Roles and interdependence of calcineurin/NFAT and ERK pathways in pulsatile GnRH effects on gonadotrophin expression

Gonadotrophin-releasing hormone (GnRH) is secreted in pulses from the brain and acts at the pituitary to control synthesis and secretion of LH and FSH. These hormones in turn control sex steroid and germ cell production in the gonads, so GnRH is absolutely essential for human reproduction. GnRH pulse frequency varies physiologically, increasing for example, through the menstrual cycle to drive ovulation. Pulsatile GnRH administration mimics the physiological situation, increasing LH/FSH secretion in treatment of some forms of infertility and in IVF. In contrast, sustained stimulation causes desensitisation so that LH/FSH secretion reduces. This reduces sex steroids and is used to treat hormone-dependent cancers (breast, ovary, prostate etc.) Given its physiological and therapeutic importance there is immense interest in understanding GnRH action. Here, the premise is that knowing how GnRH activates its target cells (the receptors and the biochemical responses involved) will provide additional therapeutic targets. A limitation of this work, however, is that most experiments have used constant stimulation so we still know remarkably little about how cells respond to pulsatile GnRH. This is largely because the techniques used to monitor responses within the cell are very labour intensive, so to resolve this research bottle-neck we have developed much more efficient systems for monitoring GnRH signalling based on imaging fluorescent proteins fused to signalling proteins. GnRH causes these fluorescent reporters to move from the cytoplasm to the nucleus. This can be quantified by automated microscopy, providing live-cell readouts for activation. Specifically, we are using extracellular signal regulated kinase (ERK)-GFP and nuclear factor for activated T-cells (NFAT)-EFP. These proteins are implicated as mediators of GnRH effects on LH and FSH expression and both decode stimulus pulse frequency in other systems. We hypothesise that the biochemical pathways activating these proteins mediate GnRH effects on LH and FSH expression with pulsatile stimulation, and that they are interdependent (i.e. that they influence one-another in the cytoplasm and converge on LH and FSH gene promoter regions in the nucleus). We have planned a series of experiments to test this, using cell imaging to test for interdependence of the cytoplasmic signals and complimentary molecular approaches to defining ERK and NFAT targets in LH and FSH genes as well as their relevance for GnRH action. We also plan to extend our imaging strategies to monitor GnRH signalling and transcription in normal pituitary cells, something that has never yet been achieved.

GnRH stimulates secretion and synthesis of gonadotropins and thereby mediates central control of reproduction. It is secreted in pulses, with pulse frequency varying under physiological conditions. Its effects are frequency-dependent as for example, LHbeta and FSHbeta expression are maximal with low-intermediate frequency pulses (0.5-1/hr) whereas alphaGSU expression is maximal at high frequency ( 2/hr). GnRH-regulated gene expression has been studied in detail but most work has involved constant stimulation. In spite of the physiological and pharmacological relevance of GnRH pulse frequency and the fact that gonadotrophs are viewed as a classic frequency decoding model, pulsatile GnRH signalling is poorly understood. This is largely because of the technical difficulty of monitoring signal dynamics with pulsatile stimulation. However, we have developed high throughput automated imaging systems (based on nuclear translocation of ERK2-GFP and NFAT2-EFP) that enable us to monitor GnRH signalling with pulsatile stimulation. These reporters were selected because a) ERK is a major mediator of GnRH effects on gonadotrophin expression with constant stimulation and is also necessary for GnRH effects with pulsatile stimuli, b) NFAT is activated by the Ca2+/CaM/calcineurin pathway and there is already evidence for involvement of this pathway in transcriptional effects of constant GnRH, and c) CaMs, ERKs and NFATs all act as frequency decoders in other systems. These pathways are also interdependent in the immune and cardiovascular systems with ERK activation influencing NFAT activity just as Ca2+, CaM and calineurin influence ERK activation. Moreover, NFAT and ERK effectors characteristically converge as co-dependent regulators at the transcriptome. Accordingly, we hypothesise that these pathways act as interdependent regulators of GnRH action by virtue of cross-talk in the cytoplasm as well as by co-dependent effects at gonadotrophin promoters. We plan to test this using automated imaging to explore functional interactions between Raf/MEK/ERK and Ca2+/CaM/calcineurin/NFAT signalling, in parallel with molecular approaches for defining ERK and NFAT response elements in gonadotrophin subunit promoters, and involvement of these regions in mediating GnRH effects on transcription. Most of the work will be with LbetaT2 cells, a well characterised gonadotroph-derived cell line but we believe that sophisticated imaging strategies will also enable similar experiments with normal gonadotrophs within the mixed cell population of dispersed pituitary cell cultures. For this we plan to develop imaging readouts for gonadotrophin subunit expression (nitroreductase reporters) so that mechanisms of GnRH signalling and transcriptional regulation can be monitored with physiologically relevant stimulation paradigms and with native receptors in normal cells.