Target Tissue Glucocorticoid Signaling: Basic and Clinical Implications

Tomoshige Kino, MD, PhD, Acting Head, Section on Pediatric Endocrinology
Nancy Nader, PhD, Visiting Fellow
Maria Pavlatou, MD, PhD, Visiting Fellow
Sin Man Ng, BS, Predoctoral Student¹
Brent Abel, Student²
Mayukh Chakrabarti, Student²

The glucocorticoids’ life-sustaining functions are many and they play an important role in the therapy of several inflammatory, autoimmune, allergic, and lymphoproliferative disorders. Changes in tissue sensitivity to glucocorticoids may develop into pathologic states and influence disease course. We thus continue to investigate the glucocorticoid signaling system and the clinical implications of changes in tissue sensitivity to glucocorticoids. In addition, we serve as the world’s referral center for analyzing pathologic mutations of the glucocorticoid receptor (GR). We also conduct research into the molecular mechanisms underlying the AIDS-related lipodystrophy and insulin resistance syndrome and strive to increase our understanding of the physiologic/pathophysiologic interactions among the endocrine, immune, and central nervous systems.

Pathologic glucocorticoid receptor mutations causing familial/sporadic glucocorticoid resistance syndrome

Kino, Nader; in collaboration with Charmandari, Chrousos

This past year, we described a glucocorticoid-resistant patient from Colombia in whom we identified a novel heterozygous point mutation in the GR gene that results in the replacement of phenylalanine (F) by leucine (L) at amino acid position 737 (GRF737L). We also indentified in another Colombian patient a heterozygotic mutation that replaced histidine (H) by aspartic acid (D) at amino acid position 401 (GRD401H). Using panels of assay methods, we characterized details of molecular defects of GRD401H and found that this mutant receptor demonstrated two to three times stronger transcriptional activity than the wild-type receptor while the patient showed a mixed phenotype characteristic of both glucocorticoid resistance and hypersensitivity. Thus, this mutant receptor causes tissue-specific alterations of glucocorticoid sensitivity. In another glucocorticoid kindred from Greece, we further identified a pathologic mutation leading to substitution of valine (V) by alanine (A) at amino acid position 423 (GRV423A) at the base of the first zinc finger of the DNA-binding domain (DBD).

Charmandari E, Ichijo T, Jubiz W, Zachman K, Chrousos GP, Kino T. A novel point mutation in the amino terminal domain of the human glucocorticoid receptor (hGR) gene enhancing hGR-mediated gene expression. J Clin Endocrinol Metab 2008;93:4963-4968.
Charmandari E, Kino T, Chrousos GP . Generalized glucocorticoid insensitivity: clinical phenotype and molecular mechanisms. In: Adcock IM, Chung KF, eds. Overcoming Steroid Insensitivity in Respiratory Disease. John Wiley & Sons, 2008;73-85.
Charmandari E, Kino T, Ichijo T, Chrousos GP. Generalized glucocorticoid resistance: clinical aspects, molecular mechanisms and implications of a rare genetic disorder. J Clin Endocrinol Metab 2008;5:1563-1572.
Charmandari E, Kino T, Ichijo T, Jubiz W, Mejia L, Zachman K, Chrousos GP. A novel point mutation in helix 11 of the ligand-binding domain of the human glucocorticoid receptor gene causing generalized glucocorticoid resistance. J Clin Endocrinol Metab 2007;92:3986-3990.

Identification of the intracellular molecules that alter tissue glucocorticoid action and their pathophysiologic implications for common diseases

Kino, Nader, Pavlatou; in collaboration with Almeida, Chrousos, Edelman, Pant, Rother, Skarulis

We discovered that the brain-specific serine/threonine kinase cyclin–dependent kinase 5 (CDK5) phosphorylates the GR at several serine residues located in the immunogenic domain and then modulates the transcriptional activity of this receptor in a target gene–specific fashion. CDK5 is essential for brain development and has implications for the pathogenesis of neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis. Given that glucocorticoids are pivotal for brain function and that excess amounts of these hormones cause neuronal death and emotional and psychiatric problems, our findings suggest that aberrant activation of CDK5 may exert its pathologic actions also by modulating glucocorticoid activity. To examine the clinical implications of our findings, we have begun to evaluate the expression/activity of CD K5 along with tissue glucocorticoid action in brain tissues from patients with major depression.

We also found that SET/TA F-Ibeta, which others identified as both a part of the SET-CAN oncogene product and a component of the inhibitor of the histone acetyltransferases (INHAT) complex, acted as a negative regulator of GR transcriptional activity. In addition, we discovered that ligandactivated GR stimulated transcription by displacing the INHAT complex from histones via physical interaction through the receptor’s DBD. In contrast to Set/TAF-Ibeta, the Set-Can fusion protein bound to glucose response elements (GRE) regardless of ligand availability and strongly suppressed GR-induced transcriptional activity/histone acetylation, possibly participating in the development of glucocorticoid insensitivity in acute undifferentiated leukemia with Set-Can translocation.

We have almost completed our examination of the non–protein-coding RNA growth arrest–specific 5 (Gas5), which accumulates in growth-arrested cells but whose physiologic roles remain unknown. We found that Gas5 has a GRE-like sequence in the 3′ portion of its intramolecular double helical structure, through which this ncRNA interacts competitively with the GR DBD by mimicking DNA GREs and suppressing GR’s transcriptional activity. Thus, Gas5 is a growth arrest–related co-repressor of the GR harboring an RNA mock “GRE,” restricting the expression of steroid-responsive genes. This novel concept suggests competition between ncRNA and genomic DNA for the DBD of steroid receptors. Our work also suggests that Gas5 contributes to the cellular adaptive reaction to starvation, preventing apoptosis and saving energy resources. In addition to these molecules (cyclin-dependent kinase 5, Set/TAF-Ibeta, and Gas5), we have examined the influence of the energy sensor AMK-activated protein kinase (AMPK) and the circadian rhythm transcription factors CLOCK/BMAL1 on GR-mediated transcriptional activity. We found that they modulate tissue glucocorticoid action in vitro and in vivo by acetylating and phosphorylating GR, respectively. We further found that the nuclear liver X receptor, which transduces biologic signals of cholesterol and fatty acid metabolism, suppresses GR transcriptional activity. These results indicate that glucocorticoid action in local tissues is adjusted, in a circadian fashion, according to cellular energy levels and the supply of energy resources.

