Rubio M A, Lopez-Rodriguez C, Nueda A, Aller P, Armesilla A L, Vega M A, Corbí A L
Hospital de la Princesa, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
Blood. 1995 Nov 15;86(10):3715-24.
To analyze the activity of the CD11c promoter during myeloid differentiation without the limitations of transient expression systems, we have stably transfected the myeloid U937 cell line with the pCD11C361-Luc plasmid, in which the expression of the firefly luciferase cDNA is driven by the CD11c promoter region -361/+43, previously shown to confer myeloid specificity to reporter genes. The stable transfectants (U937-C361) retained the ability to differentiate in response to phorbol-ester (PMA), sodium butyrate (SB), granulocyte-macrophage colony-stimulating factor (GM-CSF), and other differentiating agents. U937-C361 differentiation correlated with increased cellular luciferase levels, showing the inducibility of the CD11c promoter during myeloid differentiation and establishing the U937-C361 cells as a suitable system for studying the myeloid differentiation-inducing capacity of cytokines, growth, factors, and other biological response modifiers. Unexpectedly, the inducibility of the CD11c gene promoter showed distinct kinetics and magnitude on the PMA-, SB-, GM-CSF-triggered differentiation. Moreover, SB synergized with either PMA or GM-CSF in enhancing both the CD11c promoter activity and the cell surface expression of p150,95 on differentiating U937 cells. Furthermore, we showed the existence of a c-Myb-binding site at -85, the importance of the -99/-61 region in the CD11c promoter inducibility during PMA- or SB-triggered differentiation, and the dependency of the GM-CSF and PMA responsiveness of the CD11c promoter on an intact AP-1-binding site located at -60. These results, together with the lack of functional effect of mutations disrupting the Sp1-and Myb-binding sites within the proximal region of the CD11c promoter, indicate that the myeloid differentiation pathways indicated by SB and phorbol esters (or GM-CSF) activate a distinct set of transcription factors and show that the myeloid differentiation-inducibility of the CD11c gene maps to the -99/-53 proximal region of the promoter.
为了在不受瞬时表达系统限制的情况下分析髓系分化过程中CD11c启动子的活性，我们用pCD11C361-Luc质粒稳定转染了髓系U937细胞系，其中萤火虫荧光素酶cDNA的表达由CD11c启动子区域-361/+43驱动，该区域先前已证明能赋予报告基因髓系特异性。稳定转染子（U937-C361）保留了对佛波酯（PMA）、丁酸钠（SB）、粒细胞-巨噬细胞集落刺激因子（GM-CSF）和其他分化剂作出分化反应的能力。U937-C361的分化与细胞荧光素酶水平升高相关，表明CD11c启动子在髓系分化过程中具有诱导性，并将U937-C361细胞确立为研究细胞因子、生长因子和其他生物反应调节剂的髓系分化诱导能力的合适系统。出乎意料的是，CD11c基因启动子的诱导性在PMA、SB、GM-CSF触发的分化过程中表现出不同的动力学和幅度。此外，SB与PMA或GM-CSF协同作用，增强了分化中的U937细胞的CD11c启动子活性和p150,95的细胞表面表达。此外，我们证明了在-85处存在一个c-Myb结合位点，-99/-61区域在PMA或SB触发的分化过程中对CD11c启动子诱导性的重要性，以及CD11c启动子对GM-CSF和PMA的反应性依赖于位于-60处的完整AP-1结合位点。这些结果，连同破坏CD11c启动子近端区域内Sp1和Myb结合位点的突变缺乏功能效应，表明SB和佛波酯（或GM-CSF）指示的髓系分化途径激活了一组不同的转录因子，并表明CD11c基因的髓系分化诱导性定位于启动子的-99/-53近端区域。
