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Transient hypoxia can stimulate proliferation of endothelial cells. This effect involves generation of reactive oxygen species by mitochondria and NADPH oxidase as well as activation of the MEK/ERK pathway. Here we addressed the question whether p38 MAPK is a downstream signaling element of the hypoxia-induced proliferation. Cell proliferation (FACS), activation of ERK1/2 (ERK phosphorylation; Western blot), NADPH oxidase (Video imaging), and p38 MAPK isoforms (p38 MAPK phosphorylation; immunoprecipitation) were analysed in cultured porcine endothelial cells. The cells were exposed to hypoxia (2h; pO2<1 mmHg) or normoxia (2h; pO2=120 mmHg) as a control, followed by reoxygenation (24h; pO2=120 mmHg). During hypoxia ERK1/2, NADPH oxidase and p38 MAPK were activated. After 24h of reoxygenation cell number was increased by 65% compared to normoxic control. Inhibition of MEK1/2 by 10 µM PD 98059, a MEK inhibitor, reduced the hypoxia-induced p38 MAPK activation, indicating that p38 MAPK is downstream of MEK. Inhibition of NADPH oxidase by antisense oligonucleotides against p22phox reduced p38 MAPK activation and abolished the hypoxia-induced proliferation response, indicating that NADPH oxidase is also upstream of p38 MAPK. Immunoprecipitation of p38 MAPK showed, that hypoxia stimulated phosphorylation of α and γ isoforms of p38 MAPK by 2.5 and 1.6-fold, respectively, but neither β nor δ. Conclusion: The data of the present study show that p38 MAPK is a central signaling element of the hypoxia-induced proliferation response downstream of MEK/ERK and NADPH oxidase.
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J. Vasc. Biol. 42, Sup:2 (2005) p89