSD) are expressed in various mouse tissues in a tissue-specific, sex-specific, and developmental manner. Three distinct immunoreactive species [molecular masses, 47, 44, and 42 kilodaltons (kDa)] are detectable by Western immunoblot analysis using a 3 beta HSD antiserum. Different immunoreactive isoforms are expressed in steroidogenic (44 and 47 kDa in gonads) and nonsteroidogenic (42 and 47 kDa in liver and kidney) tissues. Two of these isoforms are sex-specific in the gonads (47 kDa) and liver (42 kDa), because they are detectable only in male mice. Sex-specific expression in the liver is developmentally regulated. Low levels of this male-specific hepatic isoform are first detectable at 23-25 days of age, but its level of expression increases progressively during sexual maturation to adult levels. NAD(+)-dependent 3 beta HSD activity is detectable in homogenates of all tissues examined, but the kinetic characteristics of this activity differ among tissues and are sexually dimorphic in the liver. Apparent Michaelis constants for dehydroepiandrosterone are much lower in steroidogenic (0.24 +/- 0.07 microM for testis) than in nonsteroidogenic (range, 10-100 microM for liver and kidney) tissues and are lower in male mouse liver (16 +/- 1 microM) than in female mouse liver (82 +/- 20 microM). Oligonucleotides with unique sequences but encoding homologous regions of the mouse type I, II, and III 3 beta HSD cDNAs were used for Northern blot analyses. A type I oligomer hybridizes with RNA from steroidogenic (adrenal, ovary, and testis) tissues, and a type III oligomer hybridizes with RNA from nonsteroidogenic (liver and kidney) tissues. A type II oligomer, however, hybridizes specifically with RNA from testis and liver of male mice, tissues that express a male-specific 3 beta HSD. These results suggest that type II-like transcripts may encode a 47-kDa sex-specific 3 beta HSD in testis and a 42-kDa sex-specific 3 beta HSD in liver of male mice. It is unclear how many members of subfamilies of the 3 beta HSD gene family will be discovered. The mouse may prove to be a valuable experimental model, as this is the first species in which multiple immunoreactive isoforms can be identified in a single tissue. This multiplicity makes it difficult to correlate the size and number of immunoreactive isoforms with the diverse kinetic characteristics of NAD(+)-dependent 3 beta HSD activities in tissue homogenates and to develop isoform-specific probes.(ABSTRACT TRUNCATED AT 400 WORDS)

Study of the alteration of gene expression in adipose tissue of diet-induced obese mice by microarray and reverse transcription-polymerase chain reaction analyses.

In the present study we developed a model of diet-induced obesity (DIO) in male C57 BL/6J mice using an 8-wk high fat diet. This model should better reflect the physiology of the majority of the human obese patients than mouse genetic models of obesity with defects in leptin or leptin signaling. At the end of the diet, DIO mice displayed an increased weight (20%) and higher leptin, insulin, glucose, and corticosterone plasma levels compared with mice fed a standard diet during the same period. Moreover, they became resistant to the central effect of peripheral administration of leptin. Oligonucleotide microarray studies were conducted in adipose tissue. They showed that a great number of genes are differentially expressed. The majority of these genes (69%) are down-regulated in DIO mice. Among those are genes encoding enzymes of the lipid metabolism or markers of adipocyte differentiation, enzymes involved in detoxification processes, as well as structural components of the cytoskeleton. Some other groups of genes displayed increased expression, such as those encoding inflammatory markers. The results of the microarray analysis were confirmed by semiquantitative RT-PCR studies run on a selected number of genes that were differentially expressed or not modified.

Transforming growth factor-beta inhibits steroid 17 alpha-hydroxylase cytochrome P-450 expression in ovine adrenocortical cells.

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of ACTH to promote the synthesis of the various steroid-metabolizing cytochrome P-450 enzymes. In the steroidogenic pathway, 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha) is a key enzyme controlling the formation of cortisol and androgens. Recently, we demonstrated that transforming growth factor-beta (TGF beta) is a potent inhibitor of steroid production in ovine adrenocortical cells. In the present study we used a polyclonal antibody to P450(17) alpha to determine adrenal cell P-450(17) alpha enzyme content by Western analysis. In addition, we used a cDNA probe encoding for bovine P-450(17) alpha mRNA to determine levels of P-450(17) alpha mRNA in sheep ovarian adrenocortical cells in primary culture. When cells were cultured in a serum-free medium in the presence of ACTH for 48 h, P-450(17) alpha activity, enzyme content, and mRNA levels for P-450(17) alpha increased by 3- to more than 10-fold. TGF beta decreased the basal level and completely blocked the stimulatory action of ACTH on P-450(17) alpha enzyme activity. The effects of TGF beta on P-450(17) alpha enzyme content and mRNA levels were manifested in a dose-dependent manner, with maximal inhibition observed using 1 ng/ml TGF beta. Importantly, the inhibitory effects of TGF beta on P-450(17) alpha were not overcome by (Bu)2cAMP. These findings indicate that TGF beta is a potent negative regulator of P-450, and the inhibitory action appears to be at the level of P-450(17) alpha gene expression. The ability of TGF beta to suppress the positive stimulatory action of ACTH suggests that TGF beta could play a role in determining the pathway of steroidogenesis and, thereby, the specific steroids secreted by adrenocortical cells.

Functional hypersomatotropism in small for gestational age (SGA) newborn infants.

Basal plasma GH levels and the GH responses to an injection of 1 microgram/kg 1-44(NH2) GHRH were determined on day 3 postnatally in 5 small gestational age (SGA) twin newborns and their appropriate gestational age (AGA) co-twins, and in 10 SGA singleton newborns and 6 AGA singleton newborns. The mean basal plasma GH level was higher in the SGA than in the AGA infants but the difference was significant only for singleton newborns (p less than 0.01). The mean peak plasma GH level was markedly increased in SGA compared to AGA infants (p less than 0.05 for twins, p less than 0.01 for singletons). Twelve SGA infants re-tested at 1 month had lower basal and peak plasma GH levels (p less than 0.001 and p less than 0.01). In 21 SGA and 17 AGA infants, serum IGF-I, measured by RIA between 12 and 96 hours after birth, was significantly higher in SGA than in AGA (p less than 0.001). These results suggest that, whatever the mechanism, functional hypersomatotropism is present at day 3 in SGA infants. This hypersomatotropism may participate in the early catch-up growth process.

Effects of transforming growth factor-beta 1 on human adrenocortical fasciculata-reticularis cell differentiated functions.

Transforming growth factor-beta 1 (TGF beta 1) has been reported to have a strong inhibitory effect on the specific function of adrenal cells of several species. In the present study, we examined the effects of TGF beta 1 on cultured human fasciculata-reticularis cells. TGF beta 1 alone had no effect on ACTH receptor messenger ribonucleic acid (mRNA) levels and was unable to reduce the strong stimulatory effects of ACTH on its own receptor. However, TGF beta 1 enhanced angiotensin-II type 1 receptor mRNA and binding sites. Treatment with TGF beta 1 increased significantly the levels of 3 beta-hydroxysteroid dehydrogenase mRNA, reduced those of cytochrome P-450 17 alpha-hydroxylase mRNA, and had no effect on cholesterol side-chain cleavage cytochrome P-450 mRNA. Whatever the experimental condition, TGF beta 1 did not reduce basal or ACTH-stimulated cortisol production, but the production of dehydroepiandrosterone sulfate of TGF beta 1-treated cells was always decreased. The effects of TGF beta 1 on 3 beta-hydroxysteroid dehydrogenase mRNA and dehydroepiandrosterone sulfate were opposite the change observed at the time of adrenarche. As adrenal cells express TGF beta 1 mRNA, it is tempting to postulate that a local diminution of TGF beta 1 might be involved in adrenarche. Our findings also illustrate the specific species differences and, therefore, the caution to extrapolate to humans the results observed in other species.

Exclusion of the adrenocorticotropin (ACTH) receptor (MC2R) locus in some families with ACTH resistance but no mutations of the MC2R coding sequence (familial glucocorticoid deficiency type 2).

