ATRA inhibits ceramide kinase transcription in a human neuroblastoma cell line, SH-SY5Y cells: the role of COUP-TFI.

Ceramide is the central lipid in the sphingolipid metabolism. Ceramide kinase (CERK) and its product, ceramide 1-phosphate, have been implicated in various cellular functions. However, the regulatory mechanism of CERK gene expression remains to be determined. Here, we examined CERK mRNA level during all-trans retinoic acid (ATRA)-induced differentiation of a human neuroblastoma cell line, SH-SY5Y. ATRA reduced CERK mRNA and protein levels. Over-expression and small interfering RNA (siRNA) of CERK revealed that CERK is inhibitory against ATRA-induced neuronal differentiation and cell growth arrest. ATRA inhibited the transcriptional activity of 5'-promoter of CERK. Truncation and mutation study suggests that ATRA-responsible region was mainly located in the tandem retinoic acid responsive elements (RARE) between -40 bp and the first exon. The electrophoresis mobility shift assay revealed that ATRA produced two retarded bands, which were erased by antibody against chicken ovalbumin upstream promoter transcription factor I (COUP-TFI), RARalpha, and RXRalpha, respectively. DNA pull-down assay confirmed increased binding of these transcription factors to RARE. Transient expression of RAR, RXR, and COUP-TFI and siRNA transfection of these genes revealed that COUP-TFI inhibited CERK mRNA. Furthermore, chromatin immunoprecipitation assay showed the recruitment of co-repressors as well as three transcription factors. These results suggest that COUP-TFI was the ATRA-responsive suppressive transcription factor of CERK gene transcription.