Response Of Renal Cells To Osmotic Stress

推理与细胞保护性转录因子TonEBP/OREBP/NFAT 5（TonEBP）物理相关的蛋白质可能调节或支持其活性，我们使用蛋白质组学来鉴定它们。 我们在HEK 293细胞中稳定表达TonEBP的氨基酸1-547，并将其与来自暴露于高NaCl的细胞核的相关蛋白免疫沉淀，从而鉴定出15种相关蛋白。相关蛋白分为几类：1）DNA依赖性蛋白激酶，包括其催化亚基和调节亚基，Ku 86和MDC 1; 2）RNA解旋酶，即RNA解旋酶A、核仁RNA解旋酶II/Gu和DEAD盒RNA解旋酶p72; 3）小的或异质的核核糖核蛋白（snRNP或hnRNP），即U 5 snRNP特异性116 kDa蛋白、U 5 snRNP特异性200 kDa蛋白、hnRNP U、hnRNP M、hnRNP K和hnRNP F;和5）聚（ADP-核糖）聚合酶-1（PARP-1）。我们通过Western分析证实了大多数蛋白质的鉴定，并通过电泳迁移率变化试验证明它们存在于与TonEBP/OREBP沿着特异性结合到其同源DNA元件的大复合物中。此外，我们发现PARP-1、MDC 1和Hsp 90调节TonEBP/OREBP活性。PARP-1表达降低TonEBP/OREBP转录活性及其反式激活结构域的活性。Hsp 90和MDC 1增强这些活性，并维持暴露于高NaCl的细胞中TonEBP/OREBP蛋白丰度的增加。 我们目前正在扩展这些蛋白质组学研究，以确定与TonEBP-1-1531相关的其他蛋白质，并确定TonEBP中被高盐磷酸化的氨基酸。 到目前为止，我们已经确定了几个磷酸化的氨基酸，并已证实与磷酸特异性抗体，其中一些磷酸化的调节。 此外，这些氨基酸的突变影响TonEBP的渗透调节。 甘油磷酸胆碱（GPC）是一种保护肾脏的相容性和抵消性有机渗透剂，在高NaCl和尿素的反应中在肾内髓细胞中积累。我们先前发现高NaCl和/或尿素增加肾（Madin-Darby犬肾，MDCK）细胞中的GPC，并且GPC来源于磷脂酰胆碱，由当时未鉴定的磷脂酶催化。神经病靶向酯酶（NTE）是一种磷脂酶B，催化磷脂酰胆碱生成GPC。因此，我们测试了NTE是否有助于肾细胞中高NaCl诱导的GPC合成增加，发现它确实如此。在小鼠内髓集合管（mIMCD 3）细胞，高NaCl增加NTE mRNA和蛋白。抑制NTE酯酶活性的氟磷酸二异丙酯减少GPC积累，特异性降低NTE蛋白丰度的siRNA也是如此。NTE mRNA的20小时半衰期不受高NaCl的影响，但通过特异性siRNA敲低TonEBP抑制高NaCl诱导的NTE mRNA增加。此外，较低的肾内髓间质NaCl浓度，慢性发生在ClCK 1-/-小鼠和急性在正常小鼠给予呋塞米与较低的NTE mRNA和蛋白质。因此，高NaCl增加了由TonEBP介导的NTE的转录，并且由此产生的NTE表达的增加有助于组织培养和体内哺乳动物肾细胞中GPC的产生和积累增加。 我们先前发现，高尿素和/或NaCl抑制磷酸二酯酶（GPC-PDE）的活性，该酶催化GPC分解为胆碱和磷酸甘油，并且这有助于GPC的渗透诱导。在目前未发表的研究中，我们已经确定磷酸二酯酶为Gdpd 5。 从mIMCD 3细胞免疫沉淀的重组Gdpd 5具有GPC-PDE活性，并且如果细胞已经暴露于高NaCl或尿素，则比活性较低。 此外，高盐会降低Gdpd 5 mRNA丰度，而针对Gdpd 5的特异性siRNA会增加GPC。 因此，Gdpd 5是GPC-PDE，其活性有助于GPC的渗透调节。高NaCl或尿素降低了Gdpd 5的磷酸化，这降低了其活性。 我们已经确定了几种调节TonEBP活性的信号分子。 c-Jun和c-Fos与许多受TonEBP调节的基因中邻近ORE的AP-1位点结合，并且该位点以及c-fos和c-Jun活性的存在对于高盐完全激活TonEBP是必需的。 高NaCl激活c-Abl，导致TonEBP-Y143的磷酸化增加。高NaCl还抑制SHP-1磷酸酶，其有助于TonEBP-Y143的磷酸化。当Y143被磷酸化时，磷脂酶C γ与TonEBP结合，增加其转录活性、核定位和反式激活活性。 最后，我们以前发现，高渗导致DNA断裂和氧化应激在细胞培养和在体内的肾脏髓质。 DNA损伤和氧化应激与细胞衰老有关，在衰老和癌症中最引人注目。 我们现在已经发现，高盐会导致组织培养中的细胞衰老，衰老细胞的年龄相关积累在正常小鼠的肾髓质中加速，在C.暴露在高盐环境中的优雅。 因此，高渗不仅导致DNA损伤和氧化应激，而且还导致细胞衰老。

Reasoning that proteins that physically associate with the osmoprotective transcription factor TonEBP/OREBP/NFAT5 (TonEBP) are likely to regulate or support its activity we used proteomics to identify them. We stably expressed amino acids 1-547 of TonEBP in HEK 293 cells and immunoprecipitated it plus associated proteins from the nuclei of cells exposed to high NaCl, thereby identifying 15 associated proteins. The associated proteins fall into several classes: 1) DNA-dependent protein kinase, both its catalytic subunit and regulatory subunit, Ku86, and MDC1; 2) RNA helicases, namely RNA helicase A, nucleolar RNA helicase II/Gu, and DEAD-box RNA helicase p72; 3) small or heterogeneous nuclear ribonucleoproteins (snRNPs or hnRNPs), namely U5 snRNP-specific 116 kDa protein, U5 snRNP-specific 200 kDa protein, hnRNP U, hnRNP M, hnRNP K, and hnRNP F; 4) heat shock proteins, namely Hsp90beta and Hsc70; and 5) poly(ADP-ribose) polymerase-1 (PARP-1). We confirmed identification of most of the proteins by Western analysis and also demonstrated by electrophoretic mobility-shift assay that they are present in the large complex that binds specifically along with TonEBP/OREBP to its cognate DNA element. In addition, we found that PARP-1, MDC1 and Hsp90 modulate TonEBP/OREBP activity. PARP-1 expression reduces TonEBP/OREBP transcriptional activity and the activity of its transactivating domain. Hsp90 and MDC1 enhance those activities and sustain the increased abundance of TonEBP/OREBP protein in cells exposed to high NaCl. We are currently extending these proteomic studies to identify additional proteins that associate with TonEBP-1-1531 and also to identify amino acids in TonEBP that are phosphorylated by high salt. Until now we have identified