Signalling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons
Signalling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons
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右美托咪定反式激活星形胶质细胞表皮生长因子受体的信号通路及其对神经元的旁分泌作用
DOI：
10.1038/bjp.2008.58
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发表时间：
2008-05-01
影响因子：
7.3
通讯作者：
Peng, L.
中科院分区：
文献类型：
--
作者：
Li, B.;Du, T.;Peng, L.
背景与目的：神经保护药物α₂ -肾上腺素能受体激动剂右美托咪定对星形胶质细胞的刺激可反式激活表皮生长因子（EGF）受体。本研究探讨了导致EGF受体配体释放的信号通路以及在EGF受体刺激过程中被激活的信号通路，以及神经元对右美托咪定和星形胶质细胞条件培养基的反应。
实验方法：通过蛋白质印迹法和免疫细胞化学法测定细胞外调节蛋白激酶1/2（ERK1/2）的磷酸化，通过免疫沉淀和蛋白质印迹法测定EGF受体的磷酸化。通过逆转录 - 聚合酶链反应（RT - PCR）测量fos家族的mRNA表达。
关键结果：百日咳毒素（0.2μg/ml）是一种Gαᵢ亚基从Gαᵢ蛋白解离的抑制剂，以及蛋白激酶C抑制剂GF 109203X（500 nM），可消除ERK1/2的磷酸化。PP1（10μM），一种抑制Src激酶的物质，以及GM 6001（10μM），一种锌依赖性金属蛋白酶抑制剂，可消除右美托咪定（50 nM）引起的ERK1/2磷酸化，但不能消除EGF（10 ng/ml）引起的磷酸化，这表明仅在第一阶段激活了Src激酶和金属蛋白酶；AG 1478（1 mM），一种EGF受体酪氨酸激酶抑制剂，可消除ERK1/2磷酸化。右美托咪定诱导的EGF受体磷酸化可被AG 1478、GM 6001、PP1和GF 109203X阻止，其对cfos和fosB的诱导可被AG 1478以及一种ERK磷酸化抑制剂U0126（10 mM）阻止，这表明ERK1/2磷酸化具有下游效应。EGF和来自右美托咪定处理的星形胶质细胞的条件培养基，但不是右美托咪定本身，可诱导小脑神经元原代培养物中的ERK磷酸化。
结论与意义：明确了右美托咪定诱导的反式激活通路。它对神经元的旁分泌作用可能是其神经保护作用的原因。
Background and purpose: Stimulation of astrocytes by the alpha(2)-adrenoceptor agonist dexmedetomidine, a neuroprotective drug, transactivates epidermal growth factor (EGF) receptors. The present study investigates signal pathways leading to release of an EGF receptor ligand and those activated during EGF receptor stimulation, and the response of neurons to dexmedetomidine and to astrocyte-conditioned medium.Experimental approach: Phosphorylation of ERK1/2 was determined by western blotting and immunocytochemistry, and phosphorylation of EGF receptors by immunoprecipitation and western blotting. mRNA expression of fos family was measured by RT-PCR.Key results: Pertussis toxin (0.2 mu g ml(-1)) an inhibitor of G alpha(i) subunit dissociation from G alpha(i) protein, and GF 109203X (500 nM), a protein kinase C inhibitor, abolished ERK1/2 phosphorylation. PP1 (10 mu M), inhibiting Src kinase and GM 6001 (10 mu M), an inhibitor of Zn-dependent metalloproteinase, abolished ERK1/2 phosphorylation by dexmedetomidine (50 nM), but not that by EGF (10 ng ml(-1)), showing Src kinase and metalloproteinase activation during the first stage only; AG 1478 (1 mM), an inhibitor of the EGF receptor tyrosine kinase, abolished ERK1/2 phosphorylation. Dexmedetomidine-induced EGF receptor phosphorylation was prevented by AG 1478, GM 6001, PP1 and GF 109203X and its induction of cfos and fosB by AG 1478 and by U0126 (10 mM), an inhibitor of ERK phosphorylation, indicating downstream effects of ERK1/2 phosphorylation. EGF and conditioned medium from dexmedetomidine-treated astrocytes, but not dexmedetomidine itself, induced ERK phosphorylation in primary cultures of cerebellar neurons.Conclusions and implications: Dexmedetomidine-induced transactivation pathways were delineated. Its paracrine effect on neurons may account for its neuroprotective effects.