ATP2A2-Regulated Keratinocyte Ca2+ Signaling Mechanisms

描述（由申请人提供）：原理：细胞内钙库在钙诱导的角质形成细胞分化的信号传导中起关键作用。角质形成细胞内质网（ER）的钙库由钙ATP酶ATP 2A 2维持，而与之密切相关的钙ATP酶ATP 2C 1定位于高尔基体并维持该细胞器中的钙库。ATP 2A 2的突变是人类皮肤病Darier病（DD）的基础，其特征是角质形成细胞分化不完全。我们和其他实验室的工作表明，ATP 2A 2和ATP 2C 1共同作用，形成由细胞外Ca 2+刺激的Ca 2+信号，使角质形成细胞成为已知的第一个受ER和高尔基体Ca 2+储存双重控制的哺乳动物细胞。我们的初步研究表明，在DD角质形成细胞的ATP 2A 2功能障碍的补偿，部分，通过ATP 2C 1上调。这些补偿性变化使DD角质形成细胞保持活力，因为DD角质形成细胞中的ATP 2C 1完全失活会导致细胞死亡。然而，补偿性ATP 2C 1上调也导致异常低的胞质Ca 2+浓度，并可能是DD角质形成细胞中观察到的异常分化的独特模式的基础。在这个建议中，我们计划集中在ATP 2A 2功能障碍和ATP 2C 1上调导致异常钙诱导分化的DD角质形成细胞的分子机制。假设：受损的角质形成细胞分化特征的DD的结果从下游异常的Ca 2+信号通路控制的突变的ATP 2A 2和其他Ca 2+信号蛋白，特别是ATP 2C 1和质膜离子通道的代偿性变化。这种异常的分化可以通过纠正Ca 2+稳态的缺陷，应用其他prodifferentiative代理人，旁路参与外皮蛋白合成的Ca 2 +-响应途径，或这些策略的组合来正常化。具体目的：目的#1：表征DD角质形成细胞中的Ca 2+信号转导缺陷和适应性反应。目标二：确定DD中受损的Ca 2+诱导分化是否由细胞质Ca 2+异常降低、细胞器Ca 2+稳态异常导致的病理性ERK信号传导或受损的SERCA 2依赖性核Ca 2+信号传导引起目的#3：通过改善Ca 2+稳态、增强Involucrin启动子激活或两者来使DD中的分化正常化。该项目的目标是确定导致Darier病异常分化的致病信号通路，从而改善患有这种疾病的患者的皮肤病理学。

DESCRIPTION (provided by applicant): RATIONALE: Intracellular Ca stores play a critical role in signaling Ca2+ -induced keratinocyte differentia- tion. Keratinocyte endoplasmic reticulum (ER) Ca2+ stores are maintained by the Ca2+ ATPase ATP2A2, while a closely related Ca2+ATPase, ATP2C1, localizes to the Golgi and maintains the Ca2+ store in this organelle. Mutations in the ATP2A2 underlie the human skin condition Darier's disease (DD), characterized by incomplete keratinocyte differentiation. Work from ours and other laboratories demonstrates that the ATP2A2 and ATP2C1 act jointly to shape Ca2+ signaling stimulated by raised extracellular Ca2+, making keratinocytes the first mammalian cells known to be under dual control of both ER and Golgi Ca2+ stores. Our preliminary studies reveal that ATP2A2 dysfunction in DD keratinocytes is compensated, in part, by ATP2C1 upregulation. These compensatory changes allow DD keratinocytes to remain viable, since completely inactivating ATP2C1 in DD keratinocytes leads to cell death. However, compensatory ATP2C1 upregulation also leads to abnormally low cytosolic Ca2+ concentrations, and may underlie the unique pattern of abnormal differentiation seen in DD keratinocytes. In this proposal, we plan to focus on the molecular mechanisms by which ATP2A2 dysfunction and ATP2C1 upregulation lead to abnormal Ca2+-induced differentiation in DD keratinocytes. HYPOTHESIS: The impaired keratinocyte differentiation characteristic of DD results both from downstream abnormalities in Ca2+ signaling pathways controlled by the mutated ATP2A2 and by the compensatory changes in other Ca2+ signaling proteins, especially the ATP2C1 and plasma membrane ion channels. This abnormal differentiation can be normalized by correcting defects in Ca2+ homeostasis, applying other prodifferentiative agents that bypass the Ca2+-responsive pathways involved in involucrin synthesis, or a combination of these strategies. SPECIFIC AIMS: Aim#1: To Characterize the Ca2+ Signaling Defects and Adaptive Responses in DD Keratinocytes. Aim #2: To Determine Whether Impaired Ca2+ -induced Differentiation in DD is Caused by Abnormally Decreased Cytosolic Ca2+, Pathologic ERK Signaling Due to Abnormal Organelle Ca2+ Homeostasis, or Impaired SERCA2-dependent Nuclear Ca2+ Signaling Aim #3: To Normalize Differentiation in DD by Improving Ca2+ Homeostasis, Enhancing Involucrin Promoter Activation, or Both. The goal of this project is to define the pathogenic signaling pathways that lead to abnormal differentiation in Darier's disease, thus ameliorating the skin pathology of patients suffering from this condition.