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-kappaB (NF-kappaB). 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 approximately 36% of the WT level. Protein expression was reduced to approximately 66% of WT levels in S651F and to approximately 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-kappaB 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-kappaB 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.
Koyano
Satoru
S
Project Team for Pharmacogenetics, National Institute of Health Sciences, Tokyo, Japan. skoyano@nihs.go.jp
Saito
Yoshiro
Y
Nagano
Michiyo
M
Maekawa
Keiko
K
Kikuchi
Yutaka
Y
Murayama
Norie
N
Fujino
Tomofumi
T
Ozawa
Shogo
S
Nakajima
Toshiharu
T
Matsumoto
Kenji
K
Saito
Hirohisa
H
Sawada
Jun-Ichi
J
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D015870:Gene Expression; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D013347:Subcellular Fractions; D013997:Time Factors; D014162:Transfection
D016328:NF-kappa B; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid