Pax5 is an essential transcription factor for B cell development, and it is reported that Pax5 expression was reduced in the IL-7 receptor (IL-7R) knockout mouse. To investigate whether signals from the IL-7R regulate Pax5 transcription, we searched the consensus sequence of signal transducers and activators of transcription (STAT) in the Pax5 promoter region, since STAT is one of the components of cytokine signal transduction. A STAT-binding motif, termed SBM, was identified at 1,118 bp upstream of the transcriptional start site, and SBM completely overlapped with the binding site for early B cell factor (EBF). STAT5 was phosphorylated in the presence of IL-7 in the IL-7-dependent preB cell line, PreBR1, and phosphorylated-STAT5 as well as EBF was found to bind to the SBM. Moreover, we also revealed STAT5 binding to SBM in PreBR1 cells by chromatin immunoprecipitation assay. Transient co-transfection of reporter genes together with expression vectors of a constitutive active form of STAT5 and EBF into NIH3T3 cells demonstrated that STAT5 enhanced EBFregulating transcription. Our results suggest that STAT5 phosphorylated by IL-7 can directly up-regulate Pax5 transcription in early B cells.
In the course of screening for immunomodulators, we found a significant blastogenic activity specific for splenic B cells in the extracts of safflower (Carthamus tinctorius L.). Active fractions termed SF1 and SF2 were purified from dried petals of safflower by boiling water extraction, ethanol precipitation and Sepharose CL-2B column chromatography. The elution profiles of the gel filtration indicated that the molecular weight of SF1 and SF2 was estimated to be more than 100 kD. Major components of SF1 and SF2 seem to be polysaccharides, and structural analysis of alditol acetate derivatives by GC-MS revealed some differences between SF1 and SF2 in the sugar component. Biological activities of SF1 and SF2 on B cells and macrophages were examined in comparison with lipopolysaccharides (LPS). SF1 and SF2 induced both the proliferation and the IgM production of B cells to the equivalent level as those induced by LPS. In macrophages, SF1 and SF2 effectively stimulated the production of NO. However, SF1 stimulated the production of IL-1, IL-6, and TNF as much as LPS, while SF2 induced them only weakly or not at all. Thus, these results suggest that SF1 and SF2 activate B cells and macrophages in different mechanisms.
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