Cellular responses to high NaCl; osmoprotective organic osmolytes

甘油磷酸胆碱(GPC)是一种具有渗透保护作用的亲和性和中和性的有机渗透压物质，在肾脏内髓细胞中积聚，以响应高盐和尿素的反应。我们先前发现，高浓度的氯化钠和/或尿素增加了肾脏(Madin-Darby Canine Renal，MDCK)细胞的GPC，GPC是由磷脂酰胆碱衍生的，由当时尚未发现的磷脂酶催化。当神经病靶标酯酶(NTE)被证明是一种磷脂酶B，催化磷脂酰胆碱产生GPC时，我们测试了NTE是否参与了氯化钠诱导的肾细胞GPC合成的增加，发现确实是这样。在小鼠内髓内集合管(MIMCD3)细胞中，高盐使NTE基因和蛋白表达增加。抑制NTE酯酶活性的二异丙基氟磷酸盐减少GPC的积累，就像siRNA特异性地降低NTE蛋白丰度一样。NTE mRNA的20h半衰期不受高盐的影响，但被特定siRNA敲除的NFAT5/TONEBP抑制了高盐诱导的NTE mRNA的增加。此外，长期服用速尿的ClCK1-/-小鼠和服用速尿的正常小鼠肾脏髓内间质氯化钠浓度降低与NTE mRNA和蛋白的降低有关。因此，高盐促进了NFAT5/TONEBP介导的NTE的转录，由此导致的NTE表达的增加有助于在组织培养和体内培养的哺乳动物肾脏细胞中增加GPC的产生和积累。 我们以前还发现，高尿素和/或氯化钠抑制了磷酸二酯酶(GPC-PDE)的活性，该酶催化GPC分解为胆碱和甘油磷酸，这有助于GPC的渗透诱导。我们鉴定该磷酸二酯酶为Gdpd5。从mIMCD3细胞获得的重组Gdpd5免疫共沉淀物具有GPC-PDE活性，当细胞暴露于高盐或高尿素时，其比活性较低，表明高盐和高尿素通过翻译后修饰(PTM)抑制GDPD5。我们目前正在鉴定PTMS中涉及的氨基酸。我们在HEK293细胞中鉴定出三种，即半胱氨酸25(C25)、C571和苏氨酸587(T587)。活性氧物种(ROS)参与C25和C571的作用。较高的氯化钠和尿素浓度增加了ROS。当这种增加被抗氧化剂N-乙酰半胱氨酸阻止时，对GDPD5的抑制要小得多。我们发现，至少有三种PTM参与了高盐和尿素诱导的HEK293细胞对GDPD5的抑制：1)ROS增加了GDPD5-C25和-C-571之间的二硫键，从而抑制了GDPD5的活性，这得到了抗氧化剂N-乙酰半胱氨酸阻止高盐和尿素诱导的对GDPD5的抑制的研究结果的支持；以及GDPD5-C27S/C571S突变或抗氧化剂过氧氧化还原蛋白的过度表达，增加了GDPD5的活性。2)GDPD5苏氨酸587呈结构性磷酸化。高氯化钠和高尿素脱磷GDPD5-T587。GDPD5-T587突变为丙氨酸，不能磷酸化，降低了GDPD5的GPC-PDE活性。3)抑制CDK1蛋白激酶使GDPD5的GDE-PDE活性降低，但不改变T587的磷酸化，并降低倍增突变的GDPD5-C27S/C571S-T587A的活性。 为了更好地了解细胞对渗透胁迫的反应，如肾髓质中存在的渗透胁迫，我们使用蛋白质质谱仪研究了高盐诱导的HEK293细胞蛋白质磷酸化和亚细胞定位的变化。 我们使用稳定同位素氨基酸细胞培养(SILAC)与质谱学相结合的方法来鉴定HEK293细胞中基于磷酸化的信号通路。我们用1%的FDR在四个生物复制样品中鉴定了30,000多个磷酸肽。对7000多个独特的磷酸肽进行了量化。80%具有单一的磷酸盐基团，20%具有两个或更多的磷酸盐基团。高浓度的氯化钠显著改变了300个磷酸肽的丰度。我们确定了这些显著变化的磷酸肽的功能类别丰富，以及受高张作用影响的细胞途径。这些结果的网络分析表明，p38MAPK可能通过磷酸化STAT1和HSSP27来激活它们，以响应高盐。我们通过用磷酸化特异性抗体进行的Western分析证实了这一点，这表明抑制p38可以减少高盐诱导的STAT1和HSP27的磷酸化。 我们使用iTRAQ对HEK293细胞在暴露于高盐1或8小时或适应于高盐几代后的细胞核和细胞质提取物中的蛋白质进行了定量。165种蛋白质的丰度在细胞核或细胞质中至少有一次发生了变化。核丰度被高盐显著改变的蛋白质包括蛋白质折叠和定位、基于微管的过程、细胞死亡的调节、细胞骨架组织、DNA代谢过程、RNA加工和细胞周期。在细胞核的显著变化中，我们发现所有6种14-3-3亚型都减少了；细胞骨架蛋白的动态变化暗示着核骨架的重组；微管蛋白的快速减少；以及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.