Sphingosine kinase 1 expression is downregulated during differentiation of Friend cells due to decreased c-MYB

• Published in: Biochimica et biophysica acta Vol. 1833; no. 5; pp. 1006 - 1016
• Main Authors: Mizutani, N., Kobayashi, M., Sobue, S., Ichihara, M., Ito, H., Tanaka, K., Iwaki, S., Fujii, S., Ito, Y., Tamiya-Koizumi, K., Takagi, A., Kojima, T., Naoe, T., Suzuki, M., Nakamura, M., Banno, Y., Nozawa, Y., Murate, T.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-05-2013
• Subjects: Animals; c-MYB; Cell Differentiation - genetics; Cell Line; ChIP; Down-Regulation; Friend cell; HMBA-induced differentiation; Humans; Mice; Phosphotransferases (Alcohol Group Acceptor) - genetics; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Promoter analysis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myb - genetics; Proto-Oncogene Proteins c-myb - metabolism; Receptors, Lysosphingolipid - antagonists & inhibitors; Receptors, Lysosphingolipid - genetics; Receptors, Lysosphingolipid - metabolism; RNA, Messenger - genetics; RNA, Small Interfering; Signal Transduction; SPHK1; S1P; c-MYB; HMBA; SPHK1; EMSA; HMBA-induced differentiation; DN; ChIP; WT; Friend cell; Promoter analysis; SPL
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/j.bbamcr.2013.01.001

Sphingosine kinase 1 (SPHK1) overexpression in malignant cells has been reported. Mouse Friend cells showed higher SPHK1 but not SPHK2 expression compared with other mouse cell lines. A Sphk1 promoter analysis demonstrated the region between −53bp and the first exon as the minimal promoter. Further promoter truncation revealed the importance of a MYB-binding site. EMSA using this region as the probe demonstrated one band containing c-MYB protein, and its intensity decreased during erythroid differentiation with hexamethylane bisacetamide (HMBA), a potent inducer of erythroid differentiation of Friend cells. ChIP assay also revealed in vivo binding of c-MYB. c-MYB overexpression and siRNA for c-Myb affected SPHK1 expression, confirming the important regulatory role of c-MYB in SPHK1 expression. HMBA reduced c-MYB expression rapidly. Induced differentiation by HMBA caused a marked and rapid reduction of SPHK1 mRNA, protein and enzyme activity leading to the rapid decrease of cellular sphingosine 1-phosphate level. Moreover, terminally differentiated cells did not resume SPHK1 expression. Compared with original Friend cells, stable overexpression of wild-type SPHK1 showed higher cell proliferation, resistance to cell death by serum depletion. Interestingly, HMBA-induced differentiation of these cells was delayed but not completely suppressed. In contrast, SPHK inhibitor and its siRNA inhibited cell growth and enhanced HMBA-induced differentiation significantly, suggesting that SPHK1 delayed HMBA-induced differentiation by its cell proliferation-promoting activity. Effects of pertussis toxin, a G-protein-coupled receptor inhibitor, and S1P receptor antagonist on Friend cell growth and differentiation were negligible, suggesting the importance of the intracellular SPHK1/S1P signaling in Friend cells.
► SPHK1 but not SPHK2 was overexpressed in a mouse Friend erythroeukemia cell line. ► SPHK1 expression of Friend cells affects their proliferation and differentiation. ► SPHK1 expression decreased during HMBA-induced differentiation. ► 5′-promoter between −53bp and the first exon is important for SPHK1 transcription. ► C-MYB regulates SPHK1 transcription by its binding with the 5′-promoter region.