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ATRA inhibits ceramide kinase transcription in a human neuroblastoma cell line, SH‐SY5Y cells: the role of COUP‐TFI
Author(s) -
Murakami Masashi,
Ito Hiromi,
Hagiwara Kazumi,
Yoshida Kayo,
Sobue Sayaka,
Ichihara Masatoshi,
Takagi Akira,
Kojima Tetsuhito,
Tanaka Kouji,
TamiyaKoizumi Keiko,
Kyogashima Mamoru,
Suzuki Motoshi,
Banno Yoshiko,
Nozawa Yoshinori,
Murate Takashi
Publication year - 2010
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2009.06486.x
Subject(s) - chromatin immunoprecipitation , microbiology and biotechnology , retinoic acid , transcription factor , chemistry , ceramide , biology , small interfering rna , electrophoretic mobility shift assay , transfection , gene expression , promoter , biochemistry , gene , apoptosis
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), RARα, and RXRα, 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.