Ire1 regulates cyp11a1 transcription to mediate steroidogenesis in the ovary

CYP11A1 transcriptional activation in response to gonadotropin hormones is critical to maintain fertility and support pregnancy. Therefore, we are interested in identifying factors that regulate CYP11A1 transcription. Our laboratory has identified several signaling proteins that activate CYP11A1 transcription. In order to identify novel factors that mediate CYP11A1 transcriptional activation, we performed a kinase screen and identified inositol-requiring enzyme 1 alpha (IRE1) as a mediator CYP11A1 transcription. IRE1 is an ER stress signal transducer, however an ER stress response is not induced in KGN cells by forskolin treatment. We hypothesize that IRE1 is necessary to mediate CYP11A1 transcription in response to forskolin, a gonadotropin mimetic, in a mechanism independent to its role in ER stress. In the human KGN granulosa cell line, IRE1 is not predominantly localized to the endoplasmic reticulum. IRE1 colocalizes with Nup98, a nuclear localized marker, in the nuclear membrane in large, oligomeric punctate structures. Since IRE is necessary to mediate CYP11A1 expression, we sought to determine if IRE1 endoribonuclease activity is required for CYP11A1 expression. KGN cells were treated with and without forskolin, or tunicamycin and co-treated with the IRE1 endoribonuclease inhibitors MKC-4485 and MKC-3946. MKC-4485 and MKC-3946 potently inhibited XBP1 splicing but had no effect CYP11A1 up-regulation in response to forskolin stimulation. Additionally, we sought to determine if XBP1u expression is necessary for up-regulating CYP11A1 in response to forskolin. Using RNA interference, XBP1 knockdown does not effect forskolin stimulated CYP11A1 expression. These data show that IRE1 mediates CYP11A1 in response to forskolin independent of its known mechanism in ER stress. In Y1 adrenal cells and JEG3 placental cells, IRE1 is required for forskolin stimulated CYP11A1 up-regulation and pregnenolone biosynthesis suggesting a general mechanism. In conclusion, IRE1 is critically important in activating CYP11A1 transcription. With global concerns increasing over declining fertility in humans, IRE1 represents a novel mechanism regulating steroidogenesis.