Transcriptional Regulation of Insulin-like Growth Factor Binding Protein 2 in Rat Granulosa Cells

Follicle stimulating hormone (FSH) is essential for normal folliculogenesis, a process that is also regulated by insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding proteins (IGFBP). As folliculogenesis progresses, the IGF/IGFBP system works in conjunction with FSH to induce maximal stimulation of steroidogenesis ultimately producing a mature oocyte. A growing body of evidence suggests that IGFBPs have antigonadotropic and atretogenic roles and that dysregulated expression of IGFBP is associated with a variety of ovarian disorders and cancers. While the differential gene expression patterns of IGFBPs during folliculogenesis and ovarian cancer progression has been extensively studied, the specific factors and mechanisms that regulate IGFBP transcription are not known. This study was designed to elucidate the mechanisms that control IGFBP2 expression in rat granulosa cells and examine the effect of FSH on the expression of this gene. FSH as well as Forskolin and dBcAMP were able to decrease IGFBP2 mRNA levels and promoter activity indicating that FSH decreases IGFBP expression through a cAMP-mediated mechanism. The physiological relevance of this finding is evidenced by the complete reversal of FSH-induced cell differentiation and steroidogenesis in cells overexpressing IGFBP2. 5’ serial deletions of the IGFBP2 promoter indicate that the fragment -176 to +1 contains all the elements necessary to sustain high basal expression of IGFBP2 and those needed for FSH inhibition as well. Further analysis of the IGFBP2 promoter revealed that it contains three highly conserved GC-rich regions known to bind Sp1, which is important for the transcription of TATA-less promoters. In addition, the promoter contained putative sites for ER and NF-1. NF-1 sites bind the Nfi transcription factors, and considering that FSH up-regulates these proteins and that they are known to inhibit Sp1-mediated transcription, we concluded that the Nfi transcription factors may mediate the inhibitory effect of FSH on IGFBP2 expression. When the PKA, MAPK, or PI3K signaling cascades were blocked, it was found that none of these FSH-stimulated pathways mediated the inhibitory effect of FSH on IGFBP2. However, this inhibitory effect was abolished by using an IGF-1R inhibitor suggesting that the decrease in IGFBP2 expression in response to FSH is dependent upon IGF-1 signaling. In summary, FSH decreases IGFBP2 expression at the transcriptional level. The inhibitory effect of FSH is mimicked by a pharmacological increase of intracellular cAMP but it was not prevented by the inhibition of the PI3K and MAPK signaling cascades. Moreover, the inhibitory effect of FSH requires IGF 1 receptor activation. We determined that a minimal promoter (-176 to +1) contains the elements necessary to maintain high IGFBP2 expression at basal conditions and to mediate FSH-inhibition. Taken together, these results suggest that follicles that express high levels of IGF-1 respond to FSH more efficiently not only by increasing steroid production but also by strongly inhibiting IGFBP2 expression. This mechanism might be crucial for the selection of the dominant follicle.