BRFAA - Biomedical Research Foundation Academy Of Athens
Biomedical Research Foundation Academy Of AthensAcademy Of Athens

Research

ONGOING RESEARCH PROJECTS

1. Molecular Mechanisms of Generalized Glucocorticoid Resistance (Evangelia Charmandari, George P. Chrousos)

2. Molecular Mechanisms of the Dominant Negative Effect of hGRβ upon the Transcriptional Activity of hGRα (Evangelia Charmandari, George P. Chrousos)

3. Molecular Mechanisms of Glucocorticoid Resistance and Hypersensitivity (Evangelia Charmandari, George P. Chrousos)

4. Metabolic Syndrome Manifestations in Classic Congenital Adrenal Hyperplasia (Evangelia Charmandari, George P. Chrousos)

5. Genetics of Obesity (Marina Doufexis, Evangelia Charmandari, George P. Chrousos)

6. Interactions Between Nuclear Receptor and G-protein Signaling System (Marina Doufexis, George P. Chrousos)

7. Studies of SELADIN-1/DHCR24 in animal models in health and disease (Athina Samara, Ema Anastasiadou, George P. Chrousos)
 



1. Molecular Mechanisms of Generalized Glucocorticoid Resistance (Evangelia Charmandari, George P. Chrousos)


Generalized Glucocorticoid Resistance is a rare, familial or sporadic condition characterized by generalized, partial, end-organ insensitivity to glucocorticoids. Affected subjects have compensatory elevations in circulating cortisol and ACTH concentrations, which maintain circadian rhythmicity and appropriate responsiveness to stressors, and resistance of the HPA axis to dexamethasone suppression, but no clinical evidence of hypo- or hypercortisolism. The molecular basis of Generalized Glucocorticoid Resistance has been ascribed to mutations in the hGR gene, which impair one or more of the molecular mechanisms of hGR action, thereby altering tissue sensitivity to glucocorticoids. Inactivating mutations within the ligand- and DNA-binding domains of the receptor, and a 4-base pair deletion at the 3'-boundary of exon 6 of the gene, have been described in five kindreds and five sporadic cases. We have identified most of these hGR mutations and have systematically investigated the molecular mechanisms through which various natural hGRα mutants affect glucocorticoid signal transduction in all described cases of Generalized Glucocorticoid Resistance. 


We have recently identified three new cases of Generalized Glucocorticoid Resistance caused by novel, heterozygous, point mutations in the hGR gene. The aim of this research project is to systematically investigate the molecular mechanisms through which these natural mutant receptors impair glucocorticoid signal transduction. In vitro studies are being performed to determine: i) the transcriptional activity of the mutant receptors; ii) the ability of the mutant receptors to exert a dominant negative effect upon the wild-type receptor; iii) the affinity of the mutant receptors for the ligand; iv) the subcellular localization of the mutant receptors and their nuclear translocation following exposure to the ligand; v) the ability of the mutant receptors to bind to GREs; vi) the interaction of the mutant receptors with the GRIP1 coactivator; and vii) the motility of the mutant receptors in the nucleus of cells. These studies will enable us to complete the molecular characterization of all natural hGR mutations identified so far and to elucidate further the molecular mechanisms underlying Generalized Glucocorticoid Resistance. They will also shed light into the specific functions of the hGRα protein, given that differential localization of various hGR mutations is associated with variable impairment of glucocorticoid signal transduction and variable clinical and biochemical phenotype and response to treatment. 



 

Figure 1:
(A) Nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Upon binding to the ligand, the activated GR dissociates from heat shock proteins and translocates into the nucleus, where it homodimerizes and binds to GREs in the promoter region of target genes.
(B) Schematic representation of the interaction of AF-1 and AF-2 of GR with coactivators. AF: activation function; DRIP/TRAP: vitamin D receptor-interacting protein/thyroid hormone-associated protein; GR: glucocorticoid receptor; GREs: glucocorticoid-response elements; HSPs: heat shock proteins; SWI/SNF: switching/sucrose non-fermenting; TF: transcription factor; TFRE: transcription factor-response element.

