Transcription factor specificity protein 1 (Sp1) is the main regulator of nerve growth factor-induced sphingosine kinase 1 gene expression of the rat pheochromocytoma cell line, PC12.

Sphingosine kinase (SPHK) is known to exert an anti-apoptic role in various cells and cell lines. We previously reported that human brain is rich in SPHK1 (Murate et al. 2001). After showing a high expression of SPHK1 in rat brain, we examined the gene expression mechanism using nerve growth factor (NGF)-stimulated rat PC12 cells. With RT-PCR, we found that both rat brain and PC12 utilized exon 1d mostly out of eight untranslated first exons. NGF induced an increase in SPHK enzyme activity and protein about double those in PC12 cells, and NGF-induced SPHK1 mRNA was three times higher than in the control. The minimal 5' promoter was determined, and TrkA specific inhibitor K252a inhibited the NGF-induced promoter activity of SPHK1. The truncation or mutation of putative transcription factor-binding motifs revealed that one specificity protein 1 (Sp1) binding motif of the 5' region of exon 1d is prerequisite. Electrophoresis mobility shift assay confirmed the promoter analysis, indicating increased Sp1 protein binding to this motif after NGF treatment. Chromatin immunoprecipitation assay also showed the binding of Sp1 and the promoter region in vivo. These results suggest the signal transduction pathway from NGF receptor TrkA to transcription factor Sp1 protein binding to the promoter Sp1-like motif in NGF-induced rat SPHK1 gene expression.