Calcium release-activated Calcium (CRAC) current: Impact on human B-cell function and its physiological role in human mesothelial cells

钙（Ca2+）是一种无处不在的信号信使，在细胞生死的各个方面起着至关重要的作用。细胞可以通过从细胞内储存释放Ca2+或通过打开质膜（PM）中的Ca2+传导通道来增加细胞内Ca2+浓度，例如由通道形成蛋白Orai（希腊神话中天堂之门的守护者）和内质网（ER） Ca2+传感器基质相互作用分子（STIM）组成的钙释放激活钙通道（CRAC）。存储耗尽后，STIM结合并激活质膜定位的Orai通道，提供Ca2+内流途径，这对几乎所有真核细胞的正常细胞功能至关重要。仔细滴定STIM和Orai的蛋白浓度，从而确定Ca2+内流的数量及其随后的下游效应，例如活化T细胞核因子（NFAT）信号传导基因转录级联，促凋亡途径如EK信号传导或B/Akt等生存途径。通道形成蛋白Orai以三个同源物Orai1、2和3的形式存在。钙敏感蛋白STIM以两个同源物STIM1和2的形式存在。已有研究表明，储存操作钙进入（SOCE）在免疫细胞功能中起着至关重要的作用，这一过程的失调导致免疫缺陷、自身免疫和癌症的发展。虽然近年来人们对Orai1的生理作用进行了深入研究，但Orai2或Orai3功能对原代细胞的影响仍然难以捉摸。因此，我建议研究Orai2在人原代b细胞和b细胞系中的生理作用（Daudi, Raji）。人类b细胞表现出一个完美的模型系统来描绘天然的Orai2功能，表达大量的Orai2，易于分离和处理。我将用电生理、基于fura2的成像和沉默方法来表征Orai介导的Ca2+内流，并研究其在抗体产生或白细胞介素分泌（如IL-10）方面的生理作用。这部分项目将获得我们有限的关于Orai2的一般知识和关于人b细胞（SA 1）中的CRAC/SOCE的知识。Ca2+信号和CRAC电流在我的第二个研究项目中也起着至关重要的作用。TNFalpha和NF-kappaB信号通路都是Ca2+依赖的过程。Haining Yang等人能够确定这些信号通路在石棉诱发的间皮瘤（一种特殊形式的癌症，每年在美国导致约2000 - 3000人死亡）发生中的重要性。潜在的Ca2+动员过程仍然是难以捉摸的，一般来说，它几乎是一无所知的Ca2+信号在人间皮细胞（HM）。因此，我建议对原代人间皮细胞中的SOCE和CRAC进行表征，并阐明这种特定的Ca2+进入途径对石棉诱导的肿瘤发生的贡献（SA 2）。

Calcium (Ca2+) is an ubiquitous signalling messenger and plays an essential role in every aspect of a cell s life and death. Cells can increase their intracellular Ca2+ concentration either by releasing Ca2+ from intracellular stores or by opening of Ca2+ conducting channels in the plasma membrane (PM), such as the Calcium release-activated calcium channel (CRAC) composed of the channel-forming protein Orai (after keeper of heaven's gate in Greek mythology) and the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule (STIM). Upon store depletion, STIM binds and activates plasma membrane localized Orai channels, providing a Ca2+ influx pathway that is essential for proper cell function in almost all eukaryotic cells. Careful titration of the protein concentration of STIM and Orai thus determines the amount of Ca2+ influx and its subsequent downstream effects e.g. nuclear factor of activated T cells (NFAT) signalling cascades of gene transcription, proapoptotic pathways such as EK signalling or survival pathways such as B/Akt. The channel forming protein Orai exists as the three homologs Orai1, 2 and 3. The calcium sensing protein STIM exists as the two homologs STIM1 and 2. It is already shown that store operated calcium entry (SOCE) plays a crucial role in immune cell function and that dysregulation in this process leads to the development of immunodeficiency, autoimmunity and cancer.While the physiological role of Orai1 was intensively researched within the last years, the impact of Orai2 or Orai3 function in primary cells remains still elusive. Thus, I propose to investigate the physiological role of Orai2 in primary human B-cells and B-cell lines (Daudi, Raji). Human B-cells exhibit a perfect model system for the delineation of the native Orai2 function, expressing remarkable amounts of Orai2 and easy to isolate and to handle. I will characterize the Orai mediated Ca2+ influx with electrophysiological, Fura2-based imaging and silencing approaches and investigate its physiological role regarding the antibody production or the interleukine secretion e.g. IL-10. This part of the project will gain our limited knowledge about Orai2 in general and about CRAC/SOCE in human B-cells (SA 1). Ca2+ signalling and the CRAC current play also a crucial role in my second research project. The TNFalpha and NF-kappaB signalling pathways are both Ca2+ dependent processes. Haining Yang et al. were able to identify the importance of these signalling pathways in the genesis of asbestos-induced mesothelioma, a special form of cancer, which causes ~2.000-3.000 deaths per year in the U.S.. The underlying Ca2+ mobilizing processes are still elusive and in general it is almost nothing known about Ca2+ signalling in human mesothelial cells (HM). So I suggest to characterize SOCE and CRAC in primary human mesothelial cells and to elucidate the contribution of this specific Ca2+ entry pathway to the development of asbestos-induced oncogenesis (SA 2).