several phosphorylated amino acids and have confirmed with phospho-specific antibodies that phosphorylation of some of them is osmotically regulated. Also, mutation of those amino acids affects osmotic regulation of TonEBP. Glycerophosphocholine (GPC) is an osmoprotective compatible and counteracting organic osmolyte that accumulates in renal inner medullary cells in response to high NaCl and urea. We previously found that high NaCl and/or urea increases GPC in renal (Madin-Darby canine kidney, MDCK) cells and that the GPC is derived from phosphatidylcholine, catalyzed by a phospholipase that was not identified at that time. Neuropathy target esterase (NTE) was recently shown to be a phospholipase B that catalyzes production of GPC from phosphatidylcholine. Therefore, we tested whether NTE contributes to the high NaCl-induced increase of GPC synthesis in renal cells, finding that it does. In mouse inner medullary collecting duct (mIMCD3) cells, high NaCl increases NTE mRNA and protein. Diisopropyl fluorophosphate, which inhibits NTE esterase activity, reduces GPC accumulation, as does an siRNA that specifically reduces NTE protein abundance. The 20-h half-life of NTE mRNA is unaffected by high NaCl, but knockdown of TonEBP by a specific siRNA inhibits the high NaCl-induced increase of NTE mRNA. Further, the lower renal inner medullary interstitial NaCl concentration that occurs chronically in ClCK1-/- mice and acutely in normal mice given furosemide is associated with lower NTE mRNA and protein. Thus, high NaCl increases transcription of NTE, mediated by TonEBP, and the resultant increase of NTE expression contributes to increased production and accumulation of GPC in mammalian renal cells in tissue culture and in vivo. We previously found that high urea and/or NaCl inhibit the activity of a phosphodiesterase (GPC-PDE) that catalyzes breakdown of GPC to choline and glycerol phosphate, and that this contributes to osmotic induction of GPC. In current unpublished studies we have identified the phosphodiesterase as Gdpd5. Recombinant Gdpd5 immunoprecipitated from mIMCD3 cells has GPC-PDE activity and the specific activity is lower if the cells have been exposed to high NaCl or urea. Further, high salt reduces Gdpd5 mRNA abundance and a specific siRNA against Gdpd5 increases GPC. Thus, Gdpd5 is a GPC-PDE, whose activity contributes to osmotic regulation of GPC. High NaCl or urea decreases phophorylation of Gdpd5, which reduces its activity. We have identified several more signaling molecules that regulate TonEBP activity. c-Jun and c-Fos bind to an AP-1 site adjacent to the OREs in many genes that are regulated by TonEBP, and presence of the site and of c-fos and c-Jun activity are necessary for full activation of TonEBP by high salt. High NaCl activates c-Abl, resulting in increased phosphorylation of TonEBP-Y143. High NaCl also inhibits SHP-1 phophatase, which contributes to phosphorylation of TonEBP-Y143. When Y143 is phosphorylated, phospholipase C gamma binds to TonEBP, increasing its transcriptional activity, nuclear localization, and transactivating activity. Finally, we previously found that hyperosmolality causes DNA breaks and oxidative stress both in cell culture and in kidney medullas in vivo. DNA damage and oxidative stress are associated with cellular senescence, most striking in aging and in cancer. We have now found that high salt causes cellular senescence in tissue culture and that age-associated accumulation of a senescent cells is accelerated in kidney medullas of normal mice, as well as in C. Elegans exposed to high salt. Thus, hyperosmolality not only causes DNA damage and oxidative stress, but also causes cellular senescence.