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

Lead Research Organisation: University of Bristol
Department Name: Henry Wellcome LINE

Abstract

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.

Technical Summary

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.

Publications

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McArdle CA (2012) Gonadotropin-releasing hormone receptor signaling: biased and unbiased. in Mini reviews in medicinal chemistry

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Pratap A (2017) Mathematical modeling of gonadotropin-releasing hormone signaling. in Molecular and cellular endocrinology

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Tsaneva-Atanasova K (2012) Decoding neurohormone pulse frequency by convergent signalling modules. in Biochemical Society transactions

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Voliotis M (2014) Information transfer by leaky, heterogeneous, protein kinase signaling systems. in Proceedings of the National Academy of Sciences of the United States of America

 
Description Decoding GnRH pulse frequency (BBSRC - project grant)
Amount £618,457 (GBP)
Funding ID BB/J014699/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2012 
End 05/2015
 
Description Novel mechanisms of oxytocin function in human myometrial cells
Amount £195,773 (GBP)
Funding ID 1797 
Organisation Action Medical Research 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2012 
End 12/2015
 
Title Cell sorting in silico 
Description We use a high content cel imaging platform to monitor cell signalling and for most experiments monitor responses in the whole cell population. However, we have developed methods for binning cells that enable us to relate signalling to reporter concentration (so that responses can be followed with effectors at physiological, sub-physiological and superphysiological levels). We have validated this by comparsion with physical sorting (by flow cytometry) and are using it to explore the relevance of ERK and NFAT expression levels for GnRH signalling. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact In silico sorting provides much higher throughput than the alternative (physical sorting) and is an excellent way to exploit the vast amount of single cell imaging data that we are acquiring. The impact is that we will be able to test mathematical model predictions about the relevance of effector concetration more efficiently. 
 
Description Andres Lopes-Bernal 
Organisation University of Bristol
Department School of Clinical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of reporter constructs (Ad NFAT-EFP, Ad NFAT-RE-luc), ideas, expertise and acccess to equipment. Co-supervision of PhD student and research assistant.
Collaborator Contribution Provision of human myometrial cells, ideas, expertise. Co-supervision of PhD student and research asssistant.
Impact Joint publication.
Start Year 2008
 
Description Clive Bowsher 
Organisation University of Bristol
Department School of Mathematics
Country United Kingdom 
Sector Academic/University 
PI Contribution provision of data from high content imaging experiments.
Collaborator Contribution statistical analysis of data from high content imaging experiments
Impact MSc project co-supervision. Manuscript in submission. This is multidisciplinary - mathematics and biology.
Start Year 2011
 
Description Jim Caunt 
Organisation University of Bath
Department Department of Biology and Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Reagents for generation of molecular tools. hardware and software for high content image data analysis.
Collaborator Contribution Generation of molecular tools - ERK/DUSP feedback
Impact co-authored publications
Start Year 2009
 
Description Krasimira Tsaneva-Atanasova 
Organisation University of Bristol
Department School of Mathematics
Country United Kingdom 
Sector Academic/University 
PI Contribution provision of data from high content imaging experiments
Collaborator Contribution Development of mathematical models based on high content imaging data
Impact BBSRC grant BB/J014699/1 manuscripts (published and in preparation)
Start Year 2009
 
Description Robert Fowkes 
Organisation Royal Veterinary College (RVC)
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration (co-PIs) on MRC grant - provision of data, reagents, training
Collaborator Contribution Collaboration (co-PIs) on MRC grant - provision of data, reagents, training
Impact joint manuscripts and grants
Start Year 2006
 
Description FP2 Medic workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Health professionals
Results and Impact 10-12 Health professionals attend tutorial and demonstration of high content cell imaging - much of the data shown derived from this MRC project.

Feedback not yet received.
Year(s) Of Engagement Activity 2011
 
Description MRC 100th Anniversary Science Cafe (Bristol) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Approx. 100 people attended the pop-up science Cafe event. This included several round-able demonstration/discussion groups - mine was one of them.

Positve feedback from attendees
Year(s) Of Engagement Activity 2013
 
Description Scientific-art advisor & Science Museum launch 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Acting as science advisor for a local artist who has been preparing an installation and a gallery exhibit inspired by HeLa cells and the Henrietta Lacks story. She has also prepared a documentary film (approx. 50 min) featuring interviews with Henrietta lack's descendents and with scientists (including CAM).

Helen has created an installation for the London Science Museum that includes artwork inspired by our cell images and footage of interviews with scientists (including CAM).

The installation launch was coupled with showing of the documentary and Q and A session with scientists (including CAM). This was at the Science Museum's Dana centre in London, and was sponsored by the Science Museum and Wellcome Trust.

Impact is ongoing as the installation will tour other UK venues. Similarly, the gallery exhibit is planned for the M-Shed museum in Bristol, to be coupled with filming of the documentary etc.
Year(s) Of Engagement Activity 2011,2012,2013
URL http://www.sciencemuseum.org.uk/onlinestuff/hela_main/