Heme oxygenase activity and hemoglobin neurotoxicity are attenuated by inhibitors of the MEK/ERK pathway

Abstract

Hemoglobin breakdown produces an iron-dependent neuronal injury after experimental CNS hemorrhage that may be attenuated by heme oxygenase (HO) inhibitors. The HO enzymes are phosphoproteins that are activated by phosphorylation in vitro. While testing the effect of kinase inhibitors in cortical cell cultures, we observed that HO activity was consistently decreased by the MEK inhibitor U0126. The present study tested the hypothesis that MEK/ERK pathway inhibitors reduce HO activity and neuronal vulnerability to hemoglobin. The MEK inhibitors U0126 and SL327 and the ERK inhibitor FR180204 reduced baseline culture HO activity by 35-50%, without altering the activity of recombinant HO-1 or HO-2; negative control compounds U0124 and FR180289 had no effect. Hemoglobin exposure for 16h produced widespread neuronal injury, manifested by release of 59.2+/-7.8% of neuronal lactate dehydrogenase and a twelve-fold increase in malondialdehyde; kinase inhibitors were highly protective. HO-1 induction after hemoglobin treatment was also decreased by U0126, SL327, and FR180204. These results suggest that reduction in HO activity may contribute to the protective effect of MEK and ERK inhibitors against heme-mediated neuronal injury.

Figure 1

MEK and ERK inhibitors reduce culture heme oxygenase (HO) activity. A) Mean HO activity (nmol CO/h/mg protein ±SEM, n = 5–12/condition) in cultures washed and then treated with DMSO vehicle only (Veh) or with U0126 (30 μM), SL327 (30 μM), FR180204 (FR, 50 μM), U0124 (negative control for U0126, 30 μM), or FR180289 (FRN, negative control for FR180204, 50 μM) for 30 minutes. ***P<0.001 and **P<0.01 v. activity in vehicle-treated cultures, Bonferroni multiple comparisons test. B) Immunoblot demonstrating effect of MEK inhibitors and negative control U0124 on kinase activity at these concentrations, as assessed by phosphorylation of ERK substrate Elk-1. Cultures were treated with hemoglobin (Hb) 10 μM alone or with inhibitors or control for 4 hours before lysis and immunoprecipation of active ERK for kinase assay. C) Immunoblot demonstrating inhibition of active ERK by FR180204 (FR, 50 μM) but not by FR180289 (FRN, 50 μM).

Figure 2

Effect of MEK and ERK inhibitors on activity of recombinant HO-1 and HO-2. HO activity of recombinant rat HO-1 and HO-2 with DMSO vehicle alone (Veh) or with U0126 (30 μM), SL327 (30 μM), FR180204 (FR, 50 μM), or zinc protoporphyrin IX (ZnPPIX, 50 μM). Reaction vial contained 0.25μg cytochrome P450 reductase with 2.5 μg HO-1 or 1 μg HO-2. **P<0.01, **P<0.001 v. corresponding vehicle condition, Bonferroni multiple comparisons test.

Figure 3

Morphologic appearance of mixed neuron-astrocyte cultures 24h after: A, C, E) Treatment with vehicle only. Neurons with phase-bright cell bodies appear primarily in clusters on the glial feeder layer monolayer, which is in a different plane and therefore slightly out of focus. Phase-bright cells stain with Alexa Fluor®488 conjugated anti-NeuN (C), confirming neuronal identity, while background glial monolayer stains with anti-GFAP (E). B, D, F) Treatment with hemoglobin 10 μM with vehicle. Most neurons have degenerated to debris, astrocyte monolayer is intact (B), NeuN immunoreactivity is diminished and limited to areas of degenerating cells (D), GFAP immunoreactivity persists and is increased, particularly near degenerating neurons (E). G, H) Unfixed cultures treated with hemoglobin 10 μM plus U0126 (30 μM) or FR180204 (50 μM), respectively; neuronal morphology is preserved. Scale bar = 100 μm.

Figure 4

MEK and ERK inhibitors protect neurons from hemoglobin. A) Cultures were treated for 16h with hemoglobin (Hb) 10 μM plus DMSO vehicle alone or with U0126 (30 μM), SL327 (30 μM), or FR180204 (FR, 50 μM). LDH values are scaled to mean values in sister cultures treated with 300 μM N-methyl-D-aspartate (NMDA), which produces near-100% neuronal death, after subtraction of mean LDH in sister cultures subjected to wash and vehicle treatment only, in order to yield the LDH signal specific for hemoglobin neurotoxicity. B) Culture malondialdehyde (MDA) after treatment as in A. ***P<0.001. v. Hb, Bonferroni multiple comparisons test, n = 8–23/condition.

Figure 5

MEK and ERK inhibitors prevent HO-1 induction after hemoglobin treatment. A) Expression of HO-1, HO-2 and actin (gel loading control) in cultures treated with vehicle (Veh) only for 4h, with hemoglobin (Hb) 10 μM plus vehicle, or with Hb plus U0126 (30 μM), SL327 (30 μM), or FR180204 (FR, 50 μM). B) HO activity in cultures treated as in A. ###P<0.001. v. vehicle, *P<0.05, **P<0.01, ***P<0.001 v. Hb, Bonferroni multiple comparisons test, n = 5–13/condition.