The orphan nuclear receptor NGFI-B regulates expression of the gene encoding steroid 21-hydroxylase.
As part of its trophic action to maintain the steroidogenic capacity of adrenocortical cells, corticotropin (ACTH) increases the transcription of the cytochrome P-450 steroid hydroxylase genes, including the gene encoding steroid 21-hydroxylase (21-OHase). We previously identified several promoter elements that regulate 21-OHase gene expression in mouse Y1 adrenocortical tumor cells. One of these elements, located at nucleotide -65, closely resembles the recognition sequence of the orphan nuclear receptor NGFI-B, suggesting that NGFI-B regulates this essential steroidogenic enzyme. To explore this possibility, we first used in situ hybridization to demonstrate high levels of NGFI-B transcripts in the adrenal cortex of the adult rat. In cultured mouse Y1 adrenocortical cells, treatment with ACTH, the major regulator of 21-OHase transcription, rapidly increased NGFI-B expression. Gel mobility shift and DNase I footprinting experiments showed that recombinantly expressed NGFI-B interacts specifically with the 21-OHase -65 element and identified one complex formed by Y1 extracts and the 21-OHase -65 element that contains NGFI-B. Expression of NGFI-B significantly augmented the activity of the intact 21-OHase promoter, while mutations of the -65 element that abolish NGFI-B binding markedly diminished NGFI-B-mediated transcriptional activation. Specific mutations of NGFI-B shown previously to impair either DNA binding or transcriptional activation diminished the effect of NGFI-B coexpression on 21-OHase expression. Finally, an oligonucleotide containing the NGFI-B response element conferred ACTH response to a core promoter from the prolactin gene, showing that this element is sufficient for ACTH induction. Collectively, these results identify a cellular promoter element that is regulated by NGFI-B and implicate NGFI-B in the transcriptional induction of 21-OHase by ACTH.
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
T E
TE
Wilson
A R
AR
Mouw
C A
CA
Weaver
J
J
Milbrandt
K L
KL
Parker
D000302:Adrenal Cortex / Q000166:cytology / Q000378:metabolism; D000324:Adrenocorticotropic Hormone / Q000502:physiology; D000818:Animals; D000911:Antibodies, Monoclonal; D015152:Blotting, Northern; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D017403:In Situ Hybridization; D051379:Mice; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D013255:Steroid 21-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; C539818:Nr4a1 protein, mouse; C539820:Nr4a1 protein, rat; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D014157:Transcription Factors; D000324:Adrenocorticotropic Hormone; D013255:Steroid 21-Hydroxylase