Wierenga Albertus T J, Schepers Hein, Moore Malcolm A S, Vellenga Edo, Schuringa Jan Jacob
Department of Hematology, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands.
Blood. 2006 Jun 1;107(11):4326-33. doi: 10.1182/blood-2005-11-4608. Epub 2006 Feb 2.
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/EBPalpha as a critical component that is down-regulated by STAT5. Microarray and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on STAT5A(16)-transduced CD34(+) cells identified C/EBPalpha as the most prominently down-regulated gene. To determine the cell-biological relevance of these observations, a 4-OHT-inducible C/EBPalpha-ER protein was co-expressed with the STAT5A(16) mutant in CB CD34(+) cells using a retroviral approach. Re-expression of C/EBPalpha in STAT5A(16) cells resulted in a marked restoration of myelopoiesis. The proliferative advantage imposed on CD34(+) cells by STAT5A(16) depended on the down-modulation of C/EBPalpha, as reintroduction of C/EBPalpha induced a quick cell-cycle arrest and the onset of myeloid differentiation. Long-term culture-initiating cell (LTC-IC) frequencies were elevated from 0.8% +/- 0.6% to 7.8% +/- 1.9% by STAT5A(16) as compared with controls, but these elevated LTC-IC frequencies were strongly reduced upon re-introduction of C/EBPalpha in STAT5A(16) cells, and no second cobble-stone 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/EBPalpha was co-expressed in STAT5A(1*6) cells. Our data indicate that down-modulation of C/EBPalpha is a prerequisite for STAT5-induced effects on self-renewal and myelopoiesis.
此前，我们证明在人脐带血（CB）来源的干/祖细胞中强制激活信号转导及转录激活因子5（STAT5A）会导致自我更新增强和髓系造血受损。本研究确定C/EBPα是一个受STAT5下调的关键成分。对转导了STAT5A(16)的CD34(+)细胞进行微阵列和逆转录聚合酶链反应（RT-PCR）分析，确定C/EBPα是下调最显著的基因。为了确定这些观察结果在细胞生物学上的相关性，使用逆转录病毒方法在CB CD34(+)细胞中与STAT5A(16)突变体共表达一种4-羟基他莫昔芬（4-OHT）诱导型C/EBPα-ER蛋白。在STAT5A(16)细胞中重新表达C/EBPα导致髓系造血显著恢复。STAT5A(16)赋予CD34(+)细胞的增殖优势依赖于C/EBPα的下调，因为重新引入C/EBPα会诱导细胞周期快速停滞并启动髓系分化。与对照组相比，STAT5A(16)使长期培养起始细胞（LTC-IC）频率从0.8%±0.6%提高到7.8%±1.9%，但在STAT5A(16)细胞中重新引入C/EBPα后，这些升高的LTC-IC频率大幅降低，并且双转导细胞无法产生第二代鹅卵石区形成细胞（CAFC）。祖细胞计数显示，当在STAT5A(1*6)细胞中共表达C/EBPα时，集落形成细胞（CFC）数量减少了20多倍。我们的数据表明，C/EBPα的下调是STAT5诱导自我更新和髓系造血作用的先决条件。
