ATP2A2-Regulated Keretinocyted Ca2+ Signaling Mechanisms

 描述(申请人提供)：Darier病(DD)，由内质网(ER)ATPase ATP2A2(蛋白质SERCA2b)突变引起，以表皮细胞与细胞之间的黏附受损、病理性细胞凋亡和角质形成细胞分化缺陷为特征。目前的治疗方式不能解决潜在的缺陷，对许多患者无效。DD还可以作为一个模型来研究钙离子对粘连连接的控制以及桥粒的形成和更新，这些知识直接适用于伤口愈合或屏障修复等过程。在之前的授予期，我们发现角质形成细胞的脂质、PKCA和钙信号通路在正常角质形成细胞和DD角质形成细胞中相交，以控制钙依赖的分化和黏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成酶(GCS)或下调鞘氨醇-1-磷酸裂解酶(SGPL1)或神经酰胺酶可挽救DD连接的形成。这些发现表明，DD的脂质缺陷集中在特定神经酰胺的合成上，如C16，或随后代谢成下游代谢产物，包括鞘氨醇/S-1-P、神经酰胺-1-磷酸(C1P)或葡萄糖神经酰胺。因此，我们已经确定了三个可以调节以改善DD的酶靶标(SGPL1、神经酰胺酶和GCS)，并建立了新的模型来研究DD表皮的基上分化、棘层松解和细胞凋亡特性。这些工具将被用来检验我们的假设：总体假设：角质形成细胞ER钙信号控制角质形成细胞的脂代谢，这反过来又反馈促进ER钙固存和电容性钙进入。DD的SERCA2b突变减少了内质网钙的滞留，损害了神经酰胺和鞘磷脂的代谢，阻碍了PKCA介导的桥粒形成和E-钙粘附素介导的黏附连接形成/电容性钙内流。脂代谢正常化将提高内质网钙水平，改善DD棘层松解、分化障碍和细胞凋亡。我们现在建议：1)识别和纠正由内质网钙离子封存缺陷(Sp.目的：1)和2)明确PKCA、细胞内神经酰胺/鞘磷脂和ER钙信号通路在正常与DD角质形成细胞信号中的相互作用(Sp。目标2)。这些研究的短期目标是开发更有效的DD治疗方法。这些研究的长期目标是了解内质网钙离子的封存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、细胞间黏附和细胞凋亡。

 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.