Direct regulation of androgen receptor-associated protein 70 by thyroid hormone and its receptors.
Thyroid hormone (T3) regulates multiple physiological processes during development, growth, differentiation, and metabolism. Most T3 actions are mediated via thyroid hormone receptors (TRs) that are members of the nuclear hormone receptor superfamily of ligand-dependent transcription factors. The effects of T3 treatment on target gene regulation was previously examined in TRalpha1-overexpressing hepatoma cell lines (HepG2-TRalpha1). Androgen receptor (AR)-associated protein 70 (ARA70) was one gene found to be up-regulated by T3. The ARA70 is a ligand-dependent coactivator for the AR and was significantly increased by 4- to 5-fold after T3 treatment by Northern blot analyses in the HepG2-TRalpha1 stable cell line. T3 induced a 1- to 2-fold increase in the HepG2-TRbeta1 stable cell line. Both stable cell lines attained the highest fold expression after 24 h treatment with 10 nM T3. The ARA70 protein was increased up to 1.9-fold after T3 treatment in HepG2-TRalpha1 cells. Similar findings were obtained in thyroidectomized rats after T3 application. Cycloheximide treatment did not suppress induction of ARA70 transcription by T3, suggesting that this regulation is direct. A series of deletion mutants of ARA70 promoter fragments in pGL2 plasmid were generated to localize the thyroid hormone response element (TRE). The DNA fragments (-234/-190 or +56/+119) gave 1.55- or 2-fold enhanced promoter activity by T3. Thus, two TRE sites exist in the upstream-regulatory region of ARA70. The TR-TRE interaction was further confirmed with EMSAs. Additionally, ARA70 could interfere with TR/TRE complex formation. Therefore, the data indicated that ARA70 suppresses T3 signaling in a TRE-dependent manner. These experimental results suggest that T3 directly up-regulates ARA70 gene expression. Subsequently, ARA70 negatively regulates T3 signaling.
Tai
Pei-Ju
PJ
Department of Biochemistry, Chang-Gung University, and First Cardiovascular Division, Chang Gung Memorial Hospital, 259 Wen-hwa 1 Road, Taoyuan, Taiwan 333, Republic of China.
Huang
Ya-Hui
YH
Shih
Chung-Hsuan
CH
Chen
Ruey-Nan
RN
Chen
Chi-De
CD
Chen
Wei-Jan
WJ
Wang
Chia-Siu
CS
Lin
Kwang-Huei
KH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015152:Blotting, Northern; D002460:Cell Line; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D056918:Nuclear Receptor Coactivators; D015513:Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013963:Thyroid Hormones / Q000494:pharmacology*; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014284:Triiodothyronine / Q000494:pharmacology
C111382:NCOA4 protein, human; D056918:Nuclear Receptor Coactivators; D015513:Oncogene Proteins; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D013963:Thyroid Hormones; D014157:Transcription Factors; D014284:Triiodothyronine; D008156:Luciferases