Depolarization-induced, Glutamate Receptor-mediated, And Transactivation-dependent Extracellular-signal Regulated Kinase Phosphorylation In Cultured Cerebellar Granule Neurons

IntroductionPhosphorylation of extracellular-signal regulated kinase (ERK) 1 and 2 (ERK_1/2) generates extracellular-signal regulated kinase 1 and extracellular-signal regulated kinase 2 phosphorylated at serine/threonine residues (p-ERK_1/2), the active form of ERK_1/2. This activation and ensuing downstream signaling is important for many neuronal functions during development and in the adult brain, where it plays a major role in plasticity and thus in memory formation. It is consistent with these functions that phosphorylation of ERK_1/2 occurs both in the intact nervous system in response to afferent stimulation and during exposure of cultured neurons, PC12 cells or brain slices to a depolarizing concentration of potassium ions (K⁺), to the excitatory neurotransmitter glutamate, or to subtype-specific glutamate agonists.Epiithelial growth factor receptor (EGFR) transactivation in which signaling to a G-protein-coupled receptor (GPCR) leads to release ('shedding') of a growth factor, which in turn stimulates receptor tyrosine-kinase (RTK) on the same of adjacent cells and evoked by directly obtained by direct RTK stimulation. It is a novel signaling pathway which plays a majoy role in neuron survival and function.In the transactivation process of the EGFR, activation of G_i/o protein-coupled receptors or a depolarization- or transmitter-mediated increase in free cytosolic calcium concentration ([Ca²⁺]_i) leads to metalloproteinase-catalyzed shedding of an EGFR agonist, which stimulates EGFRs on the same cell or its neighbor(s). Among EGFR agonists transforming growth factor-α(TGF-α) and heparin-binding epidermal growth factor (HB-EGF) are expressed in cerebellar granule cells and the cerebellar external granule layer, but there is no information whether other EGFR agonists are present.7-8-day-old primary cultures of cerebellar granule neurons constitute a well-established, well-differentiated glutamatergic preparation, expressing NMDA, AMPA and metabotropic glutamate receptors. In the present study we have studied the mechanism of ERK_1/2 phosphorylation during K⁺-mediated depolarization using 7-8-day-old primary cultures of cerebellar granule neurons. (1) K⁺-mediated depolarization is transactivation of epidermal growth factor (EGF) receptors (EGFRs). (2) K⁺-induced depolarization is mediated by glutmate receptor. (3) EGFR family expression in the primary cultures of cerebellar granule neuron.MethodsThe primary cerebellar granule neuron culture were the pre-incubate in the corresponding medium without serum at 37℃at a [K⁺]_e of 5, 10, 25 or 45 mM in the absence or presence of specific inhibitors. The reaction was stopped by washing with ice-cold phosphate-buffered saline (PBS) containing 7.5 mM glucose, and the cells were scraped off the dishes and harvested in 0.5 ml of ice-cold buffer for Western-Blot. Concentration of glutamate released were measured by high-pressure liquid chromatography. The results are analysised with one-way ANOVA by SPSS12.0 software. P<0.05 indicate the statically significant difference.Results1. [K⁺]_e induced ERK_1/2 phosphorylationExposure to raised [K⁺]_e for 20 min caused a concentration-dependent increase of p-ERK_1/2, whereas there was no significant change in total content of ERK_1/2 (results not presented). A small response of ERK_1/2 appeared to occur at an [K⁺]_e concentration of 10 mM and was larger at 25 mM and a large and statistically significant increase was seen at 45 mM [K⁺]_e. The stimulation of ERK_1/2 activities by high [K+]e occurred relatively slowly. After 10 min of exposure to 45 mM [K⁺]_e, p-ERK₁ and p-ERK₂ increased slightly and reached a statistically significant maximum after 20 min, followed by a decrease to basal values at 40 min and a second, similar increase at 60 min.2. mRNA expression of EGF-related family membersThe response to a depolarizing [K⁺]_e might be due to a transactivation of the EGFR, since mRNAs of both HB-EGF and TGF-αwere expressed in 8-day-old cerebellar granule cells in primary cultures at similar levels as in cerebellum of adult mice . In contrast, neither the cultured cells nor cerebellum of adult mice expressed EGF or amphiregulm mRNA. Betacellulin, epiregulin and epigen were not tested for, since their presence has not been reported in brain.3. Effects of inhibitors of TRK and metalloproteinase and of heparinThe effect of AG 