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

Abstract

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% O2) in the oxygen-responsive pheochromocytoma cell line. The dual specificity protein-tyrosine phosphatase MAPKphosphatase-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. 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% O2) in the oxygen-responsive pheochromocytoma cell line. The dual specificity protein-tyrosine phosphatase MAPKphosphatase-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. hypoxia-inducible factor mitogen-activated protein kinase stress-activated protein kinase subtractive suppression hybridization MAPKphosphatase-1 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester nerve growth factor epidermal growth factor polymerase chain reaction MAPK/extracellular signal-regulated kinase kinase phosphatidylinositol 3′-kinase Hypoxia is a critical physiological stimulus in a variety of disease states, including ischemia, respiratory disorders, and tumorigenesis (1Zhu H. Bunn H.F. Respir. Physiol. 1999; 115: 239-247Crossref PubMed Scopus (157) Google Scholar, 2Semenza G.L. Cell. 1999; 98: 281-284Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). In recent years, the mechanisms by which cells respond and adapt to decreased O2 levels have begun to be elucidated. For example, the hypoxia-inducible factor (HIF)1 family of proteins includes transcription factors that are stabilized and activated specifically under conditions of low O2 (for review, see Ref. 3Semenza G.L. Genes Dev. 2000; 14: 1983-1991PubMed Google Scholar). Upon activation by hypoxia, the HIF-α/β heterodimer can enhance expression of genes that contain the hypoxia response element motif (5′-RCGTG-3′) in their 5′-fl