ATP2A2-Regulated Keratinocyte Ca2+ Signaling Mechanisms

描述（由申请人提供）：理由：细胞内钙储存在Ca2+诱导的角质细胞分化信号传导中起关键作用。角化细胞内质网（ER） Ca2+储存由Ca2+ atp酶ATP2A2维持，而密切相关的Ca2+ atp酶ATP2C1定位于高尔基体并维持该细胞器中的Ca2+储存。ATP2A2基因突变是人类皮肤状况达瑞尔病（DD）的基础，其特征是角化细胞分化不完全。我们和其他实验室的工作表明，ATP2A2和ATP2C1共同作用，形成由细胞外Ca2+升高刺激的Ca2+信号，使角化细胞成为已知的第一个受到ER和高尔基Ca2+储存双重控制的哺乳动物细胞。我们的初步研究表明，DD角化细胞中的ATP2A2功能障碍部分通过ATP2C1上调来补偿。这些代偿性变化使DD角化细胞保持活力，因为完全失活DD角化细胞中的ATP2C1会导致细胞死亡。然而，代偿性ATP2C1上调也会导致异常低的胞质Ca2+浓度，并可能是DD角化细胞异常分化的独特模式的基础。在本提案中，我们计划关注ATP2A2功能障碍和ATP2C1上调导致DD角化细胞Ca2+诱导异常分化的分子机制。假设：DD的角化细胞分化受损的特征是由于由突变的ATP2A2控制的Ca2+信号通路的下游异常和其他Ca2+信号蛋白的代偿性变化，特别是ATP2C1和质膜离子通道。这种异常分化可以通过纠正Ca2+稳态缺陷来正常化，应用其他促分化剂绕过参与天合蛋白合成的Ca2+反应途径，或这些策略的组合。特定目的：目的#1：表征Ca2+信号缺陷和DD角化细胞的适应性反应。目的2：确定受损Ca2+诱导的DD分化是由细胞器Ca2+稳态异常引起的细胞质Ca2+异常减少、病理ERK信号传导或serca2依赖性核Ca2+信号传导受损引起的。目的3：通过改善Ca2+稳态、增强Involucrin启动子激活或两者同时激活来正常化DD分化。该项目的目标是明确导致Darier病异常分化的致病信号通路，从而改善该疾病患者的皮肤病理。

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.