| Abstract: |
Previously, we demonstrated that enforced activation of signal transducer and activator of transcription 5 (STAT5A) in human cord blood (CB)-derived stem/progenitor cells results in enhanced self-renewal and impaired myelopoiesis. The present study identifies C/EBP alpha as a critical component that is down-regulated by STAT5. Microarray and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on STAT5A(1.6)-transduced CD34(+) cells identified C/EBP alpha as the most prominently down-regulated gene. To determine the cell-biological relevance of these observations, a 4-OHT-inducible C/EBP alpha-ER protein was co-expressed with the STAT5A(1*6) mutant in CB COW cells using a retroviral approach. Re-expression of C/EBP alpha in STAT5A(1*6) cells resulted in a marked restoration of myelopoiesis. The proliferative advantage imposed on CD34(+) cells by STAT5A(1*6) depended on the down-modulation of C/EBP alpha, as reintroduction of C/EBPa induced a quick cell-cycle arrest and the onset of myeloid differentiation. Longterm culture-initiating cell (LTC-IC) frequencies were elevated from 0.8% +/- 0.6% to 7.8% +/- 1.9% by STAT5A(1*6) as compared with controls, but these elevated LTC-IC frequencies were strongly reduced upon re-introduction of C/EBP alpha in STAT5A(1*6) cells, and no second cobblestone area-forming cells (CAFCs) could be generated from double-transduced cells. Enumeration of progenitors revealed that the number of colony-forming cells (CFCs) was reduced more than 20-fold when C/EBP alpha was co-expressed in STAT5A(1*6) cells. Our data indicate that down-modulation of C/EBP alpha is a prerequisite for STAT5-induced effects on self-renewal and myelopoiesis. |
