Role of intracellular sphingolipids in calcium signaling

描述(由申请人提供)： 钙信号通路的改变与从肿瘤到神经退行性疾病的各种人类病理过程有关。钙信号的时空特性可以调节转录因子的活性，直接影响基因的表达。肌醇1，4，5-三磷酸(InsP3)激活含有内源性钙通道(InsP3Rs)的特异性受体，定位于内质网。值得注意的是，许多增加InsP3的细胞表面受体同时增加了细胞内鞘氨醇和鞘氨醇-1磷酸(Sphingosine-1 Phone，简称LP)的产生。这两种鞘磷脂的钙动员作用已被提出，但它们的作用机制尚不清楚，它们激活的细胞内钙通道(S)尚未确定。此外，它们与InsP3的功能相互作用还没有被表征。目前的建议旨在验证这一假设，即细胞内的鞘氨醇和鞘氨醇-LP都通过InsP3Rs来动员钙。这项建议的具体目的是：1)利用将在活细胞内被光激活的笼状前体，表征细胞内鞘氨醇和鞘氨醇-LP-单独作用或与InsP3-联合作用的钙动员作用，并用单细胞显微荧光法和数字成像分析它们的影响。2)研究InsP3R亚型的不同表达是否能决定细胞内鞘氨醇和鞘氨醇-LP激活的钙信号类型。使用表达特定InsP3R亚型的基因工程细胞的实验将与使用不同InsP3R亚型的哺乳动物细胞株进行的研究相结合；3)为了确定鞘氨醇和鞘氨醇-LP是否直接开放InsP3R通道和/或在功能上调节InsP3的作用。为此，将采用平面脂质双层中重组的天然和重组InsP3R亚型的单通道记录。这项建议的长期目标是确定鞘氨醇和鞘氨醇-LP单独或与InsP3-一起动员细胞内钙离子的机制和细胞内通道，并表征它们在质膜受体刺激后在细胞信号转导中的作用。

DESCRIPTION (provided by applicant): AIterations in calcium (Ca) signaling are implicated in human pathologies ranging from neoplastic to neurodegenerative diseases. The spatio-temporal characteristics of Ca signals can regulate the activity of transcription factors and directly affect gene expression. Ca is generally mobilized from intracellular stores by inositol 1,4,5-trisphosphate (InsP3), which activates specific receptors containing an intrinsic Ca channel (InsP3Rs) and localized to the endoplasmic reticulum. Notably, many cell-surface receptors that increase InsP3 concurrently increase the intracellular production of sphingosine and sphingosine-1 phosphate (sphingosine-lP) as well. A Ca-mobilizing role for these two sphingolipids has been proposed, but their mechanism of action is unclear and the intracellular Ca channel(s) they activate yet to be identified. Furthermore, their functional interactions with InsP3 have not been characterized. The current proposal aims to test the hypothesis that both intracellular sphingosine and sphingosine-lP mobilize Ca through InsP3Rs. The specific aims of this proposal are: 1) To characterize the Ca-mobilizing action of intracellular sphingosine and sphingosine-lP - acting separately or in combination with InsP3 - using caged-precursors that will be photo-activated inside living cells and their effects analyzed by single-cell microfluorimetry and digital imaging. The reciprocal interactions between InsP3 and the two sphingolipids during the Ca signaling following the stimulation of several plasma membrane receptors will also be investigated; 2) To establish whether the expression of different InsP3R-subtypes can determine the type of Ca signals evoked by intracellular sphingosine and sphingosine-lP. Experiments using cells genetically engineered to express specific InsP3R-subtypes will be combined with studies performed with mammalian cell lines transfected with distinct InsP3R-subtypes; 3) To ascertain whether sphingosine and sphingosine-lP directly open the InsP3R-channel and/or functionally modulate the action of InsP3. To this end, single-channel recording from native and recombinant InsP3R subtypes reconstituted in planar lipid bilayers will be employed. The long-term objective of this proposal is to identify the mechanisms and intracellular channels through which sphingosine and sphingosine-lP mobilize intracellular Ca - acting alone or in combination with InsP3 - and characterize their role in cellular signaling following the stimulation of plasma membrane receptors.