Concerted regulation of the porcine steroidogenic acute regulatory protein gene promoter activity by follicle-stimulating hormone and insulin-like growth factor I in granulosa cells involves GATA-4 and CCAAT/enhancer binding protein beta.
We previously demonstrated that FSH alone or in combination with IGF-I activated the porcine steroidogenic acute regulatory protein gene promoter in a concerted manner in primary cultures of granulosa cells. Studies were undertaken to further delineate cis- and trans-acting elements of the porcine promoter and mechanisms mediating FSH stimulation and its augmentation by IGF-I. Mutation of several putative regulatory elements localized hormone-stimulated activity to the highly conserved GATA site and identified novel nucleotides downstream as a functional CCAAT/enhancer binding protein (C/EBP)beta site. In granulosa cell nuclear extracts, GATA-4 and C/EBPbeta formed a high-molecular-weight complex with an oligonucleotide spanning -76 to -32 bp of the porcine promoter. The intensity of this high-molecular-weight complex was increased in granulosa cell nuclear extracts by treatment with FSH alone and was enhanced with the combination of FSH and IGF-I at 2-3 h of treatment. GATA-4 and C/EBPbeta proteins were uniformly expressed with all treatments at time points associated with increased DNA binding. Treatment (2 h) with FSH alone or FSH + IGF-I increased phosphorylation of GATA-4 on a protein kinase A consensus site. The 38-kDa isoform of C/EBPbeta exhibited greater phosphorylation with FSH + IGF-I treatment. Porcine luteal cell nuclear extracts also demonstrated GATA-4 and C/EBPbeta binding to the -76 to -32 bp region of the promoter providing evidence for their cooperation in vivo. These data indicate that GATA-4 and C/EBPbeta are both required for FSH +/- IGF-I stimulation of the porcine steroidogenic acute regulatory protein gene promoter in homologous granulosa cell cultures.
LaVoie
Holly A
HA
Department of Cell and Developmental Biology and Anatomy, Building 1, University of South Carolina School of Medicine, Columbia, South Carolina 29208, USA. hlavoie@med.sc.edu.
Singh
Dheer
D
Hui
Yvonne Y
YY
D000818:Animals; D001483:Base Sequence; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D050991:GATA4 Transcription Factor; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000502:physiology; D034421:Sus scrofa; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D000098682:Steroidogenic Acute Regulatory Protein
D022782:CCAAT-Enhancer-Binding Protein-beta; D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D010750:Phosphoproteins; D014157:Transcription Factors; D000098682:Steroidogenic Acute Regulatory Protein; D007334:Insulin-Like Growth Factor I; D005640:Follicle Stimulating Hormone