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2. Molecular Mechanisms of the Dominant Negative Effect of hGRβ upon the Transcriptional Activity of hGRα (Evangelia Charmandari, George P. Chrousos)


Glucocorticoids regulate a variety of biological processes and play a pivotal role in the maintenance of basal and stress-related homeostasis. At the cellular level, their actions are mediated by a 94-kDa intracellular receptor protein, the glucocorticoid receptor (GR), which belongs to the superfamily of steroid/thyroid/retinoic acid receptor proteins. Alternative splicing of the human GR (hGR) gene in exon 9 generates two highly homologous receptor isoforms, termed α and β. hGRα is ubiquitously expressed in almost all human tissues and cells and represents the classic hGR that functions as a ligand-dependent transcription factor. hGRβ is also ubiquitously expressed in tissues but it does not bind glucocorticoids, is transcriptionally inactive and exerts a dominant-negative effect on hGRα, inhibiting the hGRα-mediated transactivation of target genes in a dose-dependent manner. In previous studies, we examined the molecular mechanisms underlying the dominant negative effect of hGRβ on the transcriptional activity of hGRα. We demonstrated that these mechanisms primarily involve competition at the level of coactivators, while heterodimerization via the carboxyl-terminal domain dimerization interface and the formation of transcriptionally inactive or weakly active hGRα-hGRβ heterodimers might be additional factors contributing to this process. This research project aims to explore the interaction between hGRβ and other coactivators that enhance the transcriptional activity of hGRα, as well as the role of hGRβ in accentuating the inhibitory effect of corepressors on hGRα-mediated transactivation of target genes.



Figure 2:
Genomic and complementary DNA, and protein structures of the human (h) glucocorticoid receptor (GR), and the GRα and GRβ isoforms produced through alternative splicing. The hGR gene consists of 9 exons. Exon 1 is an untranslated region, exon 2 codes for the immunogenic domain (A/B), exon 3 and 4 for the DNA-binding domain (C), and exons 5-9 for the hinge region (D) and the ligand-binding domain (E). The GR gene contains two terminal exons 9 (exon 9α and 9β) alternatively spliced to produce the classic GRα and the non ligand-binding GRβ. These two isoforms are identical through amino acid 727, but then diverge, with GRα having an additional 50 amino acids and GRβ having an additional, nonhomologous 15 amino acids. The C-terminal gray colored domains in GRα and GRβ represent their non-homologous amino acid sequences.

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3. Molecular Mechanisms of Glucocorticoid Resistance and Hypersensitivity (Evangelia Charmandari, George P. Chrousos)

Target tissue resistance to glucocorticoids can be generalized or tissue-specific, transient or permanent, partial or complete, compensated or non-compensated. Inactivating mutations in the glucocorticoid receptor gene result in primary, Generalized Glucocorticoid Resistance (familial or sporadic), while several autoimmune/inflammatory states, such as rheumatoid arthritis, osteoarthritis, Crohn's disease, ulcerative colitis and asthma, are often associated with an immune tissue-specific form of glucocorticoid resistance. On the other hand, septic shock and respiratory distress syndrome are associated with a severe form of generalized glucocorticoid resistance. Target tissue hypersensitivity to glucocorticoids can also be generalized or tissue-specific. Visceral obesity-related insulin resistance associated with components of the metabolic syndrome is often associated with visceral fat-specific hypersensitivity to glucocorticoids, while the acquired immunodeficiency syndrome (AIDS) caused by Human Immunodeficiency Virus Type-1 (HIV-1) infection may lead to fat-tissue-specific glucocorticoid hypersensitivity and a characteristic lipodystrophic syndrome associated with insulin resistance and muscle wasting. The aim of the present study is to investigate other common human disorders that may be explained by alterations in tissue sensitivity to glucocorticoids.

Figure 3:
Glucocorticoid signaling system.

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4. Metabolic Syndrome Manifestations in Classic Congenital Adrenal Hyperplasia (Evangelia Charmandari, George P. Chrousos)

Classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is an autosomal recessive disorder characterized by impaired adrenocortical and adrenomedullary function, and adrenal hyperandrogenism. Compared to normal subjects, patients with classic CAH have increased incidence of obesity and visceral adiposity, hyperinsulinism and insulin insensitivity, hypertension and hyperandrogenism. The impaired adrenomedullary function, intermittent hypercortisolism, and adrenal and/or ovarian hyperandrogenism in the not adequately controlled patients and females with polycystic ovarian syndrome, may account for the above abnormalities and may predispose these subjects to the development of metabolic syndrome-related atherosclerotic cardiovascular disease in adulthood. The aim of the present study is to investigate whether treatment with insulin sensitizers improves the metabolic profile of patients with classic CAH.

