Calcium transients trigger switch-like discharge of prostaglandin E₂ (PGE₂) in an ERK-dependent manner

Reviewer #1 (Public Review):

This research article by Watabe T and colleagues characterizes PKA waves triggered by prostaglandin E2 (PGE2). What the author discovered is that waves of PKA occur both in vitro, in MDCK epithelial monolayers, and in vivo, in the ear epidermis in mice. The PKA waves are the consequence PGE2 discharge, that in turn is triggered by Calcium bursts. Calcium level and ERK activity intensity control that mechanism by acting at different levels.

This article is a technological tour de force using different biosensors and optogenetic actuators. What makes this article interesting is the combination of these tools together to dissect a complex, highly dynamic signaling pathway at the single-cell level. For this reason, this paper represents the essence of modern cell biology and paves the way for the cell biology of the future. However, we think that the paper in this stage is still partly descriptive in its nature, and more measurements are needed to increase the strength of the mechanistic insights. Also, the work is not conclusive, some results are over-interpreted, and more work has to be done if the authors want to support all their claims.

Reviewer #2 (Public Review):

This study visualizes a specific localized form of cell-to-cell communication and conveys very well with what dynamics and sensitivity this biological phenomenon occurs.

Using a FRET-based PKA biosensor, the authors observed that radial localized kinase activity changes spontaneously occur in adjacent cells of certain cell density. This phenomenon of radial propagation of PKA activity changes in groups of cells was further mechanistically elucidated and characterized. Interestingly, the authors found that individual cells in the cell groups form spontaneous Ca2+ transients, which at a certain strength can trigger the biosynthesis and release of prostaglandin E2 (PGE2). PGE2 then acts on the neighboring cells and triggers the increase of cAMP levels and the associated activation of the PKA via G-protein-coupled receptors (EP2 and EP4). In systematic, well-structured experiments, it was then found that the frequency of occurrence of such radial activations depends not only on the cell density but also on the activation state of the ERK MAP kinase pathway. The authors skillfully used various modern genetically encoded biosensors and other tools such as optogenetic tools to visualize and characterize an interesting biological phenomenon of cell-to-cell communication. The insights gained with these investigations produce a better understanding of the dynamics, sensitivity, and spatial extent with which such communications can occur in a cell network. It is also worth noting that the authors have not limited the studies to 2D cell culture in vitro, but were also able to confirm the findings in an animal model.

Reviewer #3 (Public Review):

The manuscript entitled "Calcium transients trigger switch-like discharge of prostaglandin E2 (PGE2) in an ERK-dependent manner" by Watabe et al. investigates the interaction between PGE2, PKA, calcium and ERK signaling in MCDK cells and in mouse epidermis. By expressing PKA, calcium and ERK activity reporters, the authors conclude that calcium transients trigger release of PGE2 that signals through GPCR receptors EP2 and EP4 to recruit PKA in neighboring cells. Determining the dynamics of signaling molecules and their interrelationships is important to fully identify the spatiotemporal aspects of signaling mechanisms. This study addresses some aspects of the calcium-PGE2-GPCR-Erk-PKA signaling pathway in a cell line and in mouse skin ex vivo.

However, the sequential recruitment of the different signaling molecules has been described in previous studies. Hence, the novelty of the findings is limited.
Additionally, the interpretation of some of the data is too speculative, more likely explanations are not considered, or not well supported by the data presented. The main conclusions the authors present are potentially artifactual (ie, cell density-dependent phenomena) and the authors need to either do further experiments to better support their conclusions or re-interpret the physiological significance of their findings.