Cellular responses to high NaCl; osmoprotective organic osmolytes

细胞对高氯化钠的反应；

• 批准号: 8558069
• 负责人: MAURICE BENJAM BURG
• 金额: $ 68.52万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8558069
• 关键词: Acetylcysteine; Affect; Alanine; Amino Acids; Antioxidants; Betaine; Biological; CDC2 Protein Kinase; Categories; Cell Culture Techniques; Cell Cycle; Cell Death; Cell Nucleus; Cell Volumes; Cells; Choline; Coupled; Cysteine; Cytoplasm; Cytoskeletal Proteins; Cytoskeleton; DNA; Duct (organ) structure; Employee Strikes; Enzymes; Furosemide; Genetic Transcription; Glycerol; Goals; HSPB1 gene; Half-Life; Heat shock proteins; Hour; Inositol; Kidney; Kidney Part; Lecithin; Lysophospholipase; MAP Kinase Gene; MAPK14 gene; MDCK cell; Mass Spectrum Analysis; Mediating; Messenger RNA; Metabolism; Microtubules; Mus; Mutate; Mutation; Nuclear; Organic Synthesis; Pathway Analysis; Pathway interactions; Phospho-Specific Antibodies; Phospholipase; Phosphopeptides; Phosphorylation; Post-Translational Protein Processing; Process; Production; Protein Isoforms; Protein Kinase; Proteins; RNA Processing; Reactive Oxygen Species; Recombinants; Regulation; Role; STAT1 gene; Sampling; Signal Pathway; Small Interfering RNA; Sodium Chloride; Sorbitol; Stress; Taurine; Testing; Threonine; Time; Tubulin; Urea; Urine; base; cell killing; disulfide bond; esterase; fluorophosphate; glycerophosphocholine phosphodiesterase; in vivo; inorganic phosphate; interstitial; kidney cell; kidney medulla; neuropathy target esterase; peroxiredoxin; phosphoric diester hydrolase; prevent; protein folding; response; solute; tissue culture; urinary

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.

甘油磷胆碱（GPC）是一种渗透保护相容和抵消的有机渗透电解质，在高NaCl和尿素的作用下在肾髓内细胞中积累。我们之前发现，高NaCl和/或尿素会增加肾（Madin-Darby犬肾，MDCK）细胞中的GPC，并且GPC来源于磷脂酰胆碱，由一种当时未发现的磷脂酶催化。当神经病变靶酯酶（NTE）被证明是一种磷脂酶B，催化磷脂酰胆碱生成GPC时，我们测试了NTE是否有助于高nacl诱导肾细胞中GPC合成的增加，发现确实如此。在小鼠髓内集管（mIMCD3）细胞中，高NaCl升高NTE mRNA和蛋白。抑制NTE酯酶活性的氟磷酸二异丙基减少了GPC的积累，特异性降低NTE蛋白丰度的siRNA也是如此。NTE mRNA的20 h半衰期不受高NaCl的影响，但通过特定siRNA敲低NFAT5/TonEBP可抑制高NaCl诱导的NTE mRNA的增加。此外，慢性ClCK1-/-小鼠和急性给予速尿的正常小鼠肾内髓间质NaCl浓度降低与NTE mRNA和蛋白水平降低有关。因此，在NFAT5/TonEBP的介导下，高NaCl增加了NTE的转录，从而导致NTE表达的增加，从而增加了组织培养和体内哺乳动物肾细胞中GPC的产生和积累。