Induced expression of STIM1 sensitizes intestinal epithelial cells to apoptosis by modulating store-operated Ca2+ influx.
Induced expression of STIM1 sensitizes intestinal epithelial cells to apoptosis by modulating store-operated Ca2+ influx.
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DOI：
10.1007/s11605-012-1876-8
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发表时间：
2012-07
影响因子：
3.2
通讯作者：
Wang, Jian-Ying
中科院分区：
文献类型：
--
作者：
Timmons, Jennifer A.;Rao, Jaladanki N.;Turner, Douglas J.;Zou, Tongtong;Liu, Lan;Xiao, Lan;Wang, Peng-Yuan;Wang, Jian-Ying
细胞凋亡在维持肠粘膜上皮细胞的稳态中起着关键作用，并受到包括细胞内Ca 2+在内的许多因素的密切调节。经典瞬时受体电位通道1（TRPC 1）在肠上皮细胞（IEC）中表达，并作为钙库操纵的钙通道发挥功能。我们最近已经证明，增加TRPC 1活性敏感的IEC细胞凋亡，但启动TRPC 1激活的上游信号仍然难以捉摸。新的蛋白质，基质相互作用分子1（STIM 1），被证明是作为一个存储Ca 2+传感器，它可以迅速转移到质膜，在那里它直接与TRPC 1相互作用。目前的研究确定了STIM 1是否通过激活TRPC 1通道活性在IEC凋亡的调节中起重要作用。在IEC-6细胞（源自大鼠肠隐窝）和稳定的TRPC 1转染的IEC（IEC-TRPC 1）中进行研究。用肿瘤坏死因子-α（TNF-α）/放线菌酮（CHX）诱导细胞凋亡，荧光数字成像分析测定细胞内游离钙浓度（[Ca 2 +] cyt）。通过特异性siRNA（siSTIM 1）和组成型活性STIM 1 EF-手突变体的异位过表达来研究STIM 1的功能。稳定的STIM 1转染的IEC-6细胞（IEC-STIM 1）显示STIM 1蛋白表达增加（~5倍），并显示Ca 2+库耗尽后Ca 2+内流持续增加（~2倍）。通过形态学特征、DNA片段化和caspase-3活性的变化来测量IEC-STIM 1细胞对TNF-α/CHX诱导的凋亡的敏感性显著增加。在暴露于TNF-α/CHX后4小时，凋亡细胞从亲本IEC-6细胞的约20%增加到稳定IEC-STIM 1细胞的约40%（p<0.05）。此外，稳定的IEC TRPC 1细胞也表现出对TNF-α/CHX诱导的凋亡的敏感性增加，这可以通过siSTIM 1转染的STIM 1沉默来阻止。siSTIM 1沉默STIM 1也减少了过表达TRPC 1的细胞中钙池耗尽后的Ca 2+内流。在STIM 1沉默的人群中，由于储存耗尽导致的Ca 2+内流水平降低了约70%。类似地，IEC-STIM 1细胞暴露于无Ca 2+培养基也阻断了对凋亡的敏感性增加。这些结果表明，1）STIM 1通过改变TRPC 1活性在IEC凋亡的调节中起重要作用，2）异位STIM 1表达通过诱导TRPC 1介导的Ca 2+内流使IEC对凋亡敏感。
Apoptosis plays a critical role in the maintenance of gut mucosal epithelial homeostasis and is tightly regulated by numerous factors including intracellular Ca2+. Canonical transient receptor potential channel-1 (TRPC1) is expressed in intestinal epithelial cells (IECs) and functions as a store-operated Ca2+ channel. We have recently demonstrated that increased TRPC1 activity sensitizes IECs to apoptosis, but the upstream signaling initiating TRPC1 activation remains elusive. The novel protein, Stromal interaction molecule 1 (STIM1), is shown to act as a store Ca2+ sensor and it can rapidly translocate to the plasma membrane where it directly interact with TRPC1. The current study determined whether STIM1 plays an important role in the regulation of IEC apoptosis by activating TRPC1 channel activity. Studies were conducted in IEC-6 cells (derived from rat intestinal crypts) and stable TRPC1-transfected IECs (IEC-TRPC1). Apoptosis was induced by tumor necrosis factor-α (TNF-α)/cycloheximide (CHX), and intracellular free Ca2+ concentration ([Ca2+] cyt) was measured by fluorescence digital imaging analysis. Functions of STIM1 were investigated by specific siRNA (siSTIM1) and ectopic overexpression of the constitutively active STIM1 EF-hand mutants. Stable STIM1-transfected IEC-6 cells (IEC-STIM1) showed increased STIM1 protein expression (~5 fold) and displayed a sustained increase in Ca2+ influx after Ca2+ store depletion (~2 fold). Susceptibility of IEC-STIM1 cells to TNF-α/CHX-induced apoptosis increased significantly as measured by changes in morphological features, DNA fragmentation, and caspase-3 activity. Apoptotic cells were increased from ~20% in parental IEC-6 cells to ~40% in stable IEC-STIM1 cells 4 h after exposure to TNF-α/CHX (p<0.05). In addition, stable IEC-TRPC1 cells also exhibited an increase sensitivity to TNF-α/CHX-induced apoptosis, which was prevented by STIM1 silencing through siSTIM1 transfection. STIM1 silencing by siSTIM1 also decreased Ca2+ influx after store depletion in cells overexpressing TRPC1. Levels of Ca2+ influx due to store depletion were decreased by ~70% in STIM1-silenced populations. Similarly, exposure of IEC-STIM1 cells to Ca2+ free medium also blocked increased sensitivity to apoptosis. These results indicate that 1) STIM1 plays an important role in the regulation of IEC apoptosis by altering TRPC1 activity and, 2) ectopic STIM1 expression sensitizes IECs to apoptosis through induction in TRPC1-mediated Ca2+ influx.
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