Derigs H G, Reifel-Miller A, Kaushansky K, Hromas R A, Boswell H S
Division of Hematology/Oncology, Indiana University School of Medicine, Indianapolis.
Exp Hematol. 1994 Aug;22(9):924-32.
We have reported modulation, by cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) and by hormonal cyclic-adenosine-monophosphate (cAMP) agonists, of hematopoietic growth factor production in the murine marrow adherent cell line +/+(-)1.LDA11. Previously, we reported that increased intracellular cAMP levels inhibited bioactive granulocyte-macrophage colony-stimulatory factor (GM-CSF) production stimulated by IL-1 or by the synergistic stimulus of IL-1 plus TNF-alpha. On the other hand, increased intracellular cAMP stimulated IL-6 synthesis in +/+(-)1.LDA11 cells. In addition, cAMP was additive with either IL-1 or IL-1 plus TNF-alpha in inducing production of soluble IL-6. In the present study, these observations were pursued mechanistically at the level of messenger RNA (mRNA) production. Northern blot analysis of steady-state mRNA for GM-CSF revealed induction by treatment of +/+(-)1.LDA11 cells with IL-1 or with TNF-alpha. The combined stimulation by IL-1 plus TNF-alpha resulted in supra-additive increases in GM-CSF expression by +/+(-)1.LDA11. Addition to stromal cells of the soluble cAMP agonist 8-bromo-cAMP (8BrcAMP) at 0.5 to 1 mM stimulated IL-6 mRNA expression acting alone, and it was additive with IL-1 or IL-1 plus TNF-alpha in stimulating IL-6 expression. On the other hand, 8BrcAMP inhibited GM-CSF mRNA expression stimulated by IL-1 or IL-1 plus TNF-alpha. Inhibition of GM-CSF mRNA by 8BrcAMP was time-dependent, starting 120 to 180 minutes posttreatment. In addition, inhibition of GM-CSF transcript expression in +/+(-)1.LDA11 by 8BrcAMP required the expression of a labile protein. Nuclear run-on assays revealed that GM-CSF and IL-6 genes were transcriptionally induced in +/+(-)