Glutathione peroxidase overexpression causes aberrant ERK activation in neonatal mouse cortex after hypoxic preconditioning

Abstract

Background: Preconditioning of neonatal mice with nonlethal hypoxia (HPC) protects the brain from hypoxic-ischemic (HI) injury. Overexpression of human glutathione peroxidase 1 (GPx1), which normally protects the developing murine brain from HI injury, reverses HPC protection, suggesting that a certain threshold of hydrogen peroxide concentration is required for activation of HPC signaling.

Methods: Activation (phosphorylation) of extracellular-regulated kinase (ERK) 1/2 and Akt, and induction of hypoxia-inducible factor (HIF)-1α were assessed in the cortex, one of the main structures affected by HI and protected by HPC, at different time points after reoxygenation in wild-type (WT) and GPx1-overexpressing animals.

Results: GPx1 overexpression prevented both the global and nuclear increase in activated ERK at 0.5 h after HPC and caused a significant decrease in phospho-ERK (pERK)/ERK levels at 24 h after HPC. In contrast, HIF-1α induction at the end of hypoxia was unaffected by GPx1 overexpression. In the cortex of preconditioned WT animals, enhanced pERK staining was primarily observed in neurons and to a lower extent in astrocytes and endothelial cells, with a nuclear prominence.

Conclusion: Aberrant activation of ERK probably explains the paradoxical reversal of HPC protection by GPx1 overexpression. The results identify hydrogen peroxide as an important mediator of neuroprotective ERK signaling.

Figure 1

Hypoxic preconditioning transiently activates extracellular-regulated kinase (ERK) in neonatal brain cortex. Postnatal day 6 mice were exposed to 45 min of global hypoxia (8% oxygen) and killed at various time points (0–24 h) after reoxygenation. Animals exposed to normoxia served as baseline controls (naive). Phosphorylation of ERK1 (white bars), ERK 2 (black bars), and Akt (gray bars), and hypoxia-inducible factor (HIF)-1α stabilization (hatched bars) were analyzed in cortical homogenates by western blotting as described in Methods. (a) Representative blots for a complete time course of one set of animals. (b) Bar graph shows a summary of the results from densitometric analysis of bands from n = 8 (phospho-ERK/ERK), n = 5 (pAkt/Akt), and n = 3 (HIF-1α) independent sets of animals. The level of phosphoprotein is expressed relative to total protein and to that of wild-type naive mice (=100%) ± SEM. *P < 0.05, †P < 0.001, by one-way ANOVA.

Figure 2

Overexpression of glutathione peroxidase 1 (GPx1) prevents hypoxic preconditioning (HPC)-mediated activation of cortical extracellular-regulated kinase (ERK). Phosphorylation of ERK was analyzed in cortical homogenates from wild-type (WT, white bars) and GPx1-overexpressing (black bars) animals exposed to preconditioning hypoxia at various time points after reoxygenation by western blotting. (a) Representative western blot of a complete time course of WT versus GPx1 transgenic (black) animals. Bar graph shows a summary of the results from densitometric analysis of bands from n ≥ 5 independent sets of animals. Phospho-ERK is expressed relative to ERK and to that of respective naive mice (=100%) ± SEM (*P < 0.05, one-way ANOVA). (b) Representative western blot of samples taken 0.5 h after reoxygenation. Bar graph represents the mean ± SEM of n = 10–11 animals (*P < 0.05, by one-way ANOVA) relative to respective naive controls. (c) Representative western blot of samples analyzed 24 h after HPC. Bar graph shows summary of densitometrically analyzed data from n = 5 animals ± SEM (*P < 0.05 by one-way ANOVA) relative to respective naive controls.

Figure 3

Overexpression of glutathione peroxidase 1 (GPx1) prevents nuclear accumulation of activated (a) extracellular-regulated kinase (ERK) but not of (b) hypoxia-inducible factor (HIF)-1α. (a) Nuclear (black bars) and cytosolic (white bars) extracts prepared at 0.5 h after reoxygenation were analyzed for phospho-ERK (pERK) and total ERK by western blotting. HIF-1β was used as a nuclear marker protein. (Top) Representative western blot of one set of samples. (Bottom) Bar graph shows a summary of the results from densitometric analysis of bands from n = 5 independent sets of animals. Data are expressed relative to cytosolic level of pERK/ERK in respective naive controls (=100%) ± SEM. *P < 0.05, by one-way ANOVA. (b) Nuclear extracts prepared at 0 h after reoxygenation were analyzed for HIF-1α by western blotting. (Top) Representative western blot of HIF-1α analysis in nuclear extracts. (Bottom) Bar graph shows summary of results from densitometric analysis of HIF-1α band from n = 5 independent sets of animals. Data are expressed relative to wild-type (WT) hypoxic preconditioned (HPC) animals (=100%). No significant difference between WT HPC and GPx1 transgenic HPC by one-way ANOVA.

Figure 4

Cellular localization of activated extracellular-regulated kinase (ERK) in the cortex at 0.5 h posthypoxia. Coronal sections were stained for phospho-ERK (pERK) in red and neuronal nuclear antigen (NeuN), isolectin B4 (IB4), glial fibrillary acidic protein (GFAP), or 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) in green. (a–c) Overviews of stitched images taken at 20× original magnification of (a) wild-type (WT) naive, (b) WT preconditioned, and (c) glutathione peroxidase 1 transgenic preconditioned stained for pERK and NeuN. Images are representative of at least n = 3 independent animals. Scale bars represent 100 µm. (d–g) Colocalization of pERK with cellular markers (d) NeuN, (e) GFAP, (f) IB4, and (g) CNPase in sections from preconditioned WT animals (0.5 h posthypoxia) at higher magnification (40× original magnification). Arrows point to (d) pERK-positive neurons, (e) astrocytes, (f) endothelial cells, and (g) oligodendrocytes. Scale bars represent 25 µm.