FLT3 Ligand Stimulation Research Articles

Flt3 Ligand Induces Tyrosine Phosphorylation of Gab1 and Gab2 and Their Association with Shp-2, Grb2, and PI3 Kinase

The receptor tyrosine kinase Flt3 has been shown to play an important role in proliferation, differentiation, and survival of hematopoietic stem and progenitor cells. Although some postreceptor signaling events of Flt3 have been characterized, the involvement of Gab family proteins in Flt3 signaling is not known. In this study, we show that both Gab1 and Gab2 are rapidly tyrosine phosphorylated after Flt3 ligand stimulation of Flt3 ligand-responsive cells. They interact with tyrosine-phosphorylated Shp-2, p85, Grb2, and Shc. The results suggest that Gab proteins are engaged in Flt3 signaling to mediate downstream activation of Shp-2 and PI3 kinase pathways and possibly the Ras/Raf/MAPK pathway.

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

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.

Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lines.

We have recently identified an internal tandem duplication of the human Flt3 gene in approximately 20% of acute myeloid leukemia (AML) cases. In the present study, the wild-type and the mutant Flt3 genes were transfected into two IL-3-dependent cell lines, 32D and BA/F3 cells. Mutant Flt3-transfected cells exhibited autonomous growth while wild-type Flt3-transfected cells with the continuous stimulation of Flt3 ligand exhibited a minimal proliferation. Cells expressing mutant Flt3 showed constitutive activation of STAT5 and MAP kinase. In contrast, Flt3 ligand stimulation caused rapid activation of MAP kinase but not STAT5 in cells expressing wild-type Flt3. Finally, we found constitutive activation of MAP kinase and STAT5 in all clinical samples of AML patients with mutant Flt3. Our study shows the significance of internal tandem duplication of Flt3 receptors for leukemia cell expansion.

Effects of FLT3 Ligand on Proliferation and Survival of Myeloid Leukemia Cells

FLT3 ligand (FL) acting through its tyrosine kinase receptor FLT3 has pleiotropic and potent effects on hematopoietic cells. The well-described involvement of this ligand-receptor pair in physiological hematopoiesis raised the question whether FL and FLT3 also play a role in the pathobiology of leukemia. Following the early discovery of high receptor expression by myeloid leukemia cells, several investigators have focused their attention on these cells, both primary acute myeloid leukemia (AML) cells and continuous human myeloid leukemia cell lines. Regardless of the morphological FAB subtype, the vast majority of AML cases were FLT3-positive both at the mRNA and protein level; among the myeloid cell lines, predominantly the monocytic and myelocytic cell lines were FLT3-positive whereas the erythrocytic and megakaryocytic cell lines were FLT3-negative. Virtually all cell lines studied expressed FL transcripts; the finding that some cell lines displayed both ligand and receptor indicates the possibility of autocrine, intracrine or paracrine stimulatory loops. In vitro growth assays showed that FL caused a proliferative response in a high percentage of AML cases. Only constitutively growth factor-dependent myelocytic cell lines increased their proliferation upon incubation with FL whereas all growth factor-independent cell lines were refractory to FL stimulation. Combinations of FL with various cytokines (e.g. G-CSF, GM-CSF, IL-3, M-CSF, PIXY-321, SCF) had synergistic or additive mitogenic effects. Finally, FL had significant anti-apoptotic, survival-promoting effects on primary AML cells and myeloid cell lines under serum-free culture conditions. On the strength of the above findings, it can be concluded that the FL-FLT3 signaling system may play a certain, albeit probably not causal role in the development of human leukemias. Dissection of the exact molecular pathways that lead to proliferation and/or anti-apoptosis of myeloid leukemia cells as well as the detailed elucidation of the possible contribution of the FL-FLT3 genes to leukemogenesis remain future challenges.