1478, a specific EGFR tyrosine kinase inhibitor, was tested in order to establish if transactivation was involved. In the presence of 1μM AG 1478 phosphorylation of ERK₁ and ERK₂ by 45 mM [K⁺]_e for 20 min was inhibited. The inhibitory effects on phosphorylation of ERK₁ and ERK₂ were not significantly different from each other and they were statistically significant in both cases. After exposure to 45 mM [K⁺]_e for 60 min, the response was indistinguishable from that seen after 20 min of exposure although the inhibition may have been less complete. Additional inhibitors of the transactivation process were therefore only tested after 20 min of K⁺ exposure.Ten micromolar GM 6001, an inhibitor of Zn²⁺-dependent metalloproteinase, affected the response to 20 min of exposure to 45 mM [K⁺]_e in exactly the same manner as AG 1478. In the presence of 1 mg/ml of heparin, an inhibitor of HB-EGF and amphiregulin, there was also a significant inhibition of the response to 45 mM [K⁺]_e, but the inhibition was less complete than in the case of the two other inhibitors. This was reflected by a statistically significant difference between the presence of the inhibitor alone and in the additional presence of elevated [K⁺]_e.4. Effects of glutamate and antagonists of NMDA and of non-NMDA receptorsTo establish if the transactivation-dependent ERK_1/2 phosphorylation resulted from K⁺-mediated depolarization as such or a glutamate release resulting from the depolarization, experiments were carried out in which 50 uM glutamate was added instead of elevating [K⁺]_e. Exposure to glutamate for 5 min caused a significant increase in ERK_1/2 phosphorylation, which was abolished by AG 1478 and GM 6001. The effect of glutamate was also inhibited by both 1μM of MK-801, an antagonist of NMDA glutamate receptors, and 10μM of CNQX, an antagonist of non-NMDA ionotropic glutamate receptors . One micromolar MK-801 also inhibited the stimulation by 45 mM [K⁺]_e, regardless whether the [K⁺]_e exposure lasted 20 or 60 min, although an apparent difference between the control value and the value in the presence of elevated [K⁺]_e plus MK-801 after 60 min of exposure suggested that the inhibition was not complete. Ten micromolar CNQX also inhibited K⁺-mediated ERK phosphorylation.5. Effects of elevated [K⁺]_e in glutamine-free mediumIn the absence of extracellular glutamine the extracellular glutamate concentration was significantly lowered (from 1.8 to 1.1μM at 5 mM [K⁺]_e), and the K⁺-induced glutamate release was inhibited (a non-significant increase to 9.3 uM in the glutamine-deprived medium versus a significant increase to 36 uM in the presence of glutamine). Nevertheless, also in this situation exposure to 45 mM [K⁺]_e stimulated ERK_1/2 phosphorylation at both 20 and 60 min, but AG 1478 had no effect, indicating that although K⁺-mediated depolarization as such was able to induce ERK_1/2 phosphorylation, this phosphorylation was not dependent upon transactivation.6. Effects of PKC inhibitionGF109203X, an inhibitor of PKC, had no effect on ERK_1/2 phosphorylation under resting condition (5 mM [K⁺]_e). It did also not affect the stimulation by 45 mM [K⁺]_e after 20 min . However, the effect of 50μM glutamate was inhibited. This inhibition was not complete, at least not in the case of ERK₂, where phosphorylation in the combined presence of 50μM [K⁺]_e and the inhibitor slightly, but significantly, exceeded that in the presence of GF 109203X without glutamate.Consistent with the sensitivity to GF109203X of the glutamate-mediated increase of ERK_1/2 phosphorylation after 20 min, K⁺-mediated stimulation of ERK_1/2 phosphorylation in glutamine-replete medium, known mainly to reflect the effect of glutamate released as a result of the depolarization was also inhibited by GF109203X. However, the similar ERK_1/2 phosphorylation after 60 min was unaffected by the presence of the PKC inhibitor.DiscussionSeven different members of the EGF growth factor superfamily have been identified. Only the first four have been demonstrated in brain tissue. Among these HB-EGF and TGF-αwere found to be present in cerebellum and in cultured cerebellar granule neurons. This is consistent with a previous observation that TGF-αis expressed in cerebellar granule cells in the brain in vivo, as shown by in situ hybridization. Also, HB-EGF is expressed at high level in brain in vivo at early developmental stages; it declines thereafter, although some HB-EGF remains in the cerebellum. In contrast, EGF and amphiregulin were undetectable both in cerebellar