Sp1 and C/EBP are necessary to activate the lactoferrin gene promoter during myeloid differentiation

In this study, we sought to identify factors responsible for the positive m odulation of lactoferrin (LF), a neutrophil-specific, secondary-granule pro tein gene. Initial reporter gene transfection assays indicated that the fir st 89 base pairs of the LF promoter are capable of directing myeloid-specif ic LF gene expression. The presence of a C/EBP site flanked by 2 Sp1 sites within this segment of the LF promoter prompted us to investigate the possi ble role of these sites in LF expression. Cotransfection studies of LF-89lu c plasmid with increasing concentrations of a C/EBP alpha expression vector in myeloid cells resulted in a linear transactivation of luciferase report er activity, Electrophoretic mobility shift assays found that the C/EBP sit e is recognized by C/EBP alpha and that both LF Sp1 binding sites bind the Sp1 transcription factor specifically in myeloid cells. Mutation of either Sp1 site markedly reduced activity of the LF-89luc plasmid in myeloid cells , and neither Sp1 mutant plasmid was transactivated by a C/EBP alpha expres sion plasmid to the same extent as wild-type LF-89luc, We also transfected LF-89luc into Drosophila Schneider cells, which do not express endogenous S p1, and demonstrated up-regulation of luciferase activity in response to a cotransfected Sp1 expression plasmid, as well as to a C/EBP alpha expressio n plasmid, Furthermore, cotransfection of LF-89luc plasmid simultaneously w ith C/EBP alpha and Sp1 expression plasmids resulted in an increase in luci ferase activity greater than that induced by either factor alone. Taken tog ether, these observations indicate a functional interaction between C/EBP a nd Sp1 in mediating LF expression. (C) 2000 by The American Society of Hema tology.