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5. Genetics of Obesity (Marina Doufexis, Evangelia Charmandari, George P. Chrousos)


Genetic, developmental and environmental factors contribute to the pathogenesis of the insulin resistance syndrome in obesity. Genetic risk factors for Diabetes type 2 have been the subject of intense research. Although it is clear that the rapidly increasing prevalence of obesity and Diabetes type 2 in the modern world is most likely due to environmental factors, including excessive nutrition and lack of exercise, these factors act upon individuals with genetic and developmental susceptibility to develop these disorders. The aim of the present study is to investigate genetic risk factors involved in overweight children by looking at genes that have been directly associated with the development of the pre-disease and disease states. Gene panels used include diabetes, obesity/metabolic syndrome, inflammation/autoimmunity and behaviour.

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6. Interactions Between Nuclear Receptor and G-protein Signaling System (Marina Doufexis, George P. Chrousos)


The hypothalamic-pituitary-adrenal (HPA) axis has major homeostatic functions and is crucial for human life. In recent years, it has been speculated that subtle changes in its function might lead to the development of various common diseases, such as obesity and the metabolic syndrome, inflammatory disease, even depression. The glucocorticoid receptor (GR), a member of the nuclear receptor family, and the melanocortin 2 receptor (MC2R, ACTH receptor), which belongs to the G-protein Coupled Receptors (GPCRs), play a major role in the regulation and effects of the HPA axis. 


Melanocortin receptors belong to the super-family of GPCRs or seven transmembrane receptors. There are 5 receptors of this subtype, and they are all activated by the melanocortin peptides to produce different effects on different tissues. The MC1R is activated by alpha-MSH and plays an active role in protection against melanoma and skin cancer. The MC2R, which is our main interest, is also called the ACTH receptor and forms an integral part of the HPA axis and steroidogenesis. It is thus, essential for life. MC3R and MC4R are fundamentally involved in energy homeostasis, obesity and diabetes. Understanding the multiple facets of the intracellular signalling of these receptors is our main goal. A novel aspect of their function was revealed with our recent publication showing direct interaction between the MC2R and a nuclear pore protein. This was the first time a seven-transmembrane receptor was found to interact with one of the nuclear pore proteins and this aspect of the receptor's function is one of our main research interests. Implementation of the same technique used successfully with the MC2R can lead to the discovery of novel interacting partners of the GR. We will be using the bacterial 2 hybrid technique to identify those partners. We are also interested in dissecting possible cross-talk between GPCRs and the GR signalling pathway.


Figure 4:
Subcellular fractionation of the MC2R-Nup50 complex. H295R cells were fractionated into four fractions (whole cell lysates, membrane, crude nuclear and purified nuclear fractions).
(A) Contents of subcellular fraction for an ER marker (GRP78/BiP) and nuclear membranes (Nup62) by immunoblotting. (fraction 1, whole cell lysate; fraction 2, membranes; fraction 3, crude nuclear extract; fraction 4, purified nuclear extract).
(B)
In membrane fractions, the MC2R-Nup50 complex is demonstrated by co-immunoprecipitation using the MC2R C-16 antibody at rest and following stimulation by ACTH (10-6M) for the initial 30 minutes. Some reappearance of the complex occurs by 150 minutes.
(C) In purified nuclear fractions, the MC2R-Nup50 complex is not detectable at rest; it begins to appear by 60 minutes and is prominent at 150 and 210 minutes.

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7. Studies of SELADIN-1/DHCR24 in animal models in health and disease
(Athina Samara, Ema Anastasiadou, George P. Chrousos)


This project aims to generate a conditional DHCR24 knock out mouse to study the brain cholesterol levels throughout development. The DHCR24 conditional knock out (-/-) mice will later be crossed with murine lines expressing Cre in a time- or tissue-specific manner. This model will have multiple research applications on the importance of DHCR24 and cholesterol in development, neurodegeneration, oxidative stress, mood disturbances and cholesterol biosynthesis disorders. The above project is funded by NARSAD, The National Alliance for Research in Schizophrenia and Associated Disorders (www.narsad.org) via a two year Young Investigator Award.

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