Differentiation-specific action of orphan nuclear receptor NR5A1 (SF-1): transcriptional regulation in luteinizing bovine theca cells.
BACKGROUND
The orphan nuclear receptor NR5A1 (steroidogenic factor-1, SF-1) is a master regulator of tissue-specific gene expression in reproductive and steroidogenic tissues. Two activating functions, AF-1 and AF-2, have been described to function in a cooperative manner to recruit transcriptional coactivators to the promoter regions of NR5A1-controlled genes.


METHODS
The role of the NR5A1 activating functions AF-1 and AF-2 was studied in primary bovine theca cells. Bovine theca cells were infected with recombinant adenovirus vectors over-expressing wild-type NR5A1 or NR5A1 mutants, in which one of the activating functions of this orphan nuclear receptor had been impaired. Under different culture conditions, theca cell-specific transcript levels were measured by reverse transcription and real-time PCR.


RESULTS
Under culture conditions optimized for cell growth, transcriptional up-regulation of CYP11A1 (P450 side chain-cleavage enzyme) and INSL3 (Insulin-like factor 3, Relaxin-like factor (RLF)) was found to be dependent on the presence of NR5A1 carrying an intact AF-2. Under conditions inducing luteal differentiation of theca cells, CYP11A1 and STAR (Steroidogenic acute regulatory protein) were up-regulated by the action of luteinizing hormone (LH), whereas the differentiation-specific up-regulation of INSL3 was suppressed by LH in luteinizing theca cells. Inhibition of insulin- or IGF1- (insulin-like growth factor I) dependent signal transduction by the RAF1 kinase inhibitor GW5074 and the mitogen-activated protein kinase kinase inhibitor PD98059 resulted in the finding that RAF1 kinase inhibition was able to counteract the LH-dependent regulation of NR5A1-controlled genes, whereas inhibition of the mitogen-activated protein kinase (MAP kinase) pathway did not have any significant effect.


CONCLUSIONS
The regulation of the three NR5A1-controlled genes CYPA11, STAR, and INSL3 in luteinizing theca cells apparently is not dependent on NR5A1 activating functions AF-1 or AF-2. Activation of AF-1 here even appears to have an impairing effect on NR5A1 transcriptional activity, implying that up-regulation of NR5A1-controlled genes uses a different pathway. Our results might be explained by the possible existence of an interconnection between the RAF1 kinase and the cyclic AMP-protein kinase A pathway. Such a non-classical regulatory pathway might play an important role in the control of gene expression in reproductive and steroidogenic tissues.
Walther
Norbert
N
Institute for Hormone and Fertility Research, University of Hamburg, Falkenried 88, D-20251 Hamburg, Germany. n.walther@uke.uni-hamburg.de
Jansen
Martina
M
Akbary
Wasima
W
Ivell
Richard
R
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002417:Cattle; D002454:Cell Differentiation; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000096:biosynthesis* / Q000235:genetics; D000242:Cyclic AMP / Q000502:physiology; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D007211:Indoles / Q000494:pharmacology; D034461:Luteinization / Q000187:drug effects / Q000502:physiology*; D007986:Luteinizing Hormone / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D036361:Peptide Hormones / Q000096:biosynthesis* / Q000235:genetics; D010636:Phenols / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015290:Second Messenger Systems / Q000502:physiology; D013799:Theca Cells / Q000187:drug effects / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects; D014161:Transduction, Genetic; D015854:Up-Regulation / Q000187:drug effects
D002352:Carrier Proteins; D005419:Flavonoids; D007211:Indoles; D036361:Peptide Hormones; D010636:Phenols; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D007986:Luteinizing Hormone; D000242:Cyclic AMP; D002786:Cholesterol Side-Chain Cleavage Enzyme; D019908:Proto-Oncogene Proteins c-raf; C489251:5-iodo-3-((3,5-dibromo-4-hydroxyphenyl)methylene)-2-indolinone; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one