Kültz D, Garcia-Perez A, Ferraris J D, Burg M B
Laboratory of Kidney and Electrolyte Metabolism, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-0951, USA.
J Biol Chem. 1997 May 16;272(20):13165-70. doi: 10.1074/jbc.272.20.13165.
In yeast glycerol-3-phosphate dehydrogenase 1 is essential for synthesis of the osmoprotectant glycerol and is osmotically regulated via the high osmolarity glycerol (HOG1) kinase pathway. Homologous protein kinases, p38, and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) are hyperosmotically activated in some mammalian cell lines and complement HOG1 in yeast. In the present study we asked whether p38 or SAPK/JNK signal synthesis of the osmoprotectant sorbitol in rabbit renal medullary cells (PAP-HT25), analogous to the glycerol system in yeast. Sorbitol synthesis is catalyzed by aldose reductase (AR). Hyperosmolality increases AR transcription through an osmotic response element (ORE) in the 5'-flanking region of the AR gene, resulting in elevated sorbitol. We tested if AR-ORE is targeted by p38 or SAPK/JNK pathways in PAP-HT25 cells. Hyperosmolality (adding 150 mM NaCl) strongly induces phosphorylation of p38 and of c-Jun, a specific target of SAPK/JNK. Transient lipofection of a dominant negative mutant of SAPK kinase, SEK1-AL, into PAP-HT25 cells specifically inhibits hyperosmotically induced c-Jun phosphorylation. Transient lipofection of a dominant negative p38 kinase mutant, MKK3-AL, into PAP-HT25 cells specifically suppresses hyperosmotic induction of p38 phosphorylation. We cotransfected either one of these mutants or their empty vector with an AR-ORE luciferase reporter construct and compared the hyperosmotically induced increase in luciferase activity with that in cells lipofected with only the AR-ORE luciferase construct. Hyperosmolality increased luciferase activity equally (5-7-fold) under all conditions. We conclude that hyperosmolality induces p38 and SAPK/JNK cascades in mammalian renal cells, analogous to inducing the HOG1 cascade in yeast. However, activation of p38 or SAPK/JNK pathways is not necessary for transcriptional regulation of AR through the ORE. This finding stands in contrast to the requirement for the HOG1 pathway for hyperosmotically induced activation of yeast GPD1.
在酵母中，甘油 - 3 - 磷酸脱氢酶1对于渗透保护剂甘油的合成至关重要，并通过高渗甘油（HOG1）激酶途径受到渗透压调节。同源蛋白激酶p38以及应激激活蛋白激酶/ Jun N - 末端激酶（SAPK / JNK）在一些哺乳动物细胞系中被高渗激活，并在酵母中补充HOG1的功能。在本研究中，我们探究了p38或SAPK / JNK是否像在酵母甘油系统中那样，在兔肾髓质细胞（PAP - HT25）中调节渗透保护剂山梨醇的合成。山梨醇的合成由醛糖还原酶（AR）催化。高渗通过AR基因5'侧翼区域的渗透反应元件（ORE）增加AR转录，从而导致山梨醇水平升高。我们测试了在PAP - HT25细胞中，AR - ORE是否受p38或SAPK / JNK途径靶向。高渗（添加150 mM NaCl）强烈诱导p38和c - Jun（SAPK / JNK的一个特定靶点）的磷酸化。将SAPK激酶的显性负性突变体SEK1 - AL瞬时脂质转染到PAP - HT25细胞中，特异性抑制高渗诱导的c - Jun磷酸化。将显性负性p38激酶突变体MKK3 - AL瞬时脂质转染到PAP - HT25细胞中，特异性抑制高渗诱导下p38的磷酸化。我们将这些突变体之一或其空载体与AR - ORE荧光素酶报告构建体共转染，并将高渗诱导的荧光素酶活性增加与仅用AR - ORE荧光素酶构建体脂质转染的细胞进行比较。在所有条件下，高渗均使荧光素酶活性同等增加（5 - 7倍）。我们得出结论，高渗在哺乳动物肾细胞中诱导p38和SAPK / JNK级联反应，类似于在酵母中诱导HOG1级联反应。然而，通过ORE对AR进行转录调控并不需要p38或SAPK / JNK途径的激活。这一发现与酵母GPD1高渗诱导激活需要HOG1途径形成对比。
