Biomedical subjects
Publications and source records attributed to D Naville.
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.
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.
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.
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.
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.
Expression of the adrenocorticotropin receptor (MC2-R) is restricted to adrenocortical cells and is up-regulated by both adrenocorticotropin and angiotensin II through the activation of protein kinase A and protein kinase C pathways, respectively. After cloning of the promoter region of the human MC2-R gene (hMC2-R), we have shown that cyclic AMP-induced regulation of transcriptional activity of the gene is achieved through two SF1 binding elements located in the proximal promoter. On the other hand, regulation by angiotensin II partly involved two AP1 binding sites. Using different primary cell cultures, we have also been able to delineate a region inside the promoter which is responsible for part of the tissue-specific expression of the gene.
Previous studies have demonstrated that angiotensin-II (A-II) increases the human adrenocorticotropin receptor (hMC2R) gene expression in adrenal cells. In the present study, we have characterized two activator protein-1 (AP-1)-binding sites involved in the A-II stimulation of hMC2R gene transcription. Vectors containing different fragments of the hMC2R gene promoter inserted upstream of the luciferase gene, have been constructed. After transfection of H295R cells with these constructs and treatment of the cells by A-II during 48 h, maximal stimulation of the luciferase activity was obtained using the construct p(-263/+22)luc. Using progressively deleted constructs, three regions responsible for the A-II-stimulated transcription of hMC2R have been delineated. Inside these regions, two sequences displayed some homology with an AP-1 binding element (AP-108 and AP-203). Mutation of either AP-108 or AP-203 site induced a decrease of A-II-stimulated luciferase activity by 40% and 25%, respectively. Gel-shift analysis showed protein binding to these sites which was increased by an A-II treatment (maximum obtained after 3 h). Moreover, A-II could rapidly increase mRNA levels of several factors belonging to the Fos and Jun families which may be components of the AP-1 complex.
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.
The ACTH receptor is the type 2 (MC2R) among the melanocortin receptor family and is expressed almost exclusively in the adrenal cortex. The human MC2R (hMC2R) was very difficult to express in heterologous cell lines. We have succeeded in transient and stable expression of hMC2R using the M3 melanoma cells. Moreover, we have found that the expressed hMC2R in M3 cells showed similar ACTH binding affinity and coupling to adenylate cyclase as the MC2R of normal human adrenal cells, contrarily to most of the other expression cell models. In these conditions, we have been able to test several mutant hMC2R described in patients with the familial glucocorticoid deficiency syndrome (FGD) using this cell line.
ACTH resistance syndromes consist of a group of rare disorders with three various molecular etiologies. However, all these diseases share the feature of severe glucocorticoid adrenal insufficiency. The simplest disorder is the isolated familial glucocorticoid deficiency (FGD) which could be divided in two different types. In FGD type 1, ACTH receptor mutations have been described and are responsible for the loss of function of the receptor, leading to the ACTH unresponsiveness. Patients with FGD type 2 show the same phenotype as in the previous syndrome but no mutation of the ACTH receptor has been reported in these cases. It has been proposed that morbidity of one or several other gene(s) could be responsible for this syndrome although there is no information about their chromosomal localization. The third molecular form of the disease corresponds to the Triple A syndrome for the triad of association "ACTH resistance, Achalasia, Alacrima", thus reflecting a large spectrum of additional symptoms. It has recently been reported that the morbid gene in this last syndrome maps to chromosome 12q13. The aim of this review is to examine the clinical aspect as well as the current knowledge of the molecular and genetic aspects of the different forms of the disease.
In the present study, we characterized two new SF-1 binding sites, SF-209 and SF-98, in the promoter of the human ACTH receptor (hACTH-R) gene. Both sites, together with the previously described SF-35 site, are required for full constitutive activity of this gene. This was demonstrated by the use of constructs containing part of the promoter upstream of the luciferase gene and carrying mutation in one of these sites, to transiently transfect H295R cells. Mutations of either SF-35, SF-98, or SF-209 induced a decrease of luciferase activity. This effect was amplified when two or three elements were mutated together in the same construct. Only SF-35 and SF-98 seem to play a major role in the cAMP-induced regulation of the hACTH-R gene, since mutation of either one of these sites reduced the forskolin induction of luciferase activity by 50%. When both elements were mutated, no stimulation was obtained over the control. This indicates that SF-1 protein must bind to both sites for the cAMP response.
We have previously shown that the promoter of the human ACTH receptor (ACTH-R) contains, at -35 bp, a binding site for the steroidogenic factor 1 (SF-1), an orphan nuclear receptor which could be responsible for the transcriptional activity of this promoter. In the present study, electrophoretic mobility shift assays demonstrated that the sequence -43/-19 bound the SF-1 protein present in the nuclear extracts of adrenocortical cells. Mutation of the SF-1 binding site markedly reduced (40%) the basal transcription of the reporter gene in Y-1 cells transfected with the mutated p(-56/+22)GH construct compared to the wild-type construct. These results demonstrate that the SF-1 binding element present in this fragment is required for the basal promoter activity of the human ACTH-R gene. In addition, other binding elements located upstream from this characterized SF-1 binding site are involved in the full basal promoter activity of the human ACTH-R since transfection studies with a longer p(-1017/+22)GH construct resulted in a higher GH release than with the p(-56/+22)GH construct.
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.
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.
Point mutations of the human ACTH receptor have been reported in some patients with a familial glucocorticoid deficiency syndrome. To demonstrate that these mutations were responsible for the disease, it was necessary to develop a model in which characteristics of normal and mutant receptors could be studied. We have developed a stable expression model in order to characterize the human ACTH receptor by binding studies and functional coupling to adenylate cyclase. After confirmation of the stable integration of receptor constructs, ACTH dose-responses for the production of cAMP were carried out. The EC50 for ACTH were 2.9 +/- 0.2 x 10(-10) M and 2.4 +/- 0.8 x 10(-10) M, respectively, for two different clones stably expressing the normal human ACTH receptor. EC50 calculated for clones expressing either one of the two studied mutant receptors (C251F and D107N) were increased: 4.1 +/- 0.9 x 10(-9) M and 6.4 +/- 1.3 x 10(-9) M respectively. These values were similar to that obtained with M3 parental cells (4.7 +/- 0.8 x 10(-9) M). Binding studies were performed on the same clones. Scatchard analysis showed that clones expressing the normal receptor possessed high affinity binding sites for ACTH, with K(d) = 5.8 +/- 2.4 x 10(-10) M and 6.9 +/- 3.6 x 10(-10) M, respectively, for the two different studied clones. A second type of sites, with low affinity (K(d) around 10(-8) M), was also present. There was no ACTH binding to the high affinity binding sites for the two clones expressing either one of the mutant receptors. An impaired binding of ACTH to its receptors is then responsible for the absence of biological response to ACTH in patients carrying these mutant ACTH receptors.
One kb of the 5'-flanking region of the human ACTH receptor gene was isolated and partially characterized. Transient transfections with hGH reporter confirmed the promoter activity in Y1 cells. Putative elements for transcription factors involved in regulation were noted in the sequence. The promoter activity of some of the constructs is responsive to a treatment by forskolin, due to the presence of several CRE-like sequences.
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 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.