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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 ß

Department of Cell and Developmental Biology and Anatomy, University of South Carolina School of Medicine, Columbia, South Carolina 29208

Address all correspondence and requests for reprints to: Dr. Holly A. LaVoie, Department of Cell and Developmental Biology and Anatomy, Building 1, University of South Carolina School of Medicine, Columbia, South Carolina 29208. E-mail: hlavoie{at}med.sc.edu.

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)ß site. In granulosa cell nuclear extracts, GATA-4 and C/EBPß 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/EBPß 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/EBPß exhibited greater phosphorylation with FSH + IGF-I treatment. Porcine luteal cell nuclear extracts also demonstrated GATA-4 and C/EBPß 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/EBPß are both required for FSH ± IGF-I stimulation of the porcine steroidogenic acute regulatory protein gene promoter in homologous granulosa cell cultures.

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