Alteration of c-mpl-mediated signal transduction in CD34(+) cells from patients with myelodysplastic syndromes.

Abstract

Megakaryocytic differentiation is frequently defective in patients with myelodysplastic syndromes (MDS). As underlying mechanisms, deregulated thrombopoietin receptor (c-mpl)-mediated signaling pathways have been suggested. This study therefore examined whether the impaired signaling in MDS and AML cells includes alterations of c-mpl itself or postsignaling events. Bone marrow-derived CD34(+) cells from healthy donors, patients with MDS (RA, RAEB-T), and patients with AML after MDS were isolated by MACS. Expression of c-mpl cDNA was studied by RT-PCR. Thrombopoietin dependent activation of STAT proteins and MAP Kinase p42(erk-2)/44(erk-1) was analyzed by Western blot. Both splicing isoforms of c-mpl (c-mpl-p and c-mpl-k) were expressed in all of the CD34(+) cells examined. Analysis of the c-mpl cDNA revealed no sequence abnormality. We show c-mpl dependent activation of the transcription factors STAT3 and STAT5 as well as MAP Kinase p42(erk-2)/44(erk-1) in CD34(+) cells from healthy individuals. Cells derived from RA patients revealed low basal levels of phosphorylated STAT3 and STAT5 molecules. This phosphorylation was enhanced by stimulation with recombinant thrombopoietin (PEG-rHuMGDF). STAT1 failed to be activated by PEG-rHuMGDF in CD34(+) cells from healthy donors as well as from patients with MDS. In RAEB-T and AML M7 the constitutive expression levels of STAT1, 3, 5, and MAPK were markedly upregulated, resulting in a strong activation of STAT3 and 5 by PEG-rHuMGDF. Despite its high expression, the level of MAPK phosphorylation was not increased in RA or RAEB-T compared to the normal control, and was completely undetectable in AML M7. These results suggest that the defective megakaryopoiesis in MDS is not caused by a lack of c-mpl and that STAT3 and STAT5 may contribute to the malignant phenotype of the leukemic cells.