Molecular characterization of a disintegrin and metalloprotease-17 (ADAM17) in granulosa cells of bovine preovulatory follicles.

A disintegrin and metalloprotease-17 (ADAM17) is thought to play a key role in the release of soluble and active epiregulin (EREG) and amphiregulin (AREG) in ovarian follicles but its transcriptional regulation in follicular cells remains largely unknown. The objectives of this study were to characterize the regulation of ADAM17 transcripts in bovine follicles prior to ovulation and to investigate its transcriptional control in bovine granulosa cells. To study the regulation of ADAM17 transcripts, RT-PCR analyses were performed using total RNA extracted from bovine follicles collected between 0 h and 24 h post-hCG. Results showed that levels of ADAM17 mRNA were low prior to hCG (0 h), markedly and transiently increased 6-12 h post-hCG (P <0.05), and returned to low baseline levels at 24 h post-hCG in granulosa and theca interna cells of preovulatory follicles. To determine the transcriptional control of ADAM17 expression, primary cultures of bovine granulosa cells were used. Forskolin (FSK) stimulation induced a pattern of ADAM17 mRNA up-regulation in vitro similar to that observed by hCG in vivo. 5'-Deletion mutagenesis studies identified a minimal region of the bovine ADAM17 promoter containing basal and FSK-inducible activities, which were dependent on the presence of a consensus AP1 cis-element. Electrophoretic mobility shift assays revealed an interaction between AP1 and the trans-acting factor Fra2. Chromatin immunoprecipitation assays confirmed an endogenous interaction between Fra2 and the ADAM17 promoter in granulosa cell cultures. FSK-inducible ADAM17 promoter activity and mRNA expression were suppressed by PKA and ERK1/2 inhibitors but not by a p38MAPK inhibitor, pointing to the importance of PKA and ERK1/2 signaling pathways in the up-regulation of bovine ADAM17 mRNA. Collectively, these findings describe the gonadotropin/FSK-dependent up-regulation of ADAM17 transcripts in bovine preovulatory follicles and unravel for the first time some of the molecular mechanisms involved in ADAM17 gene expression in granulosa cells of a monoovulatory species.