Functional Analysis of Three Genetic Polymorphisms in the Glucocorticoid Receptor Gene

Glucocorticoids are widely used as potent anti-inflammatory drugs. Glucocorticoids exert their pharmacological effects by binding to a glucocorticoid receptor (GR), which promotes expression of its target genes or suppresses transcription mediated by other transcriptional factors, such as nuclear factor-κB (NF-κB). To identify genetic polymorphisms affecting glucocorticoid responses, the GR gene was sequenced, and two novel single nucleotide alterations, 1510A>T (T504S) and 1952C>T (S651F), were identified in addition to an adenine base insertion at nucleotide 2314 (2314insA). mRNA expression levels of T504S and S651F were comparable with that of the wild type (WT), whereas the mRNA level of 2314insA was reduced to ∼36% of the WT level. Protein expression was reduced to ∼66% of WT levels in S651F and to ∼6% in 2314insA. No significant change was seen in the T504S variant levels. The instability of the 2314insA mRNA, S651F protein, and 2314insA protein was confirmed by time course experiments. The transcriptional activity of S651F and 2314insA was also reduced to approximately 63 and 2% of the WT levels, respectively, in the luciferase reporter assay. Moreover, the inhibitory effect of GR on NF-κB transactivation was reduced to approximately 81 and 12% of the WT levels for S651F and 2314insA, respectively. These results indicated that the overall transcriptional activity and inhibitory effect on NF-κB transactivation of S651F and 2314insA have partially reduced and almost abrogated, respectively, almost paralleling their reduced protein expression levels caused by mRNA and/or protein instabilities. Thus, these two variations were suggested to influence the response to glucocorticoid treatment.

[1]  A. Sousa,et al.  Glucocorticoid-resistant asthma , 2004 .

[2]  A. Foussat,et al.  Synthesis of glucocorticoid-induced leucine zipper (GILZ) by macrophages: an anti-inflammatory and immunosuppressive mechanism shared by glucocorticoids and IL-10. , 2003, Blood.

[3]  Yoshiro Saito,et al.  Substitution of arginine for cysteine 643 of the glucocorticoid receptor reduces its steroid-binding affinity and transcriptional activity. , 2002, Cancer letters.

[4]  R. Derijk,et al.  Glucocorticoid receptor variants: clinical implications , 2002, The Journal of Steroid Biochemistry and Molecular Biology.

[5]  G. Chrousos,et al.  A novel, C-terminal dominant negative mutation of the GR causes familial glucocorticoid resistance through abnormal interactions with p160 steroid receptor coactivators. , 2002, The Journal of clinical endocrinology and metabolism.

[6]  L. Huang,et al.  The phase-shift mutation in the glucocorticoid receptor gene: potential etiologic significance of neuroendocrine mechanisms in lupus nephritis. , 2001, Clinica chimica acta; international journal of clinical chemistry.

[7]  G. Chrousos,et al.  Pathologic human GR mutant has a transdominant negative effect on the wild-type GR by inhibiting its translocation into the nucleus: importance of the ligand-binding domain for intracellular GR trafficking. , 2001, The Journal of clinical endocrinology and metabolism.

[8]  J. Carlstedt-Duke,et al.  Characterization of two novel mutations in the glucocorticoid receptor gene in patients with primary cortisol resistance , 2001, Clinical endocrinology.

[9]  J. Ashwell,et al.  Inhibition of AP-1 by the Glucocorticoid-inducible Protein GILZ* , 2001, The Journal of Biological Chemistry.

[10]  K. Yamamoto,et al.  The glucocorticoid receptor inhibits NFkappaB by interfering with serine-2 phosphorylation of the RNA polymerase II carboxy-terminal domain. , 2000, Genes & development.

[11]  W. V. Berghe,et al.  Mechanisms of anti-inflammatory action and of immunosuppression by glucocorticoids: negative interference of activated glucocorticoid receptor with transcription factors , 2000, Journal of Neuroimmunology.

[12]  J. Cidlowski,et al.  CBP (CREB Binding Protein) Integrates NF-κB (Nuclear Factor-κB) and Glucocorticoid Receptor Physical Interactions and Antagonism , 2000 .

[13]  J. Cidlowski,et al.  Mechanisms of Glucocorticoid-receptor-mediated Repression of Gene Expression , 1999, Trends in Endocrinology & Metabolism.

[14]  G. Chrousos,et al.  Glucocorticoid Receptor β: View I , 1999, Trends in Endocrinology & Metabolism.

[15]  R. Haché,et al.  Discrimination between NL1- and NL2-Mediated Nuclear Localization of the Glucocorticoid Receptor , 1999, Molecular and Cellular Biology.

[16]  J. Connell,et al.  Glucocorticoid receptor polymorphism, skin vasoconstriction, and other metabolic intermediate phenotypes in normal human subjects. , 1998, The Journal of clinical endocrinology and metabolism.

[17]  K. Kaestner,et al.  DNA Binding of the Glucocorticoid Receptor Is Not Essential for Survival , 1998, Cell.

[18]  T. Schmidt,et al.  Trans-retinoic acid and glucocorticoids synergistically induce transcription from the mouse mammary tumor virus promoter in human embryonic kidney cells , 1997, The Journal of Steroid Biochemistry and Molecular Biology.

[19]  W. Pratt,et al.  Steroid receptor interactions with heat shock protein and immunophilin chaperones. , 1997, Endocrine reviews.

[20]  J. Cidlowski,et al.  Mouse Glucocorticoid Receptor Phosphorylation Status Influences Multiple Functions of the Receptor Protein* , 1997, The Journal of Biological Chemistry.

[21]  L. Williams,et al.  Tumor necrosis factor α‐induced activation of c‐jun N‐terminal kinase is mediated by TRAF2 , 1997, The EMBO journal.

[22]  K. Mackey,et al.  Analysis of RNA by Northern and Slot Blot Hybridization , 1997 .

[23]  S. Simpson,et al.  Frameshift mutations in the v-src gene of avian sarcoma virus act in cis to specifically reduce v-src mRNA levels , 1994, Molecular and cellular biology.

[24]  T. Brown.  Analysis of RNA by Northern and Slot‐Blot Hybridization , 1993, Current protocols in immunology.

[25]  N. Krett,et al.  Advances in Brief Alternatively Spliced Glucocorticoid Receptor Messenger RNAs in Glucocorticoid-resistant Human Multiple Myeloma Cells 1 , 2006 .

[26]  S. Detera-Wadleigh,et al.  The genomic structure of the human glucocorticoid receptor. , 1991, The Journal of biological chemistry.

[27]  S. Hirohashi,et al.  Selection of a monoclonal antibody reactive with a high-molecular-weight glycoprotein circulating in the body fluid of gastrointestinal cancer patients. , 1988, Cancer research.

[28]  R. Evans,et al.  Functional domains of the human glucocorticoid receptor , 1986, Cell.

[29]  H. Isaka,et al.  Establishment of a human colon adenocarcinoma cell line producing carcinoembryonic antigen. , 1980, Gan.

[30]  Y. Goto,et al.  Eleven novel single nucleotide polymorphisms in the NR1I2 (PXR) gene, four of which induce non-synonymous amino acid alterations. , 2002, Drug metabolism and pharmacokinetics.

[31]  P. Byers.  Killing the messenger: new insights into nonsense-mediated mRNA decay. , 2002, The Journal of clinical investigation.

[32]  J. Cidlowski,et al.  CBP (CREB binding protein) integrates NF-kappaB (nuclear factor-kappaB) and glucocorticoid receptor physical interactions and antagonism. , 2000, Molecular endocrinology.

[33]  Chrousos,et al.  Glucocorticoid Receptor beta: View I. , 1999, Trends in endocrinology and metabolism: TEM.

[34]  J. Gustafsson,et al.  The non-activated glucocorticoid receptor: structure and activation. , 1989, Journal of steroid biochemistry.

[35]  W. Pratt,et al.  The molybdate-stabilized glucocorticoid binding complex of L-cells contains a 98-100 kdalton steroid binding phosphoprotein and a 90 kdalton nonsteroid-binding phosphoprotein that is part of the murine heat-shock complex. , 1986, Journal of Steroid Biochemistry.