ATP2A2-Regulated Keretinocyted Ca2+ Signaling Mechanisms

 描述（由申请方提供）：由内质网（ER）ATP酶ATP 2A 2（蛋白SERCA 2b）突变引起的Darier病（DD）的特征为表皮细胞间粘附受损、病理性细胞凋亡和角质形成细胞分化缺陷。目前的治疗方式不能解决潜在的缺陷，并且在许多患者中无效。DD也作为一个模型，检查钙离子控制的粘附连接以及桥粒的形成和营业额，知识是直接适用于过程，如伤口愈合或屏障修复。在先前的研究期间，我们发现角质形成细胞脂质、PKCa和Ca 2+信号通路在正常和DD角质形成细胞中交叉，以控制Ca 2+依赖性分化和粘附或桥粒连接的形成。我们最近的实验表明，上调葡萄糖神经酰胺合酶（GCS）或下调鞘氨醇-1-磷酸裂解酶（SGPL 1）或神经酰胺酶拯救DD连接形成。这些发现表明，DD中的脂质缺陷集中于特定神经酰胺物质（如C16）的合成，或随后代谢为下游代谢物，包括鞘氨醇/S-1-P、神经酰胺-1-磷酸（C1 P）或葡萄糖神经酰胺。因此，我们已经确定了三个酶的目标（SGPL 1，神经酰胺酶，GCS），可以调节，以改善DD，并开发了新的模型来研究的基底上分化，棘层松解和细胞凋亡特性的DD表皮。这些工具将用于测试我们的假设：总体假设：角质形成细胞ER Ca 2+信号转导控制角质形成细胞脂质代谢，这反过来又反馈增强ER Ca 2+螯合和电容性Ca 2+进入。DD中的SERCA 2b突变减少ER Ca 2+螯合并损害神经酰胺和鞘脂代谢，阻碍PKCa介导的桥粒形成和E-钙粘蛋白介导的粘附连接形成/电容性Ca 2+内流。使脂质代谢正常化将增加ER Ca 2+水平，并将改善DD棘层松解、受损的分化和凋亡。我们现建议：1）鉴定和纠正由缺陷性ER Ca 2+螯合诱导的脂质信号传导缺陷（Sp.Aim#1）;和2）定义PKCa、细胞内神经酰胺/鞘脂和ER Ca 2+信号传导途径如何在正常与DD角质形成细胞信号传导中相互作用（Sp.Aim2）。这些研究的短期目标是为DD开发更有效的治疗方法。这些研究的长期目标是了解ER Ca 2+螯合和释放如何与脂质和PKCa信号相互作用以控制角质形成细胞和表皮分化、细胞间粘附和凋亡。

 DESCRIPTION (provided by applicant): Darier's Disease (DD), caused by mutations in the endoplasmic reticulum (ER) ATPase ATP2A2 (protein SERCA2b), is characterized by impaired epidermal cell-to-cell adhesion, pathologic apoptosis, and defective keratinocyte differentiation. Current treatment modalities do not address the underlying defects, and are ineffective in many patients. DD also serves as a model to examine Ca2+ control of adherens junctions as well as desmosome formation and turnover, knowledge that is directly applicable to processes such as wound healing or barrier repair. During the previous grant period we discovered that keratinocyte lipid, PKCa and Ca2+ signaling pathways intersect in normal and DD keratinocytes to control Ca2+-dependent differentiation and formation of adherens or desmosomal junctions. Our most recent experiments demonstrate that upregulating glucosyceramide synthase (GCS) or downregulating Sphingosine-1-Phosphate Lyase (SGPL1) or ceramidase rescue DD junctional formation. These findings suggest that the lipid defect in DD centers on synthesis of specific ceramide species such as C16, or subsequent metabolism to downstream metabolites, including sphingosine/S-1-P, Ceramide-1-Phosphate (C1P) or glucosylceramide. We thus have identified three enzyme targets (SGPL1, ceramidase, and GCS) that can be modulated to improve DD, and developed new models to study the suprabasal differentiation, acantholysis and apoptosis characteristic of the DD epidermis. These tools will be used to test our hypothesis: Overall Hypothesis: Keratinocyte ER Ca2+ signaling controls keratinocyte lipid metabolism, which in turn feeds back to enhance ER Ca2+ sequestration and capacitive Ca2+ entry. SERCA2b mutations in DD reduce ER Ca2+ sequestration and impair ceramide and sphingolipid metabolism, hindering PKCa -mediated desmosome formation and E-cadherin-mediated adherens junction formation/capacitive Ca2+ influx. Normalizing lipid metabolism will increase ER Ca2+ levels and will ameliorate DD acantholysis, impaired differentiation and apoptosis. We now propose to: 1) identify and correct the lipid signaling defects induced by defective ER Ca2+ sequestration (Sp. Aim #1); and 2) define how PKCa, intracellular ceramide/sphingolipid and ER Ca2+ signaling pathways interact in normal vs. DD keratinocyte signaling (Sp. Aim 2). The short-term goal of these studies is to develop more effective treatments for DD. The long term goal of these studies is to understand how ER Ca2+ sequestration and release interacts with lipid and PKCa signaling to control keratinocyte and epidermal differentiation, cell-to-cell adhesion and apoptosis.