Marasa Bernard S, Xiao Lan, Rao Jaladanki N, Zou Tongtong, Liu Lan, Wang Jian, Bellavance Emily, Turner Douglas J, Wang Jian-Ying
Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, USA.
Am J Physiol Cell Physiol. 2008 May;294(5):C1277-87. doi: 10.1152/ajpcell.90635.2007. Epub 2008 Mar 5.
Transient receptor potential canonical-1 (TRPC1) functions as a store-operated Ca2+ channel in intestinal epithelial cells (IECs), and induced TRPC1 expression sensitizes IECs to apoptosis by inhibiting NF-kappaB activation. However, the exact mechanism by which increased TRPC1 results in NF-kappaB inactivation remains elusive. Protein phosphatase 2A (PP2A) is a widely conserved protein serine/threonine phosphatase that is implicated in the regulation of a wide array of cellular functions including apoptosis. The present study tests the hypothesis that induced TRPC1 expression inhibits NF-kappaB activation by increasing PP2A activity through Ca2+ influx in IECs. The expression of TRPC1 induced by stable transfection with the wild-type TRPC1 gene increased PP2A activity as indicated by increases in levels of PP2A proteins and their phosphatase activity. Increased levels of PP2A activity in stable TRPC1-transfected IEC-6 cells (IEC-TRPC1) were associated with decreased nuclear levels of NF-kappaB proteins and a reduction in NF-kappaB-dependent transcriptional activity, although there were no changes in total NF-kappaB protein levels. Inhibition of PP2A activity by treatment with okadaic acid or PP2A silencing with small interfering RNA not only enhanced NF-kappaB transactivation but also prevented the increased susceptibility of IEC-TRPC1 cells to apoptosis induced by treatment with tumor necrosis factor-alpha (TNF-alpha)/cycloheximide (CHX). Decreasing Ca2+ influx by exposure to the Ca2+-free medium reduced PP2A mRNA levels, destabilized PP2A proteins, and induced NF-kappaB activation, thus blocking the increased sensitivity of IEC-TRPC1 cells to TNF-alpha/CHX-induced apoptosis. These results indicate that induced TRPC1 expression increases PP2A activity through Ca2+ influx and that increased PP2A sensitizes IECs to apoptosis as a result of NF-kappaB inactivation.
瞬时受体电位香草酸亚型1（TRPC1）作为肠道上皮细胞（IECs）中的一种储存性钙离子通道发挥作用，诱导TRPC1表达可通过抑制核因子κB（NF-κB）激活使IECs对凋亡敏感。然而，TRPC1增加导致NF-κB失活的确切机制仍不清楚。蛋白磷酸酶2A（PP2A）是一种广泛保守的蛋白丝氨酸/苏氨酸磷酸酶，参与包括凋亡在内的多种细胞功能的调节。本研究检验了以下假说：诱导的TRPC1表达通过IECs中钙离子内流增加PP2A活性来抑制NF-κB激活。野生型TRPC1基因稳定转染诱导的TRPC1表达增加了PP2A活性，这表现为PP2A蛋白水平及其磷酸酶活性的升高。稳定转染TRPC1的IEC-6细胞（IEC-TRPC1）中PP2A活性水平的升高与NF-κB蛋白的核水平降低以及NF-κB依赖性转录活性的降低相关，尽管NF-κB总蛋白水平没有变化。用冈田酸处理抑制PP2A活性或用小干扰RNA使PP2A沉默不仅增强了NF-κB反式激活，还阻止了IEC-TRPC1细胞对肿瘤坏死因子-α（TNF-α）/环己酰亚胺（CHX）处理诱导的凋亡敏感性增加。通过暴露于无钙培养基减少钙离子内流降低了PP2A mRNA水平，使PP2A蛋白不稳定，并诱导NF-κB激活，从而阻断了IEC-TRPC1细胞对TNF-α/CHX诱导凋亡的敏感性增加。这些结果表明，诱导的TRPC1表达通过钙离子内流增加PP2A活性，并且增加的PP2A由于NF-κB失活而使IECs对凋亡敏感。
