Hypoxia-induced regulation of MAPK phosphatase-1 as identified by subtractive suppression hybridization and cDNA microarray analysis.
Subtractive suppression hybridization was used to generate a cDNA library enriched in cDNA sequences corresponding to mRNA species that are specifically up-regulated by hypoxia (6 h, 1% O(2)) in the oxygen-responsive pheochromocytoma cell line. The dual specificity protein-tyrosine phosphatase MAPK phosphatase-1 (MKP-1) was highly represented in this library. Clones were arrayed on glass slides to create a hypoxia-specific cDNA microarray chip. Microarray, northern blot, and western blot analyses confirmed that MKP-1 mRNA and protein levels were up-regulated by hypoxia by approximately 8-fold. The magnitude of the effect of hypoxia on MKP-1 was approximately equal to that induced by KCl depolarization and much larger than the effects of either epidermal growth factor or nerve growth factor on MKP-1 mRNA levels. In contrast to the calcium-dependent induction of MKP-1 by KCl depolarization, the effect of hypoxia on MKP-1 persisted under calcium-free conditions. Cobalt and deferoxamine also increased MKP-1 mRNA levels, suggesting that hypoxia-inducible factor proteins may play a role in the regulation of MKP-1 by hypoxia. Pretreatment of cells with SB203580, which inhibits p38 kinase activity, significantly reduced the hypoxia-induced increase in MKP-1 RNA levels. Thus, hypoxia robustly increases MKP-1 levels, at least in part through a p38 kinase-mediated mechanism.
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Department of Molecular and Cellular Physiology and the Genome Research Institute, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0576, USA.
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Millhorn
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Beitner-Johnson
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D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D003035:Cobalt / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D003676:Deferoxamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D054638:Dual Specificity Phosphatase 1; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015723:Gene Library; D000860:Hypoxia; D007093:Imidazoles / Q000494:pharmacology; D017874:Immediate-Early Proteins / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016716:PC12 Cells; D010749:Phosphoprotein Phosphatases; D016133:Polymerase Chain Reaction; D011189:Potassium Chloride / Q000494:pharmacology; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D017874:Immediate-Early Proteins; D011725:Pyridines; D012333:RNA, Messenger; D003035:Cobalt; D011189:Potassium Chloride; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; C517021:Dusp1 protein, rat; D017027:Protein Tyrosine Phosphatases; D003676:Deferoxamine; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium