FLT3 Ligand induces functional effects through STAT5a activation and bcl-xl expression as evidenced through biochemical analysis and use of stat 5A −/− mice

Abstract

Flt3 ligand (FL) is an early acting co-stimulatory cytokine that regulates proliferation, differentiation and survival of hematopoietic stem and progenitor cells. Its receptor, Flt3/Flk2, belongs to the type III receptor tyrosine kinases (RTK) that also include the receptors for M-CSF, Steel factor, and PDGF. Although some post-receptor signaling events and substrate specificities of the Flt3 receptor have been characterized, involvement of the Jak/Stat pathway in Flt3 signaling has not been clearly studied. We found that FL selectively activated Stat5a, without activation of Janus kinases in Baf3/Flt3 cells, a murine IL-3-dependent hematopoietic cell line which stably expresses full-length human Flt3 receptor. FL did not activate Stats 1,2,3,5b, or 6. Activation of Stat5a required the kinase activity of Flt3. We have also examined whether FL upregulates bcl-xL, a STAT5 target gene. Expression of bcl-xL was up-regulated at both the mRNA and protein levels after FL stimulation of Baf3/Flt3 cells. To evaluate physiological relevance of these observations, we assessed responsiveness of myeloid progenitor cells from Stat 5a −/− mice and their littermate controls (+/+) to stimulation by a number of growth factors and co-stimulating molecules, alone and in combination. CFU-GM from Stat 5a −/− and +/+ mice responded similarly to stimulation by various concentrations of GM-CSF, M-CSF, G-CSF, or steel factor (SLF) and to synergism of SLF plus either GM-CSF, M-CSF or G-CSF. In contrast, CFU-GM from control, but not Stat 5a −/−, mice responded to FL and to the synergistic effects of FL with either GM-CSF, M-CSF or G-CSF. In a preliminary experiment, levels of bcl-xl were greatly reduced in Stat 5a −/− compared to +/+ cells. These results demonstrate that Stat5a is essential for the costimulatory effects of FL and suggest that a mechanism for the functional activity of FL is to upregulate expression of bcl-xL through activation of Stat5a.