Proximal HNF1 element is essential for the induction of human UDP-glucuronosyltransferase 1A1 by glucocorticoid receptor.
Previous study showed noinduction of the reporter gene (-3174/+14) of UGT1A1 in HepG2 by bilirubin, but induction by dexamethasone (DEX). This induction was enhanced seven-fold by the co-expression of human glucocorticoid receptor (GR) and was inhibited by a GR antagonist, RU486, indicating stimulation by DEX-GR. Meanwhile, we could not detect stimulation by beta-estradiol, phenobarbital or rifampicin (RIF) in the presence of GR. We investigated the position playing a role in this induction by GR in the promoter region of UGT1A1 using deletion mutants, and clarified the essential sequence (-75/-63) for the binding site of hepatocyte nuclear factor 1 (HNF1). However, GR did not bind directly to this sequence, because UGT-PE2 did not compete for binding to a glucocorticoid responsive element (GRE) probe in an electrophoretic mobility shift assay (EMSA) method. Labeled [(32)P]DNA probe of HNF1 binds with nuclear extracts as shown by the EMSA. This shift of the complex of probe-protein was not inhibited by unlabeled GRE but was inhibited by unlabeled HNF1 element. This shift was not influenced by the addition of anti-GR, but was super-shifted by the addition of anti-HNF1. GR did not stimulate the induction of HNF1, because we detected no-elevation of the mRNA level of HNF1 by reverse transcription-polymerase chain reaction (RT-PCR). Therefore, the induction of UGT1A1 by DEX-GR did not depend on the elevation of HNF1 but on the interaction of GR with HNF1 or the activation of HNF1 through the transcription of other proteins. Also given the lack of evidence of binding of DEX-GR to HNF1 in the EMSA, the data suggest that the mechanism of DEX-GRE effect on HNF1 is indirect by whatever mechanisms.
Usui
Toru
T
Graduate School of Pharmaceutical Sciences, Nagoya City University, Tababe-dori 3, Mizuhoku, Nagoya 467-8603, Japan.
Kuno
Takuya
T
Ueyama
Hisao
H
Ohkubo
Iwao
I
Mizutani
Takaharu
T
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D003907:Dexamethasone / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D004790:Enzyme Induction; D014453:Glucuronosyltransferase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000099148:UGT1A1 Enzyme
D017931:DNA Primers; D003907:Dexamethasone; D014453:Glucuronosyltransferase; D011965:Receptors, Glucocorticoid; D000099148:UGT1A1 Enzyme