Pulsatile GnRH signaling to ERK: Sensitivity to pulse frequency and robustness to pulse width

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 134-163-GnRH & Gonadotroph Biology & Signaling
Bench to Bedside
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-158
Rebecca Mary Perrett*1, Stephen P Armstrong2, Robert C Fowkes3, Margaritis Voliotis1, Krasimira Tsaneva-Atanasova1 and Craig Alexander McArdle1
1University of Bristol, Bristol, United Kingdom, 2University of Western Australia, Perth, Australia, 3The Royal Veterinary College, London, United Kingdom
GnRH is secreted in pulses and its effects on gonadotropes are dependent upon pulse frequency but little is known about the relevance of other parameters such as pulse width. We have begun to explore this using automated fluorescence microscopy and mathematical modeling, focusing on the ERK signaling cascade that mediates many transcriptional effects of GnRH (1,2). In HeLa cells transduced with GnRH receptors (GnRHR) and ERK2-GFP, 5 min pulses of GnRH cause rapid, transient and reproducible increases in nuclear:cytoplasmic ratio (N:C) of ERK2-GFP (a live cell readout for ERK activation) as well as ERK-dependent increases in transcription with an Egr1-luciferase reporter. Varying GnRH concentration (0.1–100 nM), pulse frequency (one pulse every 3 hours to one pulse every 30 minutes) or GnRHR number (40-160,000 sites/cell) reveals an approximately linear relationship between the input (integrated GnRHR occupancy) and the output (N:C ERK2-GFP or Egr1-luciferase). This implies that GnRHR-mediated ERK activation is a simple integrative tracking system. If so, increasing pulse width would have a corresponding effect on output but, in fact, increasing pulse width 10 fold (from 1-10 min) caused only a 2 fold increase in Egr1-luciferase (at all pulse intervals tested). Mathematical modelling of the GnRHR-mediated ERK cascade revealed the same behaviour as a consequence of time-delayed ERK activation and inactivation. More detailed kinetic analysis revealed that this insensitivity to pulse width could reflect the fact that MEK-dependent ERK activation outlasts the GnRH pulse (having a more pronounced effect with 1 min GnRH pulses than with 10 min pulses). Binning cells according to ERK2-GFP level also revealed dependence of GnRHR-mediated ERK activation kinetics upon ERK concentration (as another possible explanation for more complex input-output behaviours). Together our data provide a wet-lab demonstration and the mathematical basis for a system that is robust to changes in GnRH concentration and pulse width but is sensitive to GnRHR number and pulse frequency, the two variables thought most important for system control in vivo.

(1) Tsaneva-Atanasova K, Caunt CJ, Armstrong SP, Perrett RM, McArdle CA. 2012 Decoding GnRH neurohormone pulse frequency by convergent signalling modules. J Royal Soc Interface 9:170-182 (2) Armstrong SP, Caunt CJ, Fowkes RC, Tsaneva-Atanasova K, McArdle CA. 2010 Pulsatile and sustained gonadotropin-releasing hormone (GnRH) receptor signaling: does the ERK signaling pathway decode GnRH pulse frequency? J Biol Chem 285:24360-71

Nothing to Disclose: RMP, SPA, RCF, MV, KT, CAM

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: This work was funded by Project Grants from the MRC (G0901763) and BBSRC (J014699/1)