The atypical orphan nuclear receptor DAX-1 interacts with orphan nuclear receptor Nur77 and represses its transactivation.
DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome, gene 1) (NROB1) is an atypical member of the nuclear receptor family, which lacks the classical zinc finger DNA binding domain and acts as a coregulator of a number of nuclear receptors. In this study, we have found that DAX-1 is a novel coregulator of the orphan nuclear receptor Nur77 (NR4A1). We demonstrate that DAX-1 represses the Nur77 transactivation by transient transfection assays. Specific interaction between Nur77 and DAX-1 was detected by coimmunoprecipitation, yeast two-hybrid, and glutathione-S-transferase pull-down assays. The ligand binding domain of DAX-1 and the activation function-2 domain of Nur77 were determined as the direct interaction domains between DAX-1 and Nur77. In vitro competition binding assay showed that DAX-1 repressed Nur77 transactivation through the competition with steroid receptor coactivator-1 for the binding of Nur77. Moreover, DAX-1 repressed Nur77- and LH-dependent increase of cytochrome P450 protein 17 promoter activity in transient transfection assays. Furthermore, Nur77-mediated transactivation was significantly increased by down-regulation of DAX-1 expression with DAX-1 small interfering RNA in testicular Leydig cell line, K28. LH treatment induced a transient increase in Nur77 mRNA, whereas LH repressed DAX-1 expression in a time- and dose-dependent manner in K28 cells. In addition, immunohistochemical analysis showed the expression of Nur77 in mouse testicular Leydig cells. These results suggest that DAX-1 acts as a novel coregulator of the orphan nuclear receptor Nur77, and that the DAX-1 may play a key role in the regulation of Nur77-mediated steroidogenesis in testicular Leydig cells.
Song
Kwang-Hoon
KH
Hormone Research Center, Chungnam National University, Gwangju 500-757, Republic of Korea.
Park
Yun-Young
YY
Park
Ki Cheol
KC
Hong
Cheol Yi
CY
Park
Jin Hee
JH
Shong
Minho
M
Lee
Keesook
K
Choi
Hueng-Sik
HS
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D051548:Histone Acetyltransferases; D007985:Leydig Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007986:Luteinizing Hormone / Q000494:pharmacology; D008297:Male; D051379:Mice; D056920:Nuclear Receptor Coactivator 1; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism*; D011987:Receptors, Steroid; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics
D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins; C539966:Nr0b1 protein, mouse; C539818:Nr4a1 protein, mouse; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid; D012097:Repressor Proteins; D014157:Transcription Factors; D007986:Luteinizing Hormone; D013254:Steroid 17-alpha-Hydroxylase; D051548:Histone Acetyltransferases; C538861:Ncoa1 protein, mouse; D056920:Nuclear Receptor Coactivator 1