Bone morphogenetic protein 9 is a potent synergistic fatter for murine hemopoietic progenitor cell generation and colony formation in serum-free cultures

The effect of the recently cloned cytokine bone morphogenetic protein 9 (BM P-9) on colony formation and generation in vitro clonable hematopoietic pro genitors (CFU-C) in serum-free liquid cultures (LC) of both normal and post -5-fluorouracil murine bone marrow cells was studied in the presence of var ious other cytokines. In LC, BMP-9 concentrations of 100 ng or more per mi led to complete inhibition of Steel Factor (SF) + interleukin-11 (IL-11) or IL-12 supported CFU-C generation, which was partly abrogated when IL-3 was additionally included. We found this inhibitory effect of BMP-9 to be medi ated by an increased TGF-beta 1 elaboration and TGF-beta 1 mRNA expression in bone marrow cells with increasing BMP-9 concentrations. in the presence of neutralizing antibodies (Ab) against TGF-beta 1, BMP-9 concentrations of 3 ng or higher synergized with IL-3, SF+IL-3, SF+IL-11/12, or IL-3+SF+IL-1 1/12 to increase CFU-C generation. Similarly, high BMP-9 concentrations dra matically inhibited primary colony formation induced by SF+IL-11/12, wherea s in the presence of TGF-beta 1 neutralizing Ab only 3 ng or more BMP-9 per mi stimulated both the time of colony appearance, the colony size and colo ny numbers in the presence of IL-3, AA-CSF, GM-CSF, SF, SF+Flt3-L, SF+IL-3, SF+IL-11/12 or IL-3+SF+IL-11/12. BMP-9 neither stimulated CFU-C generation nor colony formation as a single factor, nor did it synergize with thrombo poietin (Tpo), erythropoietin (Epo), Flt3-L, IL-11, IL-12 or G-CSF. The eff ect of BMP-9 on its target cells was direct as demonstrated using single-so rted stem cells. These observations demonstrate that BMP-9 plays a dual rol e in regulating proliferation of primitive hemopoietic progenitor cells. Th us, in addition to its ability to enhance TGF pr elaboration in bone marrow cells, it acts as a potent synergistic activity that is different from SF, Flt3-L, IL-11 or IL-12. BMP-9 mRNA was exclusively detected in the fiver o f adult mice, whilst no expression was found in stromal cell lines propagat ed from day-16 fetal liver or neonatal or adult bone marrow. I-125-BMP-9 bo und specifically to a high percentage of blast cells in lineage-depleted po st-fluorouracil bone marrow cells and to megakaryocytes in normal and post- fluorouracil bone marrow, indicating that BMP-9R are expressed on these cel ls. The dissociation between the site of BMP-9 production and its target ce lls in the bone marrow makes BMP-9 a hemopoietic hormone.