Identification of a Novel Transport-independent Function of PiT1/SLC20A1 in the Regulation of TNF-induced Apoptosis
Identification of a Novel Transport-independent Function of PiT1/SLC20A1 in the Regulation of TNF-induced Apoptosis
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DOI：
10.1074/jbc.m110.130989
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发表时间：
2010-11-05
影响因子：
4.8
通讯作者：
Friedlander, Gerard
中科院分区：
文献类型：
--
作者：
Salauen, Christine;Leroy, Christine;Friedlander, Gerard
PiT 1/SLC 20 A1是大多数哺乳动物细胞表达的钠依赖性Pi转运蛋白。有趣的是，PiT 1转录已被证明是上调的肿瘤坏死因子α（TNF），我们现在已经调查了可能参与的PiT 1在TNF诱导的细胞凋亡。我们发现PiT 1缺失的细胞对TNF的促凋亡活性更敏感（即当抗凋亡NF κ B途径失活时）。这些观察是在通过RNA干扰瞬时或稳定耗尽PiT 1的人HeLa癌细胞系和从PiT 1敲除胚胎分离的永生化小鼠胚胎成纤维细胞中进行的。耗尽密切相关的家族成员PiT 2对TNF诱导的细胞凋亡没有影响，表明这种影响是特异于PiT 1的。无论是否存在细胞外Pi，PiT 1耗尽细胞的敏感性增加都是明显的，这表明Pi摄取的缺陷不涉及所观察到的表型。重要的是，我们表明，在PiT 1（-/-）小鼠胚胎成纤维细胞中的Pi摄取突变体的重新表达，有效地延迟细胞凋亡的WT蛋白，表明这种功能的PiT 1是无关的，其运输活动。Caspase-8在PiT 1缺失的细胞中更活化，我们的数据显示MAPK JNK的持续活化响应于TNF而上调。JNK活性实际上参与了PiT 1耗尽的细胞死亡，因为特定的JNK抑制剂延迟了细胞凋亡。
PiT1/SLC20A1 is a sodium-dependent Pi transporter expressed by most mammalian cells. Interestingly, PiT1 transcription has been shown to be up-regulated by the tumor necrosis factor alpha (TNF), and we have now investigated the possible involvement of PiT1 in TNF-induced apoptosis. We show that PiT1-depleted cells are more sensitive to the proapoptotic activity of TNF (i.e. when the antiapoptotic NF kappa B pathway is inactivated). These observations were made in the human HeLa cancer cell line either transiently or stably depleted in PiT1 by RNA interference and in immortalized mouse embryonic fibroblasts isolated from PiT1 knock-out embryos. Depletion of the closely related family member PiT2 had no effect on TNF-induced apoptosis, showing that this effect was specific to PiT1. The increased sensitivity of PiT1-depleted cells was evident regardless of the presence or absence of extracellular Pi, suggesting that a defect in Pi uptake was not involved in the observed phenotype. Importantly, we show that the re-expression of a Pi uptake mutant of PiT1 in PiT1(-/-) mouse embryonic fibroblasts delays apoptosis as efficiently as the WT protein, showing that this function of PiT1 is unrelated to its transport activity. Caspase-8 is more activated in PiT1-depleted cells, and our data reveal that the sustained activation of the MAPK JNK is up-regulated in response to TNF. JNK activity is actually involved in PiT1-depleted cell death because specific JNK inhibitors delay apoptosis.