Abstract
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. J. Cell. Biochem. 112: 3449–3458, 2011. © 2011 Wiley Periodicals, Inc.