Distinct Regulation of Osmoprotective Genes in Yeast and Mammals
ALDOSE REDUCTASE OSMOTIC RESPONSE ELEMENT IS INDUCED INDEPENDENT OF p38 AND STRESS-ACTIVATED PROTEIN KINASE/Jun N-TERMINAL KINASE IN RABBIT KIDNEY CELLS*
- From the Laboratory of Kidney and Electrolyte Metabolism, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-0951
Abstract
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 increasesAR 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 mmNaCl) 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 ARthrough the ORE. This finding stands in contrast to the requirement for the HOG1 pathway for hyperosmotically induced activation of yeastGPD1.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Laboratory of Kidney and Electrolyte Metabolism, NHLBI, NIH, Bldg. 9, Rm. 1N105, 9 Memorial Dr., MSC 0951, Bethesda, MD 20892-0951. Tel.: 301-496-1268; Fax: 301-402-1443; E-mail: kultzd{at}gwgate.nhlbi.nih.gov.
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↵1 The abbreviations used are: HOG1, high osmolarity glycerol; STRE, stress-response element; SAPK, stress-activated protein kinase; ERK, environmentally regulated protein kinase; AR, aldose reductase; GPD1, glycerol-3-phosphate dehydrogenase 1; ORE, osmotic response element; PAGE, polyacrylamide gel electrophoresis; phOx, 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; X-gal, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside; MAPK, mitogen-activated protein kinase; JNK, Jun N-terminal kinase.
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- Received January 6, 1997.
- Revision received January 30, 1997.
