Global and local cGMP signals in growth control

Robert Feil and Susanne Feil, Tübingen

The physiological outcome of cGMP signal transduction may depend on the spatiotemporal profile of the cGMP signal and the prevalence of global vs local cGMP pools. In the present project, we will test this hypothesis by correlative analysis of cGMP signals and cell behaviour in real-time in living cells and tissues of mice, with a focus on the interplay between cGMP and remodelling processes in vascular smooth muscle cells (VSMCs) and dorsal root ganglion (DRG) neurons. In the previous funding period, we generated transgenic cGMP sensor mice expressing the fluorescence resonance energy transfer (FRET)-based cGi500 sensor either globally in all tissues or selectively in specific cell types. FRET microscopy showed that cGMP signals can indeed be visualized in living primary cells and tissues isolated from cGMP sensor mice. We will now extend our studies to learn more about the mechanisms that shape cGMP signals in VSMCs and DRG neurons, for instance, the role of specific cGMP-degrading phosphodiesterases. We will also analyse the relevance of our findings for vascular remodelling during atherosclerosis and restenosis and for cGMP-mediated axon branching during embryogenesis. Two major questions that will be approached by in vitro and in vivo experiments in mice are (1) how do cGMP signals affect the growth and survival of VSMCs and DRG neurons and (2) how do cell growth and phenotype affect cGMP signalling in these cell types? To achieve our goals, we will closely interact with the other groups of this research unit to share expertise as well as mouse and cell models. The visualization of cGMP during signal transduction in a living mammalian cell or organism will be useful for both basic research on cyclic nucleotides as well as for target identification and drug development. This project will not only provide novel insights into cGMP signalling, but will also advance our general understanding of information processing in health and disease.

Fig. 1. Transgenic mice for cGMP imaging. Upper, Working principle of FRET-based cGi-type cGMP biosensors. In cGi biosensors, such as cGi500, the tandem cGMP-binding sites of the bovine cGKI (grey) are flanked by CFP and YFP. In the absence of cGMP, FRET occurs from excited CFP to YFP leading to light emission from YFP. Upon cGMP binding, the cGi biosensor undergoes a conformational change that causes a decrease in FRET efficiency. Thus, light emission from YFP at 535 nm is reduced, while emission from CFP at 480 nm is increased. The FRET efficiency of cGi-type biosensors is depicted as the CFP/YFP emission ratio, which increases upon cGMP binding to the biosensor. Lower, R26/CAG-cGi500 mice for global and tissue-specific cGMP imaging. Transgenic cGi500 mice were generated by integration of a Cre/lox-switchable cGi500 construct into the murine Rosa26 locus via homologous recombination in embryonic stem cells. Note that a strong CAG promoter was co-integrated to achieve sufficient sensor expression for in vivo FRET imaging. (C) Robert Feil

Project-related publications

PIs of this project; PIs of other FOR 2060 projects are in bold.

1.    Schmidt H, Peters S, Frank K, Wen L, Feil R, Rathjen FG. Dorsal root ganglion axon bifurcation tolerates increased cyclic GMP levels: the role of phosphodiesterase 2A and scavenger receptor Npr3. Eur J Neurosci. 2016; doi: 10.1111/ejn.13434. [Pubmed] [Project 1] [Project 6]

2.    Shuhaibar LC, Egbert JR, Norris RP, Lampe PD, Nikolaev VO, Thunemann M, Wen L, Feil R, Jaffe LA. Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles. Proc Natl Acad Sci U S A. 2015;112:5527-32. [Project 1, Project 4 and Project Z] [pubmed]

3.    Thunemann M, Schmidt K, de Wit C, Han X, Jain RK, Fukumura D, Feil R. Correlative intravital imaging of cGMP signals and vasodilation in mice. Front Physiol. 2014;5:394. [Project 1 and Project Z] [pubmed]

4.    Feil S, Fehrenbacher B, Lukowski R, Essmann, Schulze-Osthoff K, Schaller M, Feil R. Transdifferentiation of vascular smooth muscle cells to macrophage-like cells during atherogenesis. Circ Res. 2014;115:662-7. [Project 1 and Project 5] [pubmed]

5.    Corradini E, Vallur R, Raaijmakers LM, Feil S, Feil R, Heck AJ, Scholten A. Alterations in the cerebellar (phospho)proteome of a cGMP-dependent protein kinase knockout mouse. Mol Cell Proteomics. 2014;13:2004-16. [Project 1] [pubmed]

6.    Vallur R, Kalbacher H, Feil R. Catalytic activity of cGMP-dependent protein kinase type I in intact cells is independent of N-terminal autophosphorylation. PLoS One. 2014;9:e98946. [Project 1] [pubmed]

7.    Dettling J, Franz C, Zimmermann U, Lee SC, Bress A, Brandt N, Feil R, Pfister M, Engel J, Flamant F, Rüttinger LKnipper M. Autonomous functions of murine thyroid hormone receptor TRα and TRβ in cochlear hair cells. Mol Cell Endocrinol. 2014;382:26-37. [Project 1 and Project 8] [pubmed]

8.    Thunemann M, Fomin N, Krawutschke C, Russwurm M, Feil R. Visualization of cGMP with cGi Biosensors. Methods Mol Biol. 2013;1020:89-120. [Project 1 and Project Z] [pubmed]

9.    Thunemann M, Wen L, Hillenbrand M, Vachaviolos A, Feil S, Ott T, Han X, Fukumura D, Jain RK, Russwurm M, de Wit C, Feil R. Transgenic mice for cGMP imaging. Circ Res. 2013;113:365-71.      [Project 1 and Project Z] [pubmed]

10.  Jaumann M, Dettling J, Gubelt M, Zimmermann U, Gerling A, Paquet-Durand F, Feil S, Wolpert S, Franz C, Varakina K, Xiong H, Brandt N, Kuhn S, Geisler H, Rohbock K, Ruth P, Schlossmann J, Hütter J, Sandner P, Feil R, Engel J, Knipper M, Rüttiger L. cGMP-Prkg1 signaling and Pde5 inhibition shelter cochlear hair cells and hearing function. Nat Med. 2012;18:252-9. [Project 1, Project 5, Project 8 and Project 9] [pubmed]