Correlation between calcium signaling and ATP release mediated by connexin hemichannels in different mouse models of disease

Abstract

The hypothesis that adenosine triphosphate (ATP) is used as a messenger for cell-to-cell communication, formulated in the 1990’, is now widely accepted [1]. Recent studies pointed out that the alteration of the mechanisms of ATP release, with the consequent alteration of calcium (Ca2+) signaling, could lead to numerous diseases [2]. The understanding of the relation between Ca2+ signaling and ATP release is fundamental for the development of new therapeutic strategies. In this thesis, we focused on the study of purinergic signaling in the developing cochleae and tumor microenvironment (TME). During pre-hearing stages of development in mice, periodic transient elevations of cytosolic free Ca2+ concentration occur spontaneously in the greater epithelial ridge (GER) and propagate as intercellular Ca2+ waves invading variable portions of the GER [3]. Prior works indicate that intercellular Ca2+ waves in the GER rely on the interplay between inositol-1,4,5-trisphosphate (IP3), generated intracellularly, and ATP, released extracellularly. A vast body of data supports the hypothesis that, in the developing cochlea, ATP is released through connexin hemichannels [4], however a direct proof is still lacking. To test this hypothesis, we generated ATP cell-based biosensors (ATP- BCs) stably expressing P2Y2 purinergic receptors (P2Y2R) and sensitive to ATP in the nM range. We also designed and built a closed microfluidic chamber (10 µl max. volume) to put in close contact the cochlea with the ATP BCs plated on the transparent roof of the chamber and facing the fluid interior of the chamber. These ATP-BCs sited at less than 100 µm from the surface of a cochlear organotypic culture plated on the chamber bottom. After loading both biosensor cells and cochlea with Ca2+ -selective dyes, this architecture and the multi-photon microscope allowed us to monitor Ca2+ responses in ATP-BCs during propagation of stimulated Ca2+ waves in the GER of the cochlea underneath. Ca2+ signals disappeared on both cochlea and ATP-BCs upon replacing ARL67156 (100 µM, an inhibitor of ectonucleotidases) with apyrase (40 U/ml, an enzyme that catalyzes the sequential hydrolysis of ATP) in the extracellular medium, confirming that ATP mediated Ca2+ wave propagation in the GER. To determine the source of the released ATP, we tested cochlear organotypic cultures from two different mutant mice with global deletion of pannexin 1 (Panx1−/−), and of connexin 30 (Cx30−/−). Using the microfluidic chamber, we determined that Ca2+ signals in the ATP-BCs were the same irrespective of whether they faced Panx1−/− or age-matched Panx1+/+ cochlear cultures. In contrast, Ca2+ signals were strongly depressed in presence of Cx30−/− cultures. Together, these results validate our working hypothesis and confirm that connexin hemichannels are the major players of ATP release process that mediates Ca2+ wave propagation in the GER. We also explored the possibility of using ATP-BCs to detect extracellular ATP in the tumor microenvironment with promising results. Replacing the fluid trapped between the ATP-BCs and the tumor with a solution containing apyrase, the responses of ATP-BCs were abolished showing that our tool is one of the few probes suitable for the in vivo detection of extracellular ATP [5]. Finally, we investigated the role of the P2X7 purinergic receptor (P2X7R) in tumor angiogenesis through micro-computed tomography (micro-CT). For this purpose, we inoculated parental B16-F10 or B16-F10 P2X7R-silenced (shRNA P2X7R) cells in wild type (WT) and P2X7 knock out (KO) mice and we compared the micro-CT measurements after the perfusion with Microfil compound. The data suggests that tumors derived from inoculation of B16 WT cells in WT mice are more angiogenic compared with the others, thus supporting the hypothesis that this receptor is involved in the release of the vascular endothelial growth factors (VEGF) [6]. Bibliography [1] Ralevic, Vera, and Geoffrey Burnstock. Receptors for purines and pyrimidines. Pharmacological reviews 50.3 (1998): 413-492. [2] Burnstock, Geoffrey. Purinergic signaling: therapeutic developments. Frontiers in pharmacology 8 (2017): 661. [3] Ceriani, Federico, Tullio Pozzan, and Fabio Mammano. Critical role of ATP-induced ATP release for Ca2+ signaling in nonsensory cell net- works of the developing cochlea. Proceedings of the National Academy of Sciences 113.46 (2016): E7194-E7201. [4] Mammano, F. and M. Bortolozzi, Ca2+ signaling, apoptosis and autophagy in the developing cochlea: Milestones to hearing acquisition. Cell Calcium, 2018. 70: p. 117-126. [5] Pellegatti, Patrizia, et al. Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase. PloS one 3.7 (2008): e2599. [6] Adinolfi, Elena, et al. Expression of P2X7 receptor increases in-vivo tumor growth. Cancer research 72.12 (2012): 2957-2969.