Calcium signaling in the photodamaged skin

BACKGROUND The mammalian skin, the body’s largest single organ, is a highly organized tissue that forms an essential barrier against dehydration, pathogens, light and mechanical injury. Damage triggers perturbations of the cytosolic free Ca2+ concentration ([Ca2+]c) that spread from cell to cell (known as intercellular Ca2+ waves) in different epithelia, including epidermis. Ca2+ waves are considered a fundamental mechanism for coordinating multicellular responses, however the mechanisms underlying their propagation in the damaged epidermis are incompletely understood.

AIM OF THE PROJECT To dissect the molecular components contributing to Ca2+ wave propagation in murine model of epidermal photodamage.

METHODS 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 the sensitive and selective Ca2+ biosensor GCaMP6s. To dissect the molecular components contributing to Ca2+ wave propagation, we performed in vivo pharmacological interference experiments by intradermal microinjection of different drugs.

EXPERIMENTAL RESULTS The major effects of drugs that interfere with degradation of extracellular ATP or P2 purinoceptors suggest that Ca2+ waves in the photodamaged epidermis are primarily due to release of ATP from the target cell, whose plasma membrane integrity was compromised by laser irradiation. The limited effect of the Connexin 43 (Cx43) selective inhibitor TAT-Gap19 suggests ATP-dependent ATP release though connexin hemichannels (HCs) 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 the Ca2+ chelator EGTA in the extracellular medium, the corresponding transient increments of the [Ca2+]c should be ascribed primarily 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 (a well-known inhibitor of SERCA pumps) and carbenoxolone (a recently recognized inhibitor of Ca2+ release through IP3 receptors) support this conclusion.

CONCLUSIONS 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, used to treat a range of pathologies from photodamage and acne scars to hidradenitis suppurativa and posttraumatic scarring from basal cell carcinoma excision. The results of our experiments support the notion that Ca2+ waves reflect chiefly 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 chiefly to signal transduction through purinergic P2Y receptors. Several studies have highlighted fundamental roles of P2Y receptors during inflammatory and infectious diseases, and the initial phase of wound healing involves acute inflammation. In addition, hyaluronan is a major component of the extracellular matrix and its synthesis is rapidly upregulated after tissue wounding via P2Y receptor activation. 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 work.

背景：哺乳动物的皮肤，作为身体最大的单一器官，是一种高度组织化的组织，它构成了抵御脱水、病原体、光线和机械损伤的关键屏障。损伤会触发细胞质游离钙离子浓度([Ca2+]c)的扰动，这种扰动从细胞到细胞（称为细胞间钙离子波）在不同上皮组织中传播，包括表皮。细胞间钙离子波被认为是协调多细胞反应的基本机制，然而，其在大面积受损表皮中传播的机制尚不完全明了。
研究目的：本项目旨在解析导致小鼠表皮光损伤模型中钙离子波传播的分子成分。
方法：为了触发钙离子波，我们使用了强烈的聚焦脉冲激光辐射，并针对活体麻醉小鼠耳廓皮肤中的表皮基底层的单个角质形成细胞。为了追踪由光损伤引发的钙离子波，我们在具有普遍表达敏感和选择性钙离子生物传感器GCaMP6s的转基因小鼠中进行了活体多光子显微镜检查。为了解析导致钙离子波传播的分子成分，我们通过真皮微注射不同药物进行了在体药理学干扰实验。
实验结果：干扰细胞外ATP或P2嘌呤受体的降解的药物的主要作用表明，光损伤表皮中的钙离子波主要是由于靶细胞释放ATP所致，其质膜完整性因激光照射而受损。Connexin 43(Cx43)选择性抑制剂TAT-Gap19的有限作用表明，通过连接蛋白半通道(HCs)的ATP依赖性ATP释放在钙离子波传播中起次要作用，仅在较大距离上影响钙离子波传播，因为由外核苷酸酶的作用引起的被动扩散和水解作用，光损伤细胞释放的ATP浓度降低。普罗本西德的无效性表明，Pannexin通道没有作用。由于GCaMP6s信号在旁观者角质形成细胞中通过在细胞外介质中暴露于钙离子螯合剂EGTA而被增强，因此应将相应的[Ca2+]c的短暂增加主要归因于ER中的钙释放，下游于ATP结合到P2Y嘌呤受体，通过质膜通道的钙进入起着相对微不足道的作用。Thapsigargin（一种已知的SERCA泵抑制剂）和Carbenoxolone（一种最近被认可的通过IP3受体释放钙的抑制剂）的作用支持这一结论。
结论：本研究提出的是一个用于意外皮肤损伤的实验模型，也可能对激光皮肤磨削这一广泛的医疗实践提供启示，该实践用于治疗从光损伤和痤疮疤痕到汗腺炎和基底细胞癌切除后的创伤性疤痕等多种疾病。我们的实验结果支持了钙离子波主要反映旁观者角质形成细胞被激光辐射击中的细胞受损的质膜释放的ATP引发的连续激活的观点。我们将观察到的[Ca2+]c的增加主要归因于通过嘌呤能P2Y受体的信号转导。几项研究已经强调了P2Y受体在炎症和感染疾病中的基本作用，伤口愈合的初始阶段涉及急性炎症。此外，透明质酸是细胞外基质的主要成分，其合成在组织损伤后通过P2Y受体激活迅速上调。很有诱惑力地推测，表皮损伤后的反应协调是通过本工作中描述的ATP依赖性细胞间钙离子波的传播来实现的。