Follicle-stimulating hormone induction of ovarian insulin-like growth factor-binding protein-3 transcription requires a TATA box-binding protein and the protein kinase A and phosphatidylinositol-3 kinase pathways.
The current study was done to elucidate the mechanism of the FSH stimulation of IGF-binding protein 3 (IGFBP-3) expression and map the FSH response element on the pig IGFBP-3 promoter. Forskolin induced IGFBP-3 reporter activity in transiently transfected granulosa cells. The protein kinase A (PKA) inhibitor [N-[2-(p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl] (and cotransfection with a PKA inhibitor expression vector), the phosphatidylinositol-3 kinase inhibitor [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], and the ERK inhibitor [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene], all blocked FSH stimulation. Use of serial deletion constructs and site-directed mutagenesis show that a TATA box-binding protein site is required for FSH stimulation and that a specific protein 1 (Sp1) site is required for basal transcription. Gel shift assays of nuclear protein with a -61/-25 probe detected four protein-DNA complexes, with bands I and II having significantly higher intensities in FSH-treated cells than in controls. Mutation of the Sp1 site prevented formation of bands I and II whereas mutation of the TATA box-binding protein site prevented formation of band IV. Use of specific antibodies showed that Sp1 participates in formation of band I, Sp3 band II, and p300 in both I and II. Band III was nonspecifically competed out. We conclude that FSH stimulation of IGFBP-3 transcription is mediated by cAMP via the PKA pathway and requires the P1-3 kinase and likely the MAPK pathways.
Ongeri
Elimelda Moige
EM
Pennsylvania State University, College of Medicine, Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA.
Verderame
Michael F
MF
Hammond
James M
JM
D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology / Q000502:physiology*; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011401:Promoter Regions, Genetic / Q000235:genetics; D020218:Response Elements; D017384:Sequence Deletion; D015398:Signal Transduction; D013552:Swine; D016385:TATA Box / Q000235:genetics; D035181:TATA-Box Binding Protein / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D005640:Follicle Stimulating Hormone; D018972:Insulin-Like Growth Factor Binding Protein 3; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D035181:TATA-Box Binding Protein; D014157:Transcription Factors