GATA and BCLxl Downregulation in Erythropoiesis during In Vitro Lineage Specific Differentiation of MDS Hematopoietic Progenitor Cells Is Not Induced by Activated Notch Pathway.

Abstract

Notch signals have recently been shown to inhibit erythroid and megakaryocytic differentiation of hematopoietic progenitor cells. In myelodysplastic syndrome (MDS) its role in dyserythropoiesis has not been fully elucidated. Therefore we asked whether dysregulation of Notch pathway elements might be associated with impaired GATA1 and BCLxl expression and ineffective erythropoiesis being a hallmark of MDS hematopoiesis. We have generated an in-vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ bone marrow cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO and TPO. Cell harvest was at days 0, 4, 7 and 11. Expression of GATA1, BCLxl, DLK1, Notch1, HES1 and HERP2 was measured by real time RT-PCR (qPCR). RNA expression of GATA1 and of BCLxl was steadily upregulated, particularly during late normal erythropoiesis. During normal megakaryopoiesis expression of both genes was up to 50 times lower as compared to normal erythropoiesis. In contrast, during MDS erythropoiesis a loss of typical late upregulation of GATA1 and BCLxl was observed. DLK1 expression during erythropoiesis showed increased expression particularly in high risk MDS vs. normal controls. Expression of HES1 was increasing during the course of normal erythropoietic and megakaryopoietic differentiation but not in lineage specific cells from MDS patients. In conclusion our data show that the central erythropoietic transcription factor GATA1 and the associated antiapoptotic molecule BCLxl are markedly downregulated during MDS erythropoiesis which may contribute to the ineffective erythropoiesis seen in this disease. Increased DLK1 expression in differentiated stem cells from high risk MDS patients was seen. However, an upregulation of the Notch pathway leading to increased expression of the GATA1 repressor HES1 could not be detected.