|Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
To investigate proliferation and differentiation signal transduction mechanisms through granulocyte-colony stimulating factor (G-CSF) receptor, the receptor was expressed in murine myeloid precursor cell, L-GM,by transfecting its cDNA.When the obtained transformant was stimulated by G-CSF,it was differentiated to neutrophil. Several mutant G-CSF receptor cDNAs were constructed that encoded either a series of deletion receptors or the receptors where each tyrosine residue was replaced with phenylalanine. By analyzing the effects of these mutations on the G-CSF-induced signal transduction, tyrosine-phosphorylaion of p54 (Shc) was found to require the 4th tyrosine residue of the receptor and 1st and 2nd tyrosine residues were indispensable for inducing the neutrophil-differentiation phenotypes, including nuclear lobulation, growth suppression and myeloperoxidase gene expression. JAK1, JAK2, STAT3 and Shc were found to phosphorylated on their tyrosine residues upon G-CSF stimulation. Phosphorylation of JAK1 and JAK2 required the Box1 and Box2 region of the receptor, while STAT3 phosphorylation need not only the Box1 and Box2 but the region around the 1st tyrosine residue.
Furthermore, the upstream promoter and its truncated regions of myeloperoxidase (MPO) gene, which is expressed specifically in neutrophils, was connected to the reporter gene, and its G-CSF dependent expression was examined. A cis-regulatory element was identified at about 800bp upstream from the transcription start site that was responsible for the G-CSF dependent gene expression. A transcription factor, NF/G-CSF,was identified to bind to the element by gel-shift assay. Using oligonucleotide affinity chromatography, NF/G-CSF was purified and its N-terminal amino acid sequence was determined. The obtained sequence was identical to that of a known transcription factor, NF-Y.Therefore, NF-Y appears to be involved in the G-CSF dependent MPO gene expression.