Oxygen tension modulates the expression of cytokine receptors, transcription factors, and lineage-specific markers in cultured human megakaryocytes

Objective. We have recently reported that 20% O-2 significantly enhances to tal megakaryocyte (Mk) number, polyploidy, and proplatelet formation compar ed to 5% O-2 in culture. In order to further elucidate the regulatory role of pO(2) on megakaryocytopoiesis, we conducted a kinetic study of the expre ssion of surface markers CD41a and CD42a; receptors for thrombopoietin (TPO ), interleukin-3 (IL-3), and Flt3-ligand; the glutamate receptor of the N-m ethyl-D-aspartate subtype 1 (NMDAR1); and transcription factors GATA-1, NF- E2, and E2F-1. Materials and Methods. Mks were generated from mobilized peripheral blood ( PB) CD34(+) cells from normal donors in serum-free medium with TPO, IL-3, a nd Flt3-ligand at 20% and 5% O-2. Quantitative assessment of Mk surface rec eptors and nuclear transcription factors was performed using multiparameter flow cytometry. mRNA levels of the nuclear transcription factors GATA-1 an d NF-E2 were evaluated using RT-PCR. Results. The proportions of cells expressing the early Mk marker CD41a and the late Mk marker CD42a at day 15 were 4 and 5 times higher, respectively, at 20% O-2. CD41a and CD42a protein levels per cell were also higher at 20 % O-2. After day 5, c-Mpl (TPO receptor) generally followed similar kinetic s as CD41a. The proportion of IL-3 receptor (IL-3R)(++) Mks at day 5 was 1. 5 times higher at 5% O-2. The NMDAR1 protein previously known to be express ed by neuronal cells has recently been identified in Mks. NMDAR1 and the tr anscription factors were studied on days 6, 9, and II. NMDAR1 was expressed at a 1.5- to 1.8-fold higher level at 5% O-2. Twenty percent O-2 supported higher expression of the Mk-early and -late-maturation-specific transcript ion factors GATA-1 (1.2- to 2.2-fold higher) and NF-E2 (1.1- to 2.8-fold hi gher). This was consistent with RT-PCR data indicating the presence of high er levels of GATA-1 and NF-M mRNA at 20% O-2. E2F-1, a ubiquitously express ed cell cycle transcription factor, was expressed at a 1.5-fold higher leve l at 20% O-2 on day 6, but this difference did not persist by day 9. Conclusion. These findings demonstrate that cytokine receptors c-Mpl and IL -3R, and Mk differentiation-specific surface receptors CD41a, CD42a, and NM DAR1, are significantly modulated by pO(2), and suggest that one of the mec hanisms of enhanced maturation at 20% O-2 may involve regulation of transcr iption factors GATA-1 and NF-E2. (C) 2001 International Society for Experim ental Hematology. Published by Elsevier Science Inc.