Analysis of mechanism of transcriptional regulation of retinoic acid-responsive genes during the differentiation of human leukemia cells.

我们以前报道过全反式维甲酸（ATRA）和粒细胞巨噬细胞集落刺激因子（GM-CSF）协同诱导人髓性白血病ML-1细胞向粒细胞分化。为了研究分化过程中视黄酸响应基因的转录调节机制，我们使用北方或西方印迹分析检测了ATRA和/或GM-CSF处理的ML-1细胞中响应基因、视黄酸受体（RARs）、类维生素A X受体（RXR）和辅因子的表达。我们首次发现ATRA和GM-CSF协同诱导C/EBP ε mRNA的表达。我们接下来检测到GM-CSF对RAR α mRNA和蛋白表达的上调，以及两种试剂对类固醇受体共激活因子-3（SRC 3/AIB 1）表达的协同增加，所述类固醇受体共激活因子-3（SRC 3/AIB 1）被称为包括RAR、mRNA和蛋白的核受体的激活因子。这些变化也在其他髓性白血病细胞系（THP-1和KG-1）中检测到，这些细胞系显示出与ML-1细胞对ATRA和GM-CSF的反应相似的协同作用。此外，我们揭示了调节组蛋白精氨酸甲基化的基因（PRMTs和PAD 4）表达的改变。为了研究这些改变是否与分化相关，我们检测了SRC 3 siRNA对ATRA和GM-CSF处理的ML-1细胞分化的影响。SRC 3 siRNA对诱导的SRC 3/AIB 1基因表达的抑制抑制了由两种试剂处理诱导的NBT还原活性。上述结果提示，ATRA和GM-CSF协同诱导分化可能通过上调SRC 3/AIB 1基因的表达，提高细胞对维甲酸的敏感性。

We reported previously that treatment with all-trans retinoic acid (ATRA) and granulocyte macrophage colony-stimulating factor (GM-CSF) induces synergistically differentiation of human myeloblastic leukemia ML-1 cells to granulocytes. To investigate the mechanism of transcriptional regulation of retinoic acid-responsive genes during the differentiation, we examined expression of the responsive genes, retinoic acid receptors (RARs), retinoid X receptors (RXRs) and the cofactors in ML-1 cells treated with ATRA and/or GM-CSF using Northern or Western blot analysis. We first showed that expression of C/EBP ε mRNA was induced synergistically by treatment with ATRA and GM-CSF. We next detected up-regulation of expression of RAR a mRNA and protein by GM-CSF and synergistic increase of expression of steroid receptor coactivator-3 (SRC3/AIB 1), which is known as activator of nuclear receptors including RARs, mRNA and protein by both reagents. These changes were also detected in other myeloid leukemia cell lines (THP-1 and KG-1) that showed a synergistic effect similar to that seen in ML-1 cells in response to ATRA and GM-CSF. Furthermore, we revealed alteration of expression of genes regulating histone arginine methylation (PRMTs and PAD4). To investigate whether these alterations are associated with the differentiation, we examined effect of SRC3 siRNA on the differentiation in ML-1 cells treated with ATRA and GM-CSF. The inhibition of induced SRC3/AIB1 gene expression by SRC3 siRNA suppressed NBT reducing activity induced by treatment with both reagents. These results suggest that the synergistic induction of differentiation by ATRA and GM-CSF is associated with the increase of sensitiveness of cells to retinoic acid via up-regulation of expression of SRC3/AIB1 gene.