An Analog of a Dipeptide-Like Structure of FK506 Increases Glial Cell Line-Derived Neurotrophic Factor Expression through cAMP Response Element-Binding Protein Activated by Heat Shock Protein 90/Akt Signaling Pathway
An Analog of a Dipeptide-Like Structure of FK506 Increases Glial Cell Line-Derived Neurotrophic Factor Expression through cAMP Response Element-Binding Protein Activated by Heat Shock Protein 90/Akt Signaling Pathway
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DOI：
10.1523/jneurosci.5010-05.2006
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发表时间：
2006-03
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通讯作者：
X. Cen;A. Nitta;Shin Ohya;Yinglan Zhao;Naoya Ozawa;A. Mouri;D. Ibi;Li Wang;Makiko Suzuki;Kuniaki Saito;Yasutomo Ito;T. Kawagoe;Y. Noda;Yoshihisa Ito;S. Furukawa;T. Nabeshima
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作者：
X. Cen;A. Nitta;Shin Ohya;Yinglan Zhao;Naoya Ozawa;A. Mouri;D. Ibi;Li Wang;Makiko Suzuki;Kuniaki Saito;Yasutomo Ito;T. Kawagoe;Y. Noda;Yoshihisa Ito;S. Furukawa;T. Nabeshima
胶质细胞源性神经营养因子（GDNF）是一种重要的神经营养因子，对神经退行性疾病具有治疗意义。我们以前表明，亮氨酸-异亮氨酸（Leu-Ile），一种类似于FK 506（他克莫司）的二肽样结构，在体内和体外诱导GDNF表达。在这项研究中，我们试图阐明这种二肽的GDNF诱导作用的细胞机制。用异硫氰酸荧光素-Leu-Ile研究了Leu-Ile在培养的神经元中的转运，结果表明该二肽具有跨膜迁移性。通过液相色谱-质谱联用和石英晶体微天平分析，我们鉴定了热休克同源蛋白70是一个与Leu-Ile特异性结合的蛋白质，分子模拟表明ATP酶结构域是预测的结合位点。Leu-Ile刺激Akt磷酸化，热休克蛋白90（Hsp 90）抑制剂格尔德霉素（GA）可显著减弱Akt磷酸化。此外，通过免疫沉淀检测到磷酸化Akt和Hsp 90之间的相互作用增强。Leu-Ile引起cAMP反应元件结合蛋白（CREB）磷酸化的增加，这被GA抑制，表明CREB是Hsp 90/Akt信号传导的下游靶点。Leu-Ile可提高GDNF mRNA和蛋白表达水平，而CREB的抑制则阻断了这种作用。Leu-Ile促进磷酸化CREB与cAMP反应元件的结合活性。这些结果表明CREB在Leu-Ile诱导的GDNF表达的转录调控中起关键作用。总之，Leu-Ile激活Hsp 90/Akt/CREB信号通路，这有助于GDNF表达的上调。它可能代表一种新的先导化合物用于治疗多巴胺能神经元或运动神经元疾病。
Glial cell line-derived neurotrophic factor (GDNF) is an important neurotrophic factor that has therapeutic implications for neurodegenerative disorders. We previously showed that leucine-isoleucine (Leu-Ile), an analog of a dipeptide-like structure of FK506 (tacrolimus), induces GDNF expression both in vivo and in vitro. In this investigation, we sought to clarify the cellular mechanisms underlying the GDNF-inducing effect of this dipeptide. Leu-Ile transport was investigated using fluorescein isothiocyanate-Leu-Ile in cultured neurons, and the results showed the transmembrane mobility of this dipeptide. By liquid chromatography-mass spectrometry and quartz crystal microbalance assay, we identified heat shock cognate protein 70 as a protein binding specifically to Leu-Ile, and molecular modeling showed that the ATPase domain is the predicted binding site. Leu-Ile stimulated Akt phosphorylation, which was attenuated significantly by heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA). Moreover, enhanced interaction between phosphorylated Akt and Hsp90 was detected by immunoprecipitation. Leu-Ile elicited an increase in cAMP response element binding protein (CREB) phosphorylation, which was inhibited by GA, indicating that CREB is a downstream target of Hsp90/Akt signaling. Leu-Ile elevated the levels of GDNF mRNA and protein expression, whereas inhibition of CREB blocked such effects. Leu-Ile promoted the binding activity of phosphorylated CREB with cAMP response element. These findings show that CREB plays a key role in transcriptional regulation of GDNF expression induced by Leu-Ile. In conclusion, Leu-Ile activates Hsp90/Akt/CREB signaling, which contributes to the upregulation of GDNF expression. It may represent a novel lead compound for the treatment of dopaminergic neurons or motoneuron diseases.