Several mutations in the coding exon of the ACTH receptor (MC2R) gene have been reported in cases of familial glucocorticoid deficiency or FGD. However, many patients with a similar syndrome do not present any mutation in the coding region of this gene. This is the case in 11 families we have investigated. Patients in these families present the typical clinical features of FGD, but no mutation was found in the coding exon of the ACTH receptor. To determine whether mutations on MC2R gene, but outside the coding region, may be involved in FGD in these families, we have performed a linkage analysis. Using three markers flanking MC2R gene on chromosome 18, we were able to exclude linkage in a region of 12 centimorgans around the gene. This result clearly indicates that FGD is genetically heterogeneous. Defects in gene(s) different from MC2R gene are implicated in this syndrome.

Demonstration by transfection studies that mutations in the adrenocorticotropin receptor gene are one cause of the hereditary syndrome of glucocorticoid deficiency.

The hereditary syndrome of unresponsiveness to ACTH is a rare autosomal recessive disorder characterized by low levels of serum cortisol and high levels of plasma ACTH. There is no cortisol response to exogenous ACTH. Recent cloning of the human ACTH receptor gene has enabled us to study this gene in patients with glucocorticoid deficiency. By using the PCR to amplify the coding sequence of the ACTH receptor gene, we identified three mutations in two unrelated patients. One mutation present in homozygous form converted the negatively charged Asp107, located in the third transmembrane domain, to an uncharged Asn residue. The second patient was a compound heterozygote: the paternal allele contained a one-nucleotide insertion leading to a stop codon within the third extracellular loop, and the maternal allele contained a point mutation converting Cys251 to Phe, also in the third extracellular loop. Normal and mutant ACTH receptor genes were expressed in the M3 cell line, and intracellular cAMP production in response to ACTH was measured. For the mutant receptors, no response to physiological ACTH concentrations was detected, suggesting an impaired binding of ACTH to the receptors and/or an altered coupling to the adenylate cyclase effector.

Characterization and regulation of the angiotensin II type-1 receptor (binding and mRNA) in human adrenal fasciculata-reticularis cells.

The classical concept of human adrenal physiology indicates that only glomerulosa cells are the target of A-II. Herein, we demonstrated that cultured human adrenal fasciculata-reticularis cells were also responsive to this hormone. Indeed, these cells contained high affinity (Kd = 0.9-1.1 nM) and low capacity (8,000-13,000 sites/cell) A-II receptors, and more than 95% of them were of the type-1. These AT1 receptors are functional since A-II was able to increase cortisol production after 48 h of treatment. These effects were inhibited by losartan, an AT1 antagonist, but not by CGP42112A, an AT2 antagonist. The expression of the type-1 A-II receptor mRNA was detected in the whole adrenal in both adult and fetus, and in cultured human adrenal fasciculata-reticularis cells. In these cells A-II negatively regulated AT1 receptor mRNA, and this effect was also mediated through the AT1 receptor subtype.

An E-box-containing region is involved in the tissue-specific expression of the human MC2R gene.

Expression of the melanocortin receptor (MC2R) gene is limited to adrenocortical cells and the aim of this study was to determine the factors responsible for this tissue specificity. We used different fragments of the human (h) MC2R gene promoter, inserted in a vector upstream of the luciferase reporter gene, to transiently transfect either bovine adrenocortical (BAC) cells or granulosa cells from bovine ovaries (B-Gran). Similar promoter activities were obtained in both cell types using constructs containing fragments up to 1017 bp of the hMC2R gene promoter. On the contrary, a 2-fold decrease was obtained after transfection of the B-Gran cells with vectors containing 1069 bp and more of the promoter. Results obtained here using BAC cells confirmed our previous data on human cells showing that steroidogenic factor 1 is the major transactivating factor involved in the basal expression of the hMC2R gene in adrenal cells. However, we showed that this factor did not permit, by itself, the expression of the hMC2R gene in B-Gran cells despite its expression in these cells. This study demonstrated for the first time that an E-box (located at -1020 bp) is involved in the repression of hMC2R gene expression in granulosa cells through interactions with several factors, such as activator protein 4, as suggested by electrophoretic mobility shift assay analyses.