Department of Oral Disease Research, National Center for Geriatrics and Gerontology, Obu, Japan.
J Cell Biochem. 2011 Nov;112(11):3449-58. doi: 10.1002/jcb.23275.
Glial cell line-derived neurotrophic factor (GDNF) is important for the development and maintenance of dopamine neurons (Lin et al. [1993] Science 260: 1130-1132). GDNF is neuroprotective in animal models of Parkinson disease, where dopamine neurons show selective degeneration. We previously reported GDNF-induced SPHK1 gene expression in a neuroblastoma cell line, TGW (Murakami et al. [2007] J Neurochem 102: 1585-1594). In the present study, we focused on the regulatory mechanism of GAP43 (GDNF-induced neuronal phenotype) transcription to further elucidate physiological roles of GDNF-induced SPHK1 expression and activity. Stable wild-type (SPHK1-WT) but not dominant-negative SPHK1 (SPHK1-DN) overexpression increased both control- and GDNF-induced GAP43 expression. SPHK1-WT cells showed enhanced GDNF-induced sphingosine 1-phosphate (S1P) secretion compared with mock- and SPHK1-DN cells. Exogenous S1P also increased GAP43 expression. In TGW cells, PD98059, a MEK inhibitor, but not SB203580 (a p38 MAPK inhibitor) and LY294002 (a PI3K inhibitor) inhibited GDNF-induced GAP43 expression, suggesting the MEK/ERK pathway has a major role in GDNF-induced GAP43 transcription. A G-protein-coupled receptor inhibitor, pertussis toxin, and S1P(1) and S1P(3) receptor antagonists (VPC23019 and CAY10444) also inhibited ERK activation. Moreover, both S1P1 and S1P3 were serine-phosphorylated by GDNF, suggesting their activated states. C/EBPβ transcription factor was induced by GDNF, and DNA pull-down and chromatin immunoprecipitation assays revealed the C/EBP binding site between -131 bp and -98 bp from the first exon of GAP43. Taken together, our results showed that in TGW cells, GDNF increased SPHK1 transcription, leading to the production and secretion of S1P. Through MEK/ERK pathway, S1P stimulates GAP43 transcription with increased binding of C/EBPβ to the 5'-promoter.
胶质细胞源性神经营养因子 (GDNF) 对于多巴胺神经元的发育和维持非常重要（Lin 等人，[1993] Science 260:1130-1132）。GDNF 在帕金森病的动物模型中具有神经保护作用，其中多巴胺神经元表现出选择性退化。我们之前报道过 GDNF 在神经母细胞瘤细胞系 TGW 中诱导 SPHK1 基因表达（Murakami 等人，[2007] J Neurochem 102:1585-1594）。在本研究中，我们专注于 GAP43（GDNF 诱导的神经元表型）转录的调节机制，以进一步阐明 GDNF 诱导的 SPHK1 表达和活性的生理作用。稳定的野生型 SPHK1（SPHK1-WT）而非显性失活的 SPHK1（SPHK1-DN）过表达均增加了对照和 GDNF 诱导的 GAP43 表达。与 mock 和 SPHK1-DN 细胞相比，SPHK1-WT 细胞显示出增强的 GDNF 诱导的神经鞘氨醇 1-磷酸（S1P）分泌。外源性 S1P 也增加了 GAP43 的表达。在 TGW 细胞中，PD98059（一种 MEK 抑制剂），而不是 SB203580（一种 p38 MAPK 抑制剂）和 LY294002（一种 PI3K 抑制剂）抑制了 GDNF 诱导的 GAP43 表达，表明 MEK/ERK 通路在 GDNF 诱导的 GAP43 转录中起主要作用。G 蛋白偶联受体抑制剂百日咳毒素以及 S1P(1)和 S1P(3)受体拮抗剂（VPC23019 和 CAY10444）也抑制了 ERK 激活。此外，GDNF 还使 S1P1 和 S1P3 发生丝氨酸磷酸化，表明它们处于激活状态。C/EBPβ 转录因子被 GDNF 诱导，DNA 下拉和染色质免疫沉淀测定显示 GAP43 第一外显子上 -131 bp 至 -98 bp 之间存在 C/EBP 结合位点。综上所述，我们的结果表明，在 TGW 细胞中，GDNF 增加了 SPHK1 的转录，导致 S1P 的产生和分泌。通过 MEK/ERK 通路，S1P 刺激 GAP43 转录，C/EBPβ 与 5'-启动子的结合增加。
