Molecular Mediators and Regulators of Glucocorticoid Actions

糖皮质激素作用的分子介质和调节剂

• 批准号: 8941482
• 负责人: Tomoshige Kino
• 金额: $ 48.31万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8941482
• 关键词: 16p11.2; 3-Dimensional; 7 year old; Acetylation; Achievement; Affect; Alanine; Allergic; Amino Acids; Androgens; Area; Attenuated; Autoimmune Process; BHLH Protein; Beds; Binding; Biological; Biological Assay; Biological Models; Biopsy; Blood Pressure; C-terminal; Characteristics; Circadian Rhythms; Clinical; Collaborations; Computers; Cultured Cells; DNA Binding; DNA Binding Domain; Data; Deacetylase; Defect; Disease; Drosophila genus; Environment; Family; Family member; Fatigue; Fatty acid glycerol esters; Female; Force of Gravity; Gene Expression; Gene Expression Profile; Genes; Genetic; Glucocorticoid Receptor; Glucocorticoids; Glycine; Goals; Greece; Histone Deacetylase; Hormonal; Hour; Human; Hydrocortisone; In Vitro; Inflammatory; Journals; Life; Ligand Binding; Ligand Binding Domain; Longevity; Lymphocyte; Lymphoproliferative Disorders; Lysine; Manuscripts; Measures; Mediating; Mediator of activation protein; Minnesota; Molecular; Moon; Mutation; N-terminal; Neuraxis; Nuclear Receptors; Nucleotides; Obesity; Pathologic; Periodicity; Peripheral; Phase; Physiological; Play; Positioning Attribute; Proteins; Publishing; RNA Sequences; Receptor Gene; Reporter; Reporting; Resistance; Role; Sampling; Sequence Analysis; Serum; Side; Sleep; Surface; Symptoms; Syndrome; System; Thyroid Hormone Receptor; Thyroid Hormones; Tissues; Valine; Zinc Fingers; base; boys; chromatin immunoprecipitation; exome sequencing; genome-wide; glucocorticoid-induced orphan receptor; histone acetyltransferase; hormone resistance; human ZNF45 protein; human study; in vivo; interest; mRNA Expression; medical schools; member; microdeletion; mutant; next generation sequencing; novel; peripheral blood; proband; promoter; prototype; receptor; receptor binding; steroid hormone receptor; subcutaneous; transcription factor

