Hypoxia Upregulates MAPK<sup>p38</sup>/MAPK<sup>ERK</sup> Phosphorylation In Vitro: Neuroimmunological Differential Time-Dependent Expression of MAPKs

Abstract

In mammalian cells, responses to hypoxia at the molecular transduction level are hallmarks of adaptation and survival under oxygen deprivation conditions. In this study, the protein expression patterns of mitogen-activated protein kinases (MAPKs) are investigated under hypoxia in primary cortical neurons and in a model of organotypic hippocampal slices in neonatal Sprague-Dawley rats. Abrupt fluctuations in MAPK expression can occur during anoxia, hypoxia, and relative hyperoxic shifts (e.g., reoxygenation); therefore, phosphorylation and dephosphorylation states could be crucial factors in metabolic reorganization for withstanding anaerobiosis. Whole cellular protein extracts were analyzed for the phosphorylation of MAPKp(p38) and MAPK(ERK-1/2 (p44/p42)) at threonine and tyrosine residues (Thr(180)/Tyr(182)) at different time periods of hypoxic exposure relative to a fixed normoxia control. The phospho-MAPK(p38) (p-MAPK(p38)) to MAPK(p38) relative unit ratio revealed that MAPK(p38) expression increased in cortical neurons after 5 and 10 min, but decreased abruptly afterwards (20 - 120 min). The expression of phospho-MAPK(ERK-1) (p-MAPK(ERK-1/p44)), however, decreased whereas that of p-MAPK(ERK-2/p42) increased compared to normoxia. In rat hippocampal slices (RHS), the expression of p-MAPK(p38) was slightly but significantly higher in hypoxia, whereas the expression of p-MAPK(ERK-2/p42) increased and that of p-MAPK(ERK-1/p44) was intangible. This indicates that in cortical neurons hypoxia differentially upregulated the phosphorylation activation states of MAPK(p38) and MAPK(ERK-1/2 (p44/p42)), whereas in the RHS model MAPK(p38) and MAPK(ERK-2/p42), but not MAPK(ERK-1/p44), phosphorylation states were upregulated in response to hypoxia. The neuroimmunological molecular patterns of the differential MAPK phosphorylation in vitro and ex vivo in response to hypoxic shift indicated a significant role for these kinases in cellular adaptation to oxygen deprivation, and thereby may identify physiologic and neuroprotective responsive signaling cofactors and pathways in cortical and hippocampal neurons during hypoxia.