Specific role of extracellular signal-regulated kinase (ERK) in cold-induced injury to cultured liver endothelial cells

Background: Hypothermic injury, mediated by an iron-dependent increase in ROS formation that leads to mitochondrial injury, occurs in various cell types and constitutes part of preservation injury. The mitogen activated protein kinases (MAPKs) ERK, JNK and p38 have, among other factors, been implicated in preservation injury. However, their role in cold-induced injury is unclear. Therefore, we here studied activation of ERK, JNK and p38 during/after cold incubation and provide evidence that the ERK phosphorylation is involved in cold-induced injury of rat liver endothelial cells.

Methods: Rat liver endothelial cells of two cell lines were incubated at 4 ° C in Krebs-Henseleit (KH) buffer or UW solution, followed by rewarming in cell culture medium. Cell injury was determined by LDH release. Phosphorylation of MAPKs was assessed by western blot and mitochondrial membrane potential by laser scanning microscopy using the fluorescent indicator TMRM.

Results and Conclusion: During hypothermia, ERK and JNK were not phosphorylated, in fact, pre-existing phosphorylation was significantly decreased compared to control. However, both MAPKs were strongly and transiently phosphorylated during rewarming. In contrast, p38 MAPK showed an opposite pattern of phosphorylation, which was reduced during rewarming. Pretreatment of the cells with an iron chelator (deferoxamine, 10 mM/ml) or Rho-GTPases inhibitor (Toxin-B, 10 ng/ml) partially inhibited ERK phosphorylation. Inhibition of ERK by a MEK inhibitor (U0126, 10µM/ml), decreased cell injury after cold incubation/rewarming in KH and UW solution (2% vs. 42% and 3% vs. 61% LDH release) respectively, whereas the inactive analogue (U0124, 10µM/ml) was ineffective. Similar protection was observed with the MEK inhibitor PD 98059. Inhibition of ERK prevented the loss of the mitochondrial membrane potential. In conclusion, the data suggest that iron-dependent ROS and Rho-GTPases contribute to transient activation of ERK after cold incubation which appears to be involved in cold-induced cell and mitochondrial injury.