title: ATRA inhibits ceramide kinase transcription in a human neuroblastoma cell line, SH-SY5Y cells: the role of COUP-TFI.
abstract: 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.
authors.affiliation: Research Fellow of the Japanese Society for the Promotion of Sciences, Nagoya, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Murakami
authors.forename: Hiromi
authors.initials: H
authors.lastname: Ito
authors.forename: Kazumi
authors.initials: K
authors.lastname: Hagiwara
authors.forename: Kayo
authors.initials: K
authors.lastname: Yoshida
authors.forename: Sayaka
authors.initials: S
authors.lastname: Sobue
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Ichihara
authors.forename: Akira
authors.initials: A
authors.lastname: Takagi
authors.forename: Tetsuhito
authors.initials: T
authors.lastname: Kojima
authors.forename: Kouji
authors.initials: K
authors.lastname: Tanaka
authors.forename: Keiko
authors.initials: K
authors.lastname: Tamiya-Koizumi
authors.forename: Mamoru
authors.initials: M
authors.lastname: Kyogashima
authors.forename: Motoshi
authors.initials: M
authors.lastname: Suzuki
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Banno
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Nozawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Murate
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D051838:COUP Transcription Factor I / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D005091:Exons / Q000187:drug effects / Q000502:physiology; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008969:Molecular Sequence Data; D016501:Neurites / Q000187:drug effects; D009447:Neuroblastoma; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection / Q000379:methods; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D051838:COUP Transcription Factor I; C000607071:RARA protein, human; D034741:RNA, Small Interfering; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors; D014212:Tretinoin; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase