Hypoxia-induced Regulation of MAPK Phosphatase-1 as Identified by Subtractive Suppression Hybridization and cDNA Microarray Analysis

• Published in: The Journal of biological chemistry Vol. 276; no. 48; pp. 44405 - 44412
• Main Authors: Seta, K A, Kim, R, Kim, H W, Millhorn, D E, Beitner-Johnson, D
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 30-11-2001
• Subjects: Animals; Blotting, Northern; Blotting, Western; Calcium - pharmacology; Cell Cycle Proteins; Cell Nucleus - metabolism; Cobalt - pharmacology; deferoxamine; Deferoxamine - pharmacology; DNA, Complementary - metabolism; Dose-Response Relationship, Drug; Dual Specificity Phosphatase 1; Enzyme Inhibitors - pharmacology; Flavonoids - pharmacology; Gene Library; Hypoxia; hypoxia-inducible factor; Imidazoles - pharmacology; Immediate-Early Proteins - metabolism; Mitogen-Activated Protein Kinases - metabolism; MKP-1 protein; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; p38 Mitogen-Activated Protein Kinases; PC12 Cells; Phosphoprotein Phosphatases; Polymerase Chain Reaction; Potassium Chloride - pharmacology; Protein Phosphatase 1; Protein Tyrosine Phosphatases - metabolism; Pyridines - pharmacology; Rats; RNA, Messenger - metabolism; Signal Transduction; Time Factors; Up-Regulation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M103346200

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 M AP K
p hosphatase- 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 ∼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.