2003 Fiscal Year Final Research Report Summary
The significance of serum sphingosine 1-phospahte in hematological diseases and the regulatory mechanism of sphingosine kinase
| Project/Area Number |
14572180
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Laboratory medicine
|
|---|
| Research Institution | Nagoya University |
|---|
Principal Investigator |
MURATE Takeshi Nagoya University, School of Health Science, Professor, 医学部, 教授 (30239537)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KOIZUMI Keiko Nagoya University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (00118027)
TAKAGI Akira Nagoya University, School of Health Science, Research Associate, 医学部, 助手 (30135371)
KOJIMA Tesuhito Nagoya University, School of Health Science, Professor, 医学部, 教授 (40161913)
BANNNO Yoshiko Gifu University School of Medicine, Gifu University School of Medicine, Associate Professor, 医学部, 助教授 (50116852)
SUZUKI Motoshi Nagoya University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 講師 (80236017)
|
|---|
| Project Period (FY) |
2002 – 2003
|
| Keywords | sphingosine kinase 1 / polyclonal antibody / gene expression / promoter analysis |
|---|
| Research Abstract |
As the preparatory step, the polyclonal antibody against 16 peptide-fragment of human sphingosine kinase 1 was made by immunizing rabbit. 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 inhibitor prevented the PMA-induced SPHK1 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 Sp1-and two AP-2-binding motifs within this area were necessary for responsiveness. Electrophoresis mobility shift assay 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.
|
