Gonadotrophin-releasing hormone signalling: exploring the sensing of hormone concentration and dynamics

Abstract

Gonadotrophin-releasing hormone (GnRH) is a hypothalamic neuropeptide that is secreted in pulses and acts via GnRHRs on the pituitary gonadotroph. It activates signal transduction cascades, causing a largely PKC-mediated activation of extracellular signal-regulated kinase (ERK) and Ca2+-mediated activation of nuclear factor of activated T-cells (NFAT), both of which mediate GnRH effects on gonadotrophin secretion. Their activation was monitored by high content imaging (fluorescence staining for ppERK and nuclear translocation of an NFAT1c-EFP reporter). Also, GnRH effects on Egr1-zsGreen and NFAT-RE-asRed (transcriptional readouts for ERK and NFAT activation) were monitored. These responses were compared in HeLa, MCF-7 and LβT2 cells. Results revealed that effects of GnRH mediated by type I GnRHR are dependent upon cellular model, with differences in response kinetics, sensitivity to inhibitors and effects on transcription. Single cell measures of GnRH effects revealed marked cell-cell variability, implying that GnRHR signalling mechanism can be differently constructed between different cell types and between genetically identical cells within a given cell type. For further exploration of cell-cell heterogeneity, a mathematical approach that considers its impact on information transfer was used. The mutual information (MI) between GnRH concentration and measured responses (I(response; GnRH)) was used to quantify (in Bits) information transfer via GnRHRs. MI values for GnRH effects differed between HeLa and LβT2 cells, extending the idea of cell-context-dependent GnRH signalling to the amount of information transferred via the GnRHR. One bit of information can resolve two different signal values. However, the MI values for GnRH sensing were <1 Bit despite 3 Bit inputs irrespective of the cellular model used. Sensing joint pathways increased MI values, but the increase was modest, and this could be because II information transfer had been underestimated by ignoring response dynamics. This was explored by live cell imaging, tracking cells and calculating MI taking response trajectory into account in LβT2 cells. The I(NFAT1c-EFP-NF; GnRH) was ~0.4 Bits at 30 min and increased to >0.5 Bits by consideration of trajectories. The I(Ca2+; GnRH) was ~0.8 Bits at ~24 sec and increased to 1 Bit by consideration of trajectories. These responses were also tracked in cells receiving two pulses of GnRH and this revealed little information gain from the 2nd pulse (for both readouts). This implies that the unknown sources of cell-cell heterogeneity are relatively stable over the time course examined in this work. Most information loss occurs upstream of Ca2+, and when cells were stimulated with ionomycin, the MI value was greater than any MI value reported for GnRH signalling. This suggests that the GnRHRs and their upstream effectors are a signalling node at which significant information loss occurs

Date of Award	1 Oct 2019
Original language	English
Awarding Institution	University of Bristol
Supervisor	Krasimira Tsaneva-Atanasova (Supervisor) & Craig A Mcardle (Supervisor)