granule cells in primary cultures and cerebellum from adult mouse, consistent with previous findings.Transactivation of neuronal EGFRs has previously been reported in GT1-7 cells, immortalized hypothalamic neurons, treated with gonadotropin-releasing hormone. It has also been found in PC12 cells, a phaeochromocytoma cell line, during depolarization or exposure to bradykinin or cAMP. However, to our knowledge transactivation of EGFRs has not previously been reported in primary cultures of neurons. The signal pathway inducing "shedding" of growth factors is complex and may be cell type-, G-protein coupled receptor-, and developmental stage-specific.A significant portion of K⁺-mediated ERK_1/2 phosphorylation depended upon NMDA receptor activation, as indicated by its inhibition by NMDA, an antagonist of the NMDA receptor. ERK_1/2 phosphorylation in cerebellar granule cells in response to NMDA receptor stimulation has previously been demonstrated by Sato et al. and Zhu et al., but these authors did not investigate whether transactivation was involved.In the present cultures the initial K⁺-mediated glutamate release amounts to about 10 nmol/min per mg protein, but due to a time-dependent reduction of release and to concentration-dependent reuptake the glutamate concentration in the medium is only 10-15 nmol/mg protein after 30 min of incubation. With approximately 1 mg cell protein incubated in 2 ml of medium, this amount corresponds to a medium glutamate concentration of 5-8 uM, and perhaps more in the synaptic clefts. This concentration is likely to suffice for activation of NMDA receptors, which have a high affinity for glutamate, but perhaps not for AMPA receptors which have a much lower affinity. This may explain why CNQX, an antagonist at AMPA receptors, had no effect, whereas Wu et al. and Limatola et al., using addition of receptor agonists have demonstrated ERK_1/2 phosphorylation induced by AMPA receptor activation in cerebellar granule cells. Again, it was not investigated whether transactivation was involved.The present report together with our previous paper show a remarkable complexity of the signaling pathways between K⁺-mediated depolarization of cerebellar granule cells in primary cultures and ERK_1/2 phosphorylation . They include a glutamate-induced ERK_1/2 phosphorylation secondary to K⁺-evoked glutamate release at both 20 and 60 min, which is dependent upon EGFR transactivation. The present study has shown that only the former of these two increases in ERK_1/2 phosphorylation requires PKC activity. The PKC dependence of the response to glutamate after 20 min was confirmed by addition of 50 uM glutamate to glutamine-free medium, although a minor part of this effect appeared to be PKC-independent. In addition the K+-mediated depolarization per se causes ERK_1/2 phosphorylation at the same time points. However, this phosphorylation occurs independently of transactivation and is also independent of PKC activation, at least at 20 min. PKC inhibition was not tested in glutamine-free medium at 60 min but there was no indication of any PKC-dependent component at 60 min in the glutamine-replete medium.Accumulating evidence suggests that ERK phosphorylation may play an important role in at least certain types of long-term potentiation (LTP) and learning. Recently, NMDA- and PKC-dependent LTP has been demonstrated at the cerebellar mossy fiber-granule cell synapse. Also, investigation of cytosolic [Ca²⁺] dynamics during repetitive neurotransmission in cerebellar slices showed that glutamate receptor-mediated [Ca²⁺]j increase in granule cell dendrites was locally amplified by opening of voltage-dependent Ca²⁺ channels and Ca²⁺ release from intracellular stores. Moreover, granule cell firing contributed to elevating cytosolic [Ca²⁺] through voltage-dependent channels also in the dendrites that were not synaptically stimulated. These findings attest to the importance of both glutamate-mediated and depolarization-mediated ERK phosphorylation in cerebellar granule cells but it is not known where PKC activity fits into the picture.ConclusionIn conclusion, depolarization of 7-8-day old cerebellar granule neurons, obtained from 7-day-old mice, by exposure to 45 mM extracellular [K⁺]_e leads to phosphorylation of ERK_1/2, mediated by NMDA receptor activation and partly by a transactivation process, leading to release of HB-EGF and perhaps also of TGF-α. The subsequent action of these EGFR agonists may play a major role for the effects of NMDA receptor activation on brain plasticity.