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 upregulated 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 ϳ8-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.

Okadaic Acid-Sensitive Protein Phosphatases Constrain Phrenic Long-Term Facilitation after Sustained Hypoxia

“…For example, sustained hypoxia may increase protein phosphatase activity in the cervical spinal cord, thereby suppressing pLTF. However, available literature reports are inconsistent with respect to this possibility, because some studies report that sustained hypoxia increases protein phosphatase activity and expression (Seta et al, 2001;Arsham et al, 2003;Yung and Tolkovsky, 2003;Zhou et al, 2004), whereas others report the opposite Mishra and DelivoriaPapadopoulos, 2004;Truttmann et al, 2004).…”

“…Indeed, multiple protein kinases and phosphatases are hypoxia sensitive (Seta et al, 2001(Seta et al, , 2002Truttmann et al, 2004;Liu et al, 2005), although the effects of the hypoxia pattern on protein kinase/phosphatase activity are not known. One possibility is that intermittent and sustained hypoxia differentially regulate protein kinase/phosphatase activity by differences in the generation of reactive oxygen species (ROSs) (Yuan et al, 2004).…”

In Vivo Evidence for Interferon-γ-mediated Homeostatic Mechanisms in Small Intestine of the NHE3 Na+/H+ Exchanger Knockout Model of Congenital Diarrhea

“…Reverse-transcribed cDNA was amplified using gene-specific primers for IFN-␥ (5Ј-TCAAGTGGCATA-GATGTGGAAGAA-3Ј and 5Ј-TGGCTCTGCAGGATTTTCATG-3Ј, 92-base pair product) (28) and IL-1␤ (5Ј-ACACTCCTTAGTCCTCGGCC-A-3Ј and 5Ј-CCATCAGAGGCAAGGAGGAA-3Ј, 82-base pair product) (29). PCRs were carried out in a Cepheid Smart Cycler (Cepheid, Sunnyvale, CA) as described previously (30) and contained the following components in a total volume of 25 l: 1ϫ LightCycler-DNA Master SYBR Green I (Roche Diagnostics), 10 pmol of each primer, and 0.05-1 g of RNA-equivalent cDNA. The annealing temperature was 60°C for both primer sets, fluorescence was recorded during the 72°C extension step of each cycle, and threshold cycle values were calculated from the second derivative of the growth curve as described previously (30).…”

“…PCRs were carried out in a Cepheid Smart Cycler (Cepheid, Sunnyvale, CA) as described previously (30) and contained the following components in a total volume of 25 l: 1ϫ LightCycler-DNA Master SYBR Green I (Roche Diagnostics), 10 pmol of each primer, and 0.05-1 g of RNA-equivalent cDNA. The annealing temperature was 60°C for both primer sets, fluorescence was recorded during the 72°C extension step of each cycle, and threshold cycle values were calculated from the second derivative of the growth curve as described previously (30). The line equations of the linear regions for each curve were used to calculate the relative -fold change between Nhe3 Ϫ/Ϫ and Nhe3 ϩ/ϩ tissues.…”

Mitogen-Activated Protein Kinase Phosphatase-1 Is a Key Regulator of Hypoxia-Induced Vascular Endothelial Growth Factor Expression and Vessel Density in Lung

“…1 MKP-1 expression in the lung is considerably higher than in other tissues, and it has been shown to protect arteries from a proinflammatory state by suppressing the activities of p38 and JNK MAP kinases in the vascular endothelium. 2,3 Although MKP-1 is a hypoxia-inducible phosphatase, and hypoxia triggers angiogenesis, 4,5 the role of MKP-1 in the formation and maintenance of vascular network in hypoxic lung remains unexplored.…”