Cellular responses to high NaCl; osmoprotective organic osmolytes

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。当神经病变靶向酯酶（NTE）被证明是一种磷脂酶B，催化生产的GPC从磷脂酰胆碱，我们测试是否NTE有助于高NaCl诱导增加的GPC合成在肾细胞中，发现它。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NFAT 5/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NTE的转录，由NFAT 5/TonEBP介导，和NTE表达的结果增加有助于增加的生产和积累的GPC在哺乳动物肾细胞在组织培养和体内。 我们以前还发现，高尿素和/或NaCl抑制磷酸二酯酶（GPC-PDE）的活性，催化GPC分解为胆碱和磷酸甘油，这有助于渗透诱导GPC。我们将磷酸二酯酶鉴定为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，表明高NaCl和高尿素通过翻译后修饰（PTM）抑制GDPD 5。我们目前正在鉴定PTM中涉及的氨基酸。我们在HEK 293细胞中鉴定了三种，即半胱氨酸25（C25）、C571和苏氨酸587（T587）。活性氧（ROS）参与C25和C571的作用。高浓度的NaCl和尿素会增加ROS。当这种增加被抗氧化剂N-乙酰半胱氨酸阻止时，GDPD 5的抑制作用要小得多。我们发现至少有三种PTM有助于HEK 293细胞中NaCl和尿素诱导的GDPD 5的高抑制作用：1）ROS增加GDPD 5-C25和-C-571之间的二硫键，这抑制GDPD 5活性，如由抗氧化剂N-乙酰半胱氨酸，防止高NaCl和尿素诱导的GDPD 5抑制; GDPD 5-C27 S/C571 S突变或抗氧化剂过氧化物氧还蛋白的过表达增加GDPD 5活性。2)GDPD 5苏氨酸587是组成型磷酸化的。高NaCl和高尿素使GDPD 5-T587去磷酸化。GDPD 5-T587突变为不能磷酸化的丙氨酸，降低了GDPD 5的GPC-PDE活性。3)抑制CDK 1蛋白激酶降低GDPD 5的GDE-PDE活性，而不改变T587处的磷酸化，并降低多重突变的GDPD 5-C27 S/C571 S-T587 A的活性。 为了更好地了解细胞对渗透压的反应，如肾髓质中存在的反应，我们正在使用蛋白质质谱法研究高NaCl诱导的HEK 293细胞中蛋白质磷酸化和亚细胞定位的变化。 我们使用细胞培养中的稳定同位素氨基酸（SILAC）结合质谱法来鉴定HEK 293细胞中基于磷酸化的信号传导途径。我们用1%FDR在四个生物重复样品中鉴定了超过30，000个磷酸肽。超过7，000个独特的磷酸肽被定量。80%有一个磷酸基团，20%有两个或更多个磷酸基团。高NaCl显著改变了300个磷酸肽的丰度。我们确定了这些显著改变的磷酸肽的功能类别富集，以及受高渗影响的细胞途径。这些结果的网络分析表明，p38 MAPK可能激活STAT 1和HSSP 27磷酸化他们响应高NaCl。我们通过磷酸特异性抗体的Western分析证实了这一点，该分析表明p38的抑制降低了高NaCl诱导的STAT 1和HSP 27的磷酸化。 我们使用iTRAQ对暴露于高NaCl 1或8小时或适应高NaCl多次传代的HEK 293细胞的细胞核和细胞质提取物中的蛋白质进行定量。165种蛋白质的丰度在细胞核或细胞质中至少有一次发生变化。高盐显著改变核丰度的蛋白质包括蛋白质折叠和定位、微管基础过程、细胞死亡调控、细胞骨架组织、DNA代谢过程、RNA加工和细胞周期等。在细胞核的显著变化中，我们发现所有6种14-3-3亚型的减少;细胞骨架蛋白的动态变化，提示核骨架重组;微管蛋白的快速减少;热休克蛋白的动态变化。

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. When neuropathy target esterase (NTE) was shown to be a phospholipase B that catalyzes production of GPC from phosphatidylcholine, 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 NFAT5/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 NFAT5/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 also 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. We 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 indicating that high NaCl and high urea inhibit GDPD5 by post translational modification (PTM). We are currently identifying the amino acids involved in the PTMs. We identify three in HEK293 cells, namely cysteine 25 (C25), C571, and threonine 587 (T587). Reactive oxygen species (ROS) are involved in the role of C25 and C571. High NaCl and urea increase ROS. When this increase is prevented by the antioxidant, N-acetyl cysteine, inhibition of GDPD5 is much less. We find that at least three PTMs contribute to high NaCl- and urea-induced inhibition of Gdpd5 in HEK293 cells: 1) ROS increase disulfide bonding between GDPD5-C25 and -C-571, which inhibits GDPD5 activity, as supported by the findings that the antioxidant N-acetylcysteine, prevents high NaCl- and urea-induced inhibition of GDPD5; and GDPD5-C27S/C571S mutation or over expression of the antioxidant, peroxiredoxin, increases GDPD5 activity. 2) GDPD5 threonine 587 is constitutively phosphorylated. High NaCl and high urea dephosphorylate GDPD5-T587. Mutation of GDPD5-T587 to alanine, which cannot be phosphorylated, decreases GPC-PDE activity of GDPD5. 3) Inhibition of CDK1 protein kinase reduces GDE-PDE activity of GDPD5 without altering phosphorylation at T587, and reduces activity of multiply mutated GDPD5-C27S/C571S-T587A. In order to understand better the cellular response to osmotic stress, like that that exists in the renal medulla, we are using protein mass spectrometry to study high NaCl-induced changes in protein phosphorylation and subcellular localization in HEK293 cells. We used Stable Isotopic Amino acids in Cell culture (SILAC) coupled to mass spectrometry to identify phosphorylation based signaling pathways in HEK293 cells. We identified more than 30,000 phosphopeptides in four biological replicate samples with 1% FDR. More than 7,000 unique phosphopeptides were quantified. 80% have a single phosphate group and 20% two or more phosphate groups. High NaCl signifiicantly changes the abundance of 300 phosphopeptides. We identified functional category enrichment for these significantly changed phosphopeptides, and cellular pathways affected by hypertonicity. Network analysis of these results suggested that p38 MAPK might activate STAT1 and HSSP27 by phosphorylating them in response to high NaCl. We confirmed this by Western analysis with phosphospecific antibodies which showed that inhibition of p38 reduces high-NaCl-induced phosphorylation of STAT1 and HSP27. We used iTRAQ to quantify proteins in nuclear and cytoplasmic extracts from HEK293 cells exposed to high NaCl for one or eight hours or adapted to high NaCl for several passages. The abundance of 165 proteins changed in the nucleus or cytoplasm at at least one of the times. The proteins whose nuclear abundance is significanty altered by high NaCl include ones involved in protein folding and localization, microtubule-based process, regulation of cell death, cytoskeleton organization, DNA metabolic process, RNA processing, and cell cycle. Among striking changes in the nucleus, we found a decrease of all six 14-3-3 isoforms; dynamic changes of cytoskeletal proteins, suggestive of nucleoskeletal reorganization; rapid decrease of tubulins; and dynamic changes of heat shock proteins.