title: Regulation of sphingosine kinase 1 gene expression by protein kinase C in a human leukemia cell line, MEG-O1.
abstract: The prolonged treatment with phorbol 12-myristate 13-acetate (PMA) of a human megakaryoblastic leukemia cell line, MEG-O1, induced increase of sphingosine kinase (SPHK) enzyme activity and SPHK1 protein expression as well as SPHK1 message. Protein kinase C (PKC) inhibitor prevented the PMA-induced SPHK1 gene expression. To elucidate the regulatory mechanism of this gene expression, we examined the promoter area (distal to the first exon) and its binding proteins. Luciferase analyses showed that the area of 300 bp from the first exon was sufficient for PMA-responsiveness, and that specificity protein 1 (Sp1)- and two activator protein 2 (AP-2)-binding motifs within this area were necessary for responsiveness. Inhibitors for PKC and MEK1 decreased this PMA-induced promoter activity. Electrophoresis mobility shift assay (EMSA) showed that Sp1 protein was originally bound to the Sp1 site and that two additional bands bound to the two AP-2 motifs were observed only when stimulated with PMA in MEG-O1 cells. The appearance of these bands resulted from binding to an unknown protein rather than AP-2. These results indicated that PMA up-regulates SPHK1 gene expression through PMA-responsive elements of the 5' promoter area of the gene, and suggested that PMA-mediated SPHK1 gene expression would be mediated via PKC- and ERK-dependent signal transduction pathway by binding the transcription factor to AP-2 motifs.
authors.affiliation: Nagoya University Graduate School of Medicine, Nagoya University School of Health Sciences, Higashi, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nakade
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Banno
authors.forename: Keiko
authors.initials: K
authors.lastname: T-Koizumi
authors.forename: Kazumi
authors.initials: K
authors.lastname: Hagiwara
authors.forename: Sayaka
authors.initials: S
authors.lastname: Sobue
authors.forename: Masahiro
authors.initials: M
authors.lastname: Koda
authors.forename: Motoshi
authors.initials: M
authors.lastname: Suzuki
authors.forename: Tetsuhito
authors.initials: T
authors.lastname: Kojima
authors.forename: Akira
authors.initials: A
authors.lastname: Takagi
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Asano
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Nozawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Murate
mesh_terms: D001665:Binding Sites; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007938:Leukemia; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D015345:Oligonucleotide Probes; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology*; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D000099157:Sphingosine Kinase
chemical_list: D004268:DNA-Binding Proteins; D020928:Mitogen-Activated Protein Kinases; D015345:Oligonucleotide Probes; D017853:Phosphotransferases (Alcohol Group Acceptor); D011493:Protein Kinase C; D016329:Sp1 Transcription Factor; D013755:Tetradecanoylphorbol Acetate; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D000099157:Sphingosine Kinase