Homocysteine Stimulates Activation Of ERK By Epidermal Growth Factor Receptor Transactivation In Mouse Cultured Cerebellar Granule Cells

IntroductionHomocysteine (Hcy) is a non-essential sulfur-containing amino acid that is derived from methionine metabolism. Dietary deficiency in folate, vitamin B6 or B12 or mutations in enzymes regulating the catabolism of homocysteine may increase the levels of plasma homocysteine (hyperhomocysteinemia). In recent years, there are accumulated evidences that hyperhomocysteinemia is not only an increased risk for atherosclerotic vascular diseases, but also associated with various neurodegenerative disorders. Its molecular mechanisms of neurotoxicity have not been fully understood.By now, a few studies have shown that homocysteine actives ERK1/2 via various signaling transduction pathways in different cell lines. ERK1 and ERK2 are members of mitogen-activated protein kinase(MAPK) superfamily, and are known to be involved in many cellular processes, such as development, proliferation ,differentiation and survival. Thus, this cascade can be activated by numerous signaling transduction pathways.Recently, it has been reported that transactivation of epidermal growth factor receptor (EGFR) can occur secondary to activation of several G protein-coupled receptors (GPCRs) , which subsequently promote the release of some kinds of EGF. This has been considered as a novel signaling transduction pathway, acting as an upstream of extracellular signal-regulated kinase signaling pathways. As known to all, Hcy acts as an endogenous agonist for glutamate receptors and can induce glutamate release into the synapse. Several toxicity experiments of Hcy have showed that antagonists of glutamate receptors can block the activation of ERK1/2 induced by Hcy. Moreover, glutamate receptor is a kind of GPCRs. Consequently, the most important aim of the present study is to verify if the glutamate receptor-mediated transactivation of epidermal growth factor receptor is involved in ERK1/2 phosphorylation by Hcy in primary cultures of mouse cerebellar granule cell, which may provide insight into the neurotoxicity mechanisms of homocysteine.MethodPrimary cultures of cerebellar granule cells were prepared from 7-day-old mice. After 7 days, the culturing medium was gently removed to corresponding medium without serum and divided randomly into four groups. For investigation the pathway of ERK1/2 phosphorylation, the specific inhibitors or appropriate controls were added to the cultures 15 min before exposure to L-homocysteine for pretreatment. Then the cultures were treated with L-homocysteine at working concentrations for 20 min and corresponding vehicle solutions were used in control cultures. The cells were homogenized in ice-cold lysis buffer. Then sigals of ERK1/2 phosphorylation of all the samples were measured by western blotting. And statistical analysis was performed by one-way analysis of variance (ANOVA). The significance level was P<0.05.Results1. Homocysteine led to a transient activation of ERK1/2 rapidly in cerebellar granule cells. Time course indicated that statistically significant (P<0.05) difference from control group and other groups for p-ERK1 and p-ERK2, and it reached a statistically significant maximum at 20 min of treatment.2. The effect of ERK1/2 phosphorylation was inhibited by both MK-801, an antagonist of NMDA glutamate receptors, and CNQX, an antagonist of non-NMDA ionotropic glutamate receptors, which indicated that NMDA receptors and non-NMDA receptors were involved in homocysteine-induced ERK1/2 phosphorylation in cerebellar granule cells.3. AG1478, an inhibitor of phosphorylation of the epidermal growth factor receptor (EGFR), could block homocysteine-mediated activation of ERK1/2 in cerebellar granule cells. And GM601, an inhibitor of Zn²⁺-dependent metalloproteinase, affected the response to homocysteinein exactly the same manner as AG 1478. Thus, we showed that homocysteine-induced phosphorylation of ERK1/2 in cerebellar granule cells depended on transactivation of the epidermal growth factor receptor, and metalloproteinase was involved in the this course. ConclusionHomocysteine led to a rapid and transient activation of ERK1/2 in cultured cerebellar granule cells by the glutamate receptor-mediated transactivation pathway of epidermal growth factor receptor, and metalloproteinase was involved in the this course.