We have investigated the pathophysiologic mechanism of familial/sporadic generalized glucocorticoid resistance syndrome, a prototype of glucocorticoid resistance caused by mutations in the glucocorticoid receptor (GR) gene. We found three new heterozygotic cases with mutations in the GR gene (GR V423A, GR V575G and GR H726R) in collaboration with Dr. Evangelia Charmandari, Univ. of Athens Medical School, Athens, Greece. We characterized molecular defects of the mutant receptors GR V423A and V575G: V423A is located close to the 2nd zinc finger of GR DNA-binding domain. Replacement of valine to alanine at amino acid position 423 reduced hydrophobic environment around this amino acid, which is necessary for GR DNA-binding domain to bind DNA GREs. This resulted in reduction of the mutant receptor for binding to GREs in vivo, and reduced transcriptional activity of this mutant receptor. On the other hand, V575G is located in the 5th helix of the GR ligand-binding domain and its side chain is exposed to the activation function 2 (AF2) surface of the receptor, which physically interacts with the LXXLL motif of the p160 type nuclear receptor coactivators. Replacement of valine to glycine at this amino acid position destroys the AF2 surface, leading to reduction in the transcriptional activity of the mutant receptor by attenuating its binding to p160 type nuclear receptor coactivators. In this fiscal year, we published 2 manuscripts explaining clinical characteristics of the subjects carrying the GR V423A and GR V575G and molecular defects of these mutant receptors. We have also completed the computer-based 3-dimensional structural analysis for the ligand-binding domain of all pathologic GR mutations ever reported and found that there are novel rules for the mutations to damage ligand-binding pocket and AF2 surface. The manuscript explaining these findings is ready for submitting to Journal. We encountered a 7-year old boy with mild resistance to glucocorticoids, androgens and thyroid hormones who carried 1.1 Mb size heterozygotic 16p11.2 microdeletion. We found and published that heterozygotic deletion of the ZNF764 gene by his microdeletion is responsible for his multi-hormone resistance, as this protein acts as a specific coactivator for the glucocorticoid, androgen and thyroid hormone receptors. We have examined molecular mechanisms underlying ZNF764-mediated coactivation of steroid hormone receptors by employing GR as a model system. We found that ZNF764 physically interacts with the ligand-binding domain of GR through its N-terminal half containing a KRAB domain in vitro and in vivo, while C-terminal portion of this molecule containing seven C2H2-type zinc fingers was required for its enhancement of GR transcriptional activity. To reveal molecular action of ZNF764 on GR-induced transcriptional activity genome-wide, we performed the chromatin immunoprecipitation (ChIP) followed by next generation sequence. Preliminary results indicated that binding regions of GR and ZNF764 significantly overlapped with each other. We are now under analysis of obtained results. Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion, and regulate the transcriptional activity of GR in target tissues. The basic helix-loop-helix protein CLOCK, a histone acetyltransferase (HAT), and its heterodimer partner BMAL1 are self-oscillating transcription factors that generate circadian rhythms both in the central nervous system and periphery. We previously reported that CLOCK/BMAL1 repressed GR-induced transcriptional activity by acetylating GR at several lysine residues located in its hinge region and by suppressing binding of GR to promoter GREs. These findings indicate that CLOCK/BMAL1 functions as a reverse phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing to the biologic actions of diurnally fluctuating circulating glucocorticoids. We focused on the longevity-associated and histone deacetylase sirtuin Sirt1 as a candidate of deaceylating the GR acetylated by CLOCK, as this protein is known to interact with the CLOCK/BMAL1 heterodimer and to deaceylate BMAL1. In non-synchronized culture cells, Sirt1 bound GR at latters ligand-binding domain and enhanced its transcriptional activity of both glucocorticoid-responsive reporter constructs and endogenous glucocorticoid-responsive genes. However, this action of Sirt1 on GR was independent to its deacetylase activity, suggesting that Sirt1 is not the deacetylase that deaceylates the GR acetylated by CLOCK. As biological impact of longevity-associated Sirt1 to glucocorticoid hormonal action is worth pursuing, we performed an RNA sequence assay to examine genome-wide effects of Sirt1 on glucocorticoid-responsive transcriptome. We also performed a human study in which we sampled peripheral blood in the morning and evening from normal subjects, and measured mRNA expression of known glucocorticoid-responsive genes and GR acetylation in circulating lymphocytes. We found that GR was acetylated higher in the morning than in the evening, positively correlating with mRNA expression of CLOCK and BMAL1, while circulating glucocorticoid-stimulated mRNA expression of glucocorticoid-responsive genes were repressed by CLOCK/BMAL1 in a gene-specific fashion. These results indicate that the peripheral CLOCK system negatively regulates GR transcriptional activity through acetylation of GR not only in cultured cells but also in humans. In another study where we measured mRNA expression of 190 GR action-regulating and glucocorticoid-responsive genes in subcutaneous fat biopsied from 25 obese subjects, we found that levels of evening cortisol is much more important than those in the morning to regulate mRNA expression of glucocorticoid responsive genes. Ratios of morning/evening serum cortisol levels also have a unique effect. It appears that higher sensitivity of tissues to circulating glucocorticoids in the evening due to reduced GR acetylation by CLOCK underlies stronger impact of evening serum cortisol levels to glucocorticoid-regulated gene expression compared to its morning levels. In connection with our study on circadian rhythms, we found one interesting family with seasonal alteration of circadian rhythmicity in collaboration with Dr. F. Halberg, the Univ. of Minnesota. The proband, 61-year old female, has suffered from an annual cycle of severe fatigue including inability to get out of bed, which lasts 2-3 months in summer and winter. She, however, is free from symptoms with high achievement during the unaffected months between these bad periods. The proposita demonstrated elongation of circadian rhythmicity (24.84 hours) in blood pressure and vigor rates during the affected periods, while she was in normal 24-hour rhythms in the unaffected months. The cycle of 24.84 hours is exactly double of the tidal cycle, which is hypothesized to come about gravity-changes caused by revolution of the moon around the earth. To identify genetic cause(s) of these manifestations, we have completed the whole exome sequence analysis for 3 affected subjects and 5 unaffected members. We found several gene areas associated with high LOD score values, as well as 4 nucleotide replacements specific to affected subjects. One of such replacements is located in the gene known to have impact on circadian rhythms and sleep in drosophila. We also performed SNP array analysis for an additional 3 family members to increase LOD scores of the gene areas identified in the whole exosome sequence. We are now analyzing obtained raw data.

