Calcium signaling in mouse skin: an intravital multiphoton microscopy approach

The mammalian skin is the body’s largest single organ, that forms an essential barrier against dehydration, pathogens, light and mechanical injury. Damage triggers perturbations of the cytosolic free Ca2+ concentration that spread from cell to cell in different epithelia. Ca2+ waves are considered a fundamental mechanism for coordinating multicellular responses, however the mechanisms underlying their propagation in the damaged epidermis are incompletely understood. The aim of the thesis is to dissect the molecular components contributing to Ca2+ wave propagation in murine model of epidermal photodamage. To trigger Ca2+ waves, we used intense and focused pulsed laser radiation and targeted a single keratinocyte of the epidermal basal layer in the earlobe skin of live anesthetized mice. To track photodamage-evoked Ca2+ waves, we performed intravital multiphoton microscopy in transgenic mice with ubiquitous expression of GCaMP6s. To dissect the molecular components contributing to Ca2+ wave propagation, we performed in vivo pharmacological interference experiments by intradermal microinjection of different drugs. Finally we formulated a mathematical model that accounts for the observed Ca2+ dynamics. The major effects of drugs that interfere with degradation of extracellular ATP or P2 purinoceptors suggest that Ca2+ waves are primarily due to release of ATP from the target cell, whose plasma membrane integrity was compromised by laser irradiation. The limited effect of TAT-Gap19 suggest ATP-dependent ATP release though connexin hemichannels plays a minor role, affecting Ca2+ wave propagation only at larger distances, where the concentration of ATP released from the photodamaged cell was reduced by the combined effect of passive diffusion and hydrolysis due to the action of ectonucleotidases. The ineffectiveness of probenecid suggests pannexin channels have no role. As GCaMP6s signals in bystander keratinocytes were augmented by exposure to EGTA in the extracellular medium, the corresponding transient increments of the [Ca2+]c should be ascribed to Ca2+ release from the ER, downstream of ATP binding to P2Y purinoceptors, with Ca2+ entry through plasma membrane channels playing a comparatively negligible role. The effect of thapsigargin and CBX support this conclusion. We modeled the dynamics of Ca2+ waves elicited by photodamage beginning with ATP release from the irradiated cell, including ATP diffusion and degradation and ATP release from connexin hemichannels. We considered IP3-dependent Ca2+ release from the ER as the main mechanism responsible for the observed increase of the [Ca2+]c. The model included also Ca2+-ATPase and Ca2+ leakage through the endoplasmic reticulum and the cell plasma membrane. The one presented here is an experimental model for accidental skin injury that may also shed light on the widespread medical practice of laser skin resurfacing. The results of our experiments and numerical simulations support the notion that Ca2+ waves reflect the sequential activation of bystander keratinocytes by the ATP released through the compromised plasma membrane of the cell hit by laser radiation. We attributed the observed increments of the [Ca2+]c to signal transduction through purinergic P2Y receptors. It is tempting to speculate that response coordination after injury in the epidermis occurs via propagation of the ATP-dependent intercellular Ca2+ waves described in this thesis work.