Mignacca R C, Lee H J, Kwon E M, Sakamoto K M
Gwynne Hazen Cherry Memorial Laboratories, Department of Pediatrics, UCLA School of Medicine 90024-1752, USA.
Blood. 1996 Aug 1;88(3):848-54.
Studies with the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin-3 (IL-3) fusion protein, PIXY321, demonstrated enhanced biological activity of this molecule in comparison with GM-CSF or IL-3 alone or in combination. Experiments were performed to study the mechanisms resulting in PIXY321-induced egr-1 expression in human myeloid leukemic cells (TF-1). Transfections of egr-1 promoter constructs revealed that PIXY321 stimulation resulted in fourfold induction of the -116 and -600 nucleotide (nt) constructs. We transfected a -116 nt construct containing a deletion of the cyclic AMP response element (CRE) or mutation in the serum response element (SRE) and demonstrated that both the SRE and CRE are necessary for maximal induction. However, PIXY321 stimulation resulted in 2.5-fold induction of a SRE-CRE-containing construct (P < .05), suggesting that the SRE and CRE are sufficient for PIXY321 responsiveness. Electrophoretic mobility shift assays (EMSA) revealed that the CRE binding protein (CREB) was phosphorylated on serine 133 in PIXY321-stimulated but not -unstimulated extracts from cells cultured in GM-CSF. By Western analysis and EMSA, CREB was constitutively phosphorylated in TF-1 cells grown on PIXY321 before growth factor and serum starvation. However, in TF-1 cells grown on GM-CSF before starvation, CREB phosphorylation was observed 10 minutes after PIXY321 stimulation. Further-more, ENSAs with PIXY321-stimulated and -unstimulated extracts demonstrated the presence of specific proteins that recognize the SRE. Our data demonstrate that transcriptional regulation of egr-1 by PIXY321 is mediated by the CRE and SRE.
对粒细胞巨噬细胞集落刺激因子（GM-CSF）/白细胞介素-3（IL-3）融合蛋白PIXY321的研究表明，与单独的GM-CSF或IL-3或两者联合使用相比，该分子具有增强的生物学活性。进行实验以研究导致PIXY321诱导人髓系白血病细胞（TF-1）中早期生长反应基因-1（egr-1）表达的机制。egr-1启动子构建体的转染显示，PIXY321刺激导致-116和-600核苷酸（nt）构建体诱导四倍。我们转染了一个缺失环磷酸腺苷反应元件（CRE）或血清反应元件（SRE）发生突变的-116 nt构建体，并证明SRE和CRE对于最大诱导都是必需的。然而，PIXY321刺激导致含SRE-CRE的构建体诱导2.5倍（P <.05），表明SRE和CRE足以实现PIXY321反应性。电泳迁移率变动分析（EMSA）显示，在GM-CSF中培养的细胞的PIXY321刺激但未刺激的提取物中，CRE结合蛋白（CREB）在丝氨酸133处被磷酸化。通过蛋白质印迹分析和EMSA，在生长因子和血清饥饿之前在PIXY321上生长的TF-1细胞中CREB被组成型磷酸化。然而，在饥饿前在GM-CSF上生长的TF-1细胞中，PIXY321刺激后10分钟观察到CREB磷酸化。此外，用PIXY321刺激和未刺激的提取物进行的EMSA显示存在识别SRE的特异性蛋白质。我们的数据表明，PIXY321对egr-1的转录调控是由CRE和SRE介导的。