我们研究了家族性/散发性广泛性糖皮质激素耐药综合征的病理生理机制，这是由糖皮质激素受体（GR）基因突变引起的糖皮质激素耐药的原型。我们与希腊雅典大学医学院的Evangelia Charmandari博士合作，发现了三例新的GR基因突变的杂合子病例（GR V423A， GR V575G和GR H726R）。我们分析了突变受体GR V423A和V575G的分子缺陷：V423A位于GR dna结合域的第二锌指附近。在423位氨基酸上缬氨酸被丙氨酸取代，减少了该氨基酸周围的疏水环境，这是GR DNA结合域与DNA GREs结合所必需的。这导致体内与GREs结合的突变受体减少，并降低了该突变受体的转录活性。另一方面，V575G位于GR配体结合域的第5螺旋，其侧链暴露于受体的活化功能2 （activation function 2, AF2）表面，与p160型核受体共激活因子的LXXLL基序发生物理相互作用。缬氨酸在这个氨基酸位置被甘氨酸取代，破坏了AF2表面，通过减弱其与p160型核受体共激活因子的结合，导致突变受体的转录活性降低。在本财政年度，我们发表了2篇论文，解释了GR V423A和GR V575G突变体受体的临床特征和分子缺陷。我们还完成了对所有已报道的病理性GR突变的配体结合域的计算机三维结构分析，发现突变对配体结合袋和AF2表面的破坏有新的规律。解释这些发现的手稿已经准备好提交给《华尔街日报》。

项目成果

Primary generalized familial and sporadic glucocorticoid resistance (Chrousos syndrome) and hypersensitivity.

• DOI: 10.1159/000342505
• 发表时间: 2013
• 期刊: Endocrine development
• 作者: Charmandari E;Kino T;Chrousos GP
• 通讯作者: Chrousos GP

Glucocorticoid receptor (GR) beta has intrinsic, GRalpha-independent transcriptional activity.

• DOI: 10.1016/j.bbrc.2009.02.110
• 发表时间: 2009-04-17
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Kino, Tomoshige;Manoli, Irini;Kelkar, Sujata;Wang, Yonghong;Su, Yan A.;Chrousos, George P.
• 通讯作者: Chrousos, George P.

Plasma soluble α-klotho protein levels in premature and term neonates: correlations with growth and metabolic parameters.

• DOI: 10.1530/eje-12-0476
• 发表时间: 2012-09
• 期刊: European journal of endocrinology
• 影响因子: 5.8
• 作者: Siahanidou T;Garatzioti M;Lazaropoulou C;Kourlaba G;Papassotiriou I;Kino T;Imura A;Nabeshima Y;Chrousos G
• 通讯作者: Chrousos G

A novel point mutation of the human glucocorticoid receptor gene causes primary generalized glucocorticoid resistance through impaired interaction with the LXXLL motif of the p160 coactivators: dissociation of the transactivating and transreppressive acti

人类糖皮质激素受体基因的一个新点突变通过与 p160 共激活因子 LXXLL 基序的相互作用受损，导致原发性全身性糖皮质激素抵抗：反式激活和反式抑制活性的解离。

• DOI: 10.1210/jc.2013-3005
• 发表时间: 2014
• 期刊: The Journal of clinical endocrinology and metabolism
• 作者: Nicolaides,NicolasC;Roberts,MichaelL;Kino,Tomoshige;Braatvedt,Geoffrey;Hurt,DarrellE;Katsantoni,Eleni;Sertedaki,Amalia;Chrousos,GeorgeP;Charmandari,Evangelia
• 通讯作者: Charmandari,Evangelia

Human glucocorticoid receptor isoform beta: recent understanding of its potential implications in physiology and pathophysiology.

• DOI: 10.1007/s00018-009-0098-z
• 发表时间: 2009-11
• 期刊: CELLULAR AND MOLECULAR LIFE SCIENCES
• 影响因子: 8
• 作者: Kino, Tomoshige;Su, Yan A.;Chrousos, George P.
• 通讯作者: Chrousos, George P.