PMID- 9140079
OWN - NLM
STAT- MEDLINE
DCOM- 19970805
LR  - 20190813
IS  - 0303-7207 (Print)
IS  - 0303-7207 (Linking)
VI  - 128
IP  - 1-2
DP  - 1997 Apr 4
TI  - The function of retinoid X receptors on negative thyroid hormone response 
      elements.
PG  - 85-96
AB  - Retinoid X receptors (RXRs) form heterodimers with thyroid hormone receptors 
      (TRs). RXRs increase DNA binding affinity of TRs and T3-mediated transactivation 
      on positive T3 response elements (TREs). However, the role of RXRs on negative 
      TREs, and the relation of RXRs to the dominant negative effect of mutant TRs, are 
      not defined. To clarify the function of RXRs on negative TREs, we performed 
      transient cotransfection studies using the rat glycoprotein hormone alpha 
      promoter fused to luciferase gene (alphaLuc), and human TRH promoter fused to 
      luciferase gene (TRH-Luc) as reporters. We found that the JEG-3 cell-alphaLuc 
      system was very sensitive to TR regulation. Using TRbeta1 wild-type (WT) 
      expression vector, 6.2 ng/well (170 ng/10 cm dish), and 0.2 ng/well (11 ng/10 cm 
      dish) caused maximal, and half maximal, inhibition of Luc activities in the 
      presence of 1 nM T3. A T3 dose dependent inhibition study was also performed. 
      From these studies, we determined that the appropriate conditions in which to 
      study alphaLuc transactivation, in a linear portion of the dose response curve, 
      was using 0.8 ng/well TRbeta1 expression vector and 0.1 nM T3. Under these 
      conditions, TRbeta1 mutant R316H (GH), but not G345R (Mf), showed a weak dominant 
      negative effect at a 1:1 ratio in the presence of 0.1 nM T3 although neither 
      mutant had detectable T3 binding affinity. Moreover this dominant negative effect 
      of R316H on the alphaLuc reporter was enhanced in the presence of RXRgamma. 
      Mutant G345R showed a stronger dominant negative effect than did R316H when using 
      a double palindromic TRE fused to herpes simplex thymidine kinase-Luc reporter as 
      a positive TRE. These results conform to the clinical features of R316H which is 
      associated with apparent pituitary resistance of thyroid hormone (PRTH). Mutant 
      R316H also showed a weak dominant negative effect with TRH-Luc at a 1:1 ratio in 
      the absence or presence of RXRgamma. However RXRgamma did not enhance the 
      dominant negative effect as it did using alphaLuc reporter gene. Electrophoretic 
      gel mobility shift assay (EMSA) showed that RXR alpha augmented the DNA binding 
      affinity of wild type and R316H TRs as heterodimers on the previously reported 
      negative TREs of glycoprotein hormone alpha promoter, suggesting that RXR does 
      not produce its response by removing TRs from these TREs. RXR alpha augmented DNA 
      binding affinity of TRbeta1WT, and R316H showed a weaker heterodimer band than 
      did the wild type in EMSA. Using the TRH-Luc reporter, basal activity was 
      increased by wild type TRbeta1. However a TRbeta1 DNA binding domain mutant, 
      (C127S) which can not bind to DNA, did not increase the basal activity. This 
      indicates that DNA binding of the TR is required for increasing basal activity of 
      TRH promoter. These results indicate that (1) RXR-TR heterodimers play a role in 
      basal transactivation and T3 suppression of negatively regulated genes, and (2) 
      RXRs increase the dominant negative effect of some mutant TRs on specific 
      negative TREs. (3) This effect occurs without removing TRs from the TRE. (4) The 
      differential dominant negative effect of mutant R316H (negative TRE > positive 
      TRE) may explain, at least in part, the presentation of R316H as PRTH. (5) 
      Augmentation of basal activity by wild type TRs on a negative TRE requires DNA 
      binding.
FAU - Takeda, T
AU  - Takeda T
AD  - Department of Medicine, The University of Chicago, IL 60637, USA.
FAU - Nagasawa, T
AU  - Nagasawa T
FAU - Miyamoto, T
AU  - Miyamoto T
FAU - Hashizume, K
AU  - Hashizume K
FAU - DeGroot, L J
AU  - DeGroot LJ
LA  - eng
GR  - DK 13377/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - Ireland
TA  - Mol Cell Endocrinol
JT  - Molecular and cellular endocrinology
JID - 7500844
RN  - 0 (DNA-Binding Proteins)
RN  - 0 (Glycoprotein Hormones, alpha Subunit)
RN  - 0 (Receptors, Retinoic Acid)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (Retinoid X Receptors)
RN  - 0 (Transcription Factors)
RN  - 06LU7C9H1V (Triiodothyronine)
RN  - EC 1.13.12.- (Luciferases)
RN  - EC 3.2.1.23 (beta-Galactosidase)
SB  - IM
MH  - Animals
MH  - Base Sequence
MH  - Binding Sites
MH  - COS Cells
MH  - DNA-Binding Proteins/biosynthesis/*physiology
MH  - Dimerization
MH  - Genes, Reporter
MH  - Glycoprotein Hormones, alpha Subunit/*biosynthesis/genetics
MH  - Humans
MH  - Luciferases/biosynthesis
MH  - Mutagenesis, Site-Directed
MH  - Point Mutation
MH  - Promoter Regions, Genetic/*drug effects
MH  - Rats
MH  - Receptors, Retinoic Acid/biosynthesis/*physiology
MH  - Recombinant Fusion Proteins/biosynthesis
MH  - Retinoid X Receptors
MH  - Transcription Factors/biosynthesis/*physiology
MH  - Transcriptional Activation/drug effects
MH  - Transfection
MH  - Triiodothyronine/*pharmacology
MH  - Tumor Cells, Cultured
MH  - beta-Galactosidase/biosynthesis
EDAT- 1997/04/04 00:00
MHDA- 1997/04/04 00:01
CRDT- 1997/04/04 00:00
PHST- 1997/04/04 00:00 [pubmed]
PHST- 1997/04/04 00:01 [medline]
PHST- 1997/04/04 00:00 [entrez]
AID - S0303-7207(97)04025-2 [pii]
AID - 10.1016/s0303-7207(97)04025-2 [doi]
PST - ppublish
SO  - Mol Cell Endocrinol. 1997 Apr 4;128(1-2):85-96. doi: 
      10.1016/s0303-7207(97)04025-2.