Functional Analysis of Three Genetic Polymorphisms in the Glucocorticoid Receptor Gene

Abstract

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-B (NF-B). To identify genetic polymorphisms affecting glucocorticoid responses, the GR gene was sequenced, and two novel single nucleotide alterations, 1510AϾT (T504S) and 1952CϾT (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 ϳ36% of the WT level. Protein expression was reduced to ϳ66% of WT levels in S651F and to ϳ6% 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-B 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-B 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.Glucocorticoid receptor (GR) is a transcriptional factor activated by glucocorticoids and a regulator of the expression of various genes. Human GR␣ (hGR␣) is encoded by nine exons, including exon 9␣ (Encio and Detera-Wadleigh, 1991). GR␤ is an alternatively spliced form with exon 9␤ replacing exon 9␣ that was identified in glucocorticoid-resistant human multiple myeloma cells and functions as an hGR␣ dominant negative type (Moalli et al., 1993). The 777-amino acid hGR␣ has many functional domains, which include DNA binding (amino acid residues 421-486), ligand binding (528 -720), homodimerization (456 -777), Hsp90 binding (568 -653), nuclear translocation (479 -506 and 526 -777), and transactivation domains (77-262, 404 -491, and 526 -556) (Savory et al., 1999;Vottero and Chrousos, 1999). In the cytosol, GRs are associated with heat-shock and other proteins (Pratt and Toft, 1997), and the binding of glucocorticoid leads to their nuclear translocation (Webster and Cidlowski, 1999).Glucocorticoid treatments are effective in many inflammatory diseases and some types of cancers. As for anti-inflammatory effects, GR is thought to exert its pharmacological effects through the activation of transcription, such as activating the glucocorticoid-induced leucine zipper gene, and/or through suppression of nuclear factor-B (NF-B) ...

FUNCTIONAL CHARACTERIZATION OF FOUR NATURALLY OCCURRING VARIANTS OF HUMAN PREGNANE X RECEPTOR (PXR): ONE VARIANT CAUSES DRAMATIC LOSS OF BOTH DNA BINDING ACTIVITY AND THE TRANSACTIVATION OF THE CYP3A4 PROMOTER/ENHANCER REGION

“…However, it was reported that hPXR ligands induced the enhanced binding of coactivators, such as steroid receptor coactivator-1, resulting in transactivation of the CYP3A4 promoter/enhancer consisting of PXR-responsive element and XREM Honkakoski and Negishi, 2000). Thus, our data support that hPXR ligands, such as rifampicin, promote the activation of nuclear hPXR rather than the translocation of the receptor to the nucleus, in contrast to the cases of VDR and glucocorticoid receptor (Racz and Barsony, 1999;Koyano et al, 2003).…”

Pharmacogenetic approaches in the treatment of asthma

“…Glucocorticoids bind to a cytoplasmic receptor, the glucocorticoid receptor, which moves to the nucleus and activates steroid-responsive genes containing the glucocorticoid response element (GRE). Several polymorphisms have been described in the glucocorticoid receptor (GR) gene with functional consequences-for example, a Val641Asp polymorphism influences the binding affinity for dexamethasone [39], a Val729Ile polymorphism confers a fourfold decrease in dexamethasone activity [40], an Asn363Ser polymorphism was associated with higher sensitivity to dexamethasone [41], and 2314insA and S651F variants had a suppressed glucocorticoid receptor messenger RNA and protein levels in a recombinant system [42]. These data demonstrate that multiple polymorphisms exist in the glucocorticoid receptor; however, several of the polymorphisms are rare, and their functional significance is questionable [4].…”

Functional Analysis of Six Human Aryl Hydrocarbon Receptor Variants in a Japanese Population

“…HeLa cells (2.8 ϫ 10 4 cells) were cotransfected with 100 ng of the wild-type hAhR expression plasmid pcDNA 3.2-DEST hAhR or the variant hAhR expression plasmid together with 100 ng of the hArnt expression plasmid, 30 ng of pGL3-hCYP1A1 promoter plasmid, and 10 ng of phRL-TK plasmid as an internal control. Four hours after transfection, the cells were treated with a vehicle (DMSO) or various concentrations of BNF, 3MC, or OME, and then cultured for an additional 48 h. The cells were washed with PBS, and the lysates were prepared using the dual luciferase reporter assay system (Promega) described previously (Koyano et al, 2003. All transfection efficiencies were normalized according to the Renilla luciferase activity.…”

“…Immunocytochemistry was performed as described previously (Ikuta et al, 1998;Koyano et al, 2003). Briefly, HeLa cells (1.4 ϫ 10 5 cells) were transfected with 600 ng of the expression plasmid for empty, wild-type, or variant AhR together with 600 ng of the Arnt expression plasmid, and then cultured for 4 h. The cells were treated with a vehicle (DMSO) or 1 M 3MC for 48 h. Transfected HeLa cells were washed twice with PBS, fixed with 3.7% formaldehyde in PBS for 15 min at room temperature, and then permeabilized with 0.5% Triton X-100 for 15 min.…”