To integrate our novel findings on regulators of tissue glucocorticoid action into clinically meaningful information, we have begun collaborative work that is examining expression of the genes known to regulate the transcriptional activity of the GR in tissues obtained from anorexic, lean, and obese individuals and diabetic patients. Our clinically oriented research will reveal factor(s) and mechanism(s) responsible for alterations of target tissue glucocorticoid action in these common diseases.

Chrousos GP, Kino T. Glucocorticoid signaling in the cell: expanding clinical implications to complex human behavioral and somatic disorders. Ann NY Acad Sci, in press.
Ichijo T, Chrousos GP , Kino T. Activated glucocorticoid receptor interacts with the INHAT component Set/TA F-Ibeta and releases it from a glucocorticoid-responsive gene promoter, relieving repression: implications for the pathogenesis of glucocorticoid resistance in acute undifferentiated leukemia with Set-Can translocation. Mol Cell Endocrinol 2008;283:19-31.
Kino T, Ichijo T, Amin ND, Kesavapany S, Wang Y, Kim N, Rao S, Player A, Zheng YL, Garabedian MJ, Kawasaki E, Pant HC , Chrousos GP . Cyclin-dependent kinase 5 differentially regulates the transcriptional activity of the glucocorticoid receptor through phosphorylation: clinical implications for the nervous system response to glucocorticoids and stress. Mol Endocrinol 2007;21:1552-1568.

Viral effects on host endocrine systems

Kino, Ng; in collaboration with Chrousos, Kopp, Ozato

This year, we examined the effect of the HIV-1 accessory protein Vpr on the activity of the nuclear receptor PPARgamma; PPARgamma is a critical regulator of lipid and glucose metabolism. We discovered that Vpr, whether administered extracellularly or expressed in cells, suppressed PPARgamma agonist-induced adipocyte differentiation by suppressing the receptor’s transcriptional activity. The results suggest that circulating Vpr, or, alternatively, Vpr produced as a consequence of direct infection of adipocytes, alters insulin sensitivity by suppressing PPARgamma activity, possibly contributing to the development of the lipodystrophy and insulin resistance observed in HIV-1–infected patients.

In collaboration with Keiko Ozato, we have begun to examine the expression of all nuclear receptors, including the GR and numerous transcriptional co-regulators, in dendritic cells in response to viral infection. These cells play a central role in the initiation of both innate and acquired immunity by capturing/presenting viral antigens to regulatory T cells and B cells. In addition, they secrete a variety of cytokines that further activate downstream defense mechanisms. Our preliminary studies suggest that the endocrine nuclear receptor system immediately responds to viral infection and changes its profile in response to specific viruses. Our research will provide information relevant to the hypothesis that some viruses alter host pathogenesis to their advantage by changing the activities of the endocrine/nuclear receptor system in immune cells. We believe that our work will provide a new understanding of chronic infection, inflammation, allergy, autoimmunity, senescence, and cancer and will allow us to explore rational therapeutic approaches to treating virus-related pathologies through nuclear hormone receptor ligands.

Kino T, Chrousos GP. Virus-mediated modulation of the host endocrine signaling systems: clinical implications. Trends Endocrinol Metab 2007;18:159-166.
Shrivastav S, Kino T, Cunningham T, Ichijo T, Schubert U, Heinklein P, Chrousos GP, Kopp JB. HIV-1 Vpr suppresses transcriptional activity of PPARgamma and inhibits adipocyte differentiation: implications for HIV-associated lipodystrophy. Mol Endocrinol 2008;22:234-247.

¹Graduate Exchange Program between NICHD and the Chinese University of Hong Kong
²Howard Hughes Medical Institute/Montgomery County Public School Internship, MD

Collaborators

Osborne F.X. Almeida, PhD, Max Planck Institute of Psychiatry, Munich, Germany
Ashok Balasubramanyam, MD, Baylor College of Medicine, Houston, TX
Evangelia Charmandari, MD, Biomedical Research Foundation of the Academy of Athens, Athens, Greece
George P. Chrousos, MD,³ Athens University Medical School, Athens, Greece
Daniel C. Edelman, PhD, Cancer Genetics Branch, NCI, Gaithersburg, MD
Philip W. Gold, MD, Clinical Neuroendocrinology Branch, NIMH, Bethesda, MD
Jeffrey B. Kopp, MD, Metabolic Diseases Branch, NIDDK, Bethesda, MD
Keiko Ozato, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Harish C. Pant, PhD, Laboratory of Neurochemistry, NINDS, Bethesda, MD
George N. Pavlakis, MD, PhD, Vaccine Branch, NCI-Frederick, Frederick, MD
Kenner C. Rice, PhD, Chemical Biology Research Branch, NIDA, Bethesda, MD
Kristina I. Rother, MD, Clinical Endocrinology Branch, NIDDK, Bethesda, MD
Monica C. Skarulis, MD, Clinical Endocrinology Branch, NIDDK, Bethesda, MD

³Chief, Section on Pediatric Endocrinology, Program in Reproductive and Adult Endocrinology, NICHD, with limited appointment.

For further information, contact kinot@mail.nih.gov.