Eilers A, Seegert D, Schindler C, Baccarini M, Decker T
Fraunhofer Institute for Toxicology and Molecular Biology, Hannover, Germany.
Mol Cell Biol. 1993 Jun;13(6):3245-54. doi: 10.1128/mcb.13.6.3245-3254.1993.
Gamma interferon activation factor (GAF) rapidly induces transcriptional activation of gamma interferon (IFN-gamma)-responsive genes. Conversion of the GAF from a latent cytoplasmic to an activated, DNA-binding form is an immediate step in the cellular response to IFN-gamma. The amount of IFN-gamma-activated GAF, measured by exonuclease III protection or gel shift assays, increased strongly upon monocytic differentiation of U937 cells. Activated GAF contained the IFN-responsive 91-kDa protein as its DNA-binding activity in gel shift or exonuclease III assays could be inhibited through direct addition of specific antiserum, and it was not present in p91-immunodepleted extracts. There was a differentiation-induced increase in the amount of nonphosphorylated (latent) p91. Transcription rate measurement demonstrated a strong induction of the p91 gene during monocytic differentiation of U937 cells. The amount of p91 which was rapidly phosphorylated in response to IFN-gamma was found to be much higher in the differentiated cells and suggested a differentiation-controlled increase in the signaling leading to p91 phosphorylation. Concomitantly with a better GAF response, transcriptional activation of IFN-gamma-induced genes and the expression of GAF-dependent, transfected reporter plasmids increased in differentiated U937 monocytes. The promonocyte-monocyte transition also affected the IFN-alpha-responsive transcription factor ISGF-3. Differentiated U937 cells contained more of both the alpha-component p91 and the gamma-component p48, which constitutes the DNA-binding subunit of the complex. Our study thus provides evidence that the synthesis of specific transcription factors can be a regulated event to control the cytokine responsiveness of cells during development.
γ干扰素激活因子（GAF）可迅速诱导γ干扰素（IFN-γ）反应性基因的转录激活。GAF从潜在的细胞质形式转变为活化的、具有DNA结合能力的形式，是细胞对IFN-γ反应的第一步。通过核酸外切酶III保护或凝胶迁移分析测定，U937细胞单核细胞分化时，IFN-γ激活的GAF量显著增加。在凝胶迁移或核酸外切酶III分析中，活化的GAF含有IFN反应性91 kDa蛋白，其DNA结合活性可通过直接添加特异性抗血清而被抑制，且在p91免疫耗尽的提取物中不存在。未磷酸化（潜在）的p91量在分化过程中有所增加。转录速率测量表明，在U937细胞单核细胞分化过程中，p91基因被强烈诱导。发现分化细胞中对IFN-γ快速磷酸化的p91量要高得多，这表明在导致p91磷酸化的信号传导中，分化控制的增加。与更好的GAF反应同时，分化的U937单核细胞中，IFN-γ诱导基因的转录激活以及GAF依赖性转染报告质粒增加。前单核细胞向单核细胞的转变也影响了IFN-α反应性转录因子ISGF-3。分化的U937细胞中，构成复合物DNA结合亚基的α组分p91和γ组分p48都更多。因此，我们的研究提供了证据，表明特定转录因子的合成可能是一个受调控的事件，以控制细胞在发育过程中的细胞因子反应性。
