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 e nriched in cDNA sequences corresponding to mRNA species that are specifical ly upregulated by hypoxia (6 h, 1% O-2) in the oxygen-responsive pheochromo cytoma cell line. The dual specificity protein-tyrosine phosphatase (M) und er bar AP (K) under bar (p) under bar hosphatase-(1) under bar (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 similar to8-fold. The magnitude of the effect o f hypoxia on MKP-1 was approximately equal to that induced by KCl depolariz ation 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-depen dent induction of MKP-1 by KCl depolarization, the effect of hypoxia on MKP -1 persisted under calcium-free conditions. Cobalt and deferoxamine also in creased MKP-1 mRNA levels, suggesting that hypoxia-inducible factor protein s may play a role in the regulation of MKP-1 by hypoxia. Pretreatment of ce lls with SB203580, which inhibits p38 kinase activity, significantly reduce d 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 mech anism.