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% 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.
Highlights
Hypoxia is a critical physiological stimulus in a variety of disease states, including ischemia, respiratory disorders, and tumorigenesis [1, 2]
As a first step toward verifying regulation of the genes in the suppression hybridization (SSH) library, polymerase chain reaction (PCR) products derived from each clone in the library were spotted onto glass slides and evaluated by cDNA microarray analysis (Fig. 1) as described under “Experimental Procedures.” cDNA microarray analysis revealed that tyrosine hydroxylase, JunB, and vascular endothelial growth factor were all strongly regulated by hypoxia (6 h, 1% O2), as expected
To further confirm that mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) was regulated by hypoxia, MKP-1 mRNA levels were determined by northern blot analysis
Summary
Hypoxia is a critical physiological stimulus in a variety of disease states, including ischemia, respiratory disorders, and tumorigenesis [1, 2]. Pretreatment of cells with SB203580, which inhibits p38 kinase activity, significantly reduced the hypoxia-induced increase in MKP-1 RNA levels. It has been suggested that an increase in MKP gene expression represents another level of negative feedback regulation on MAPK signaling pathways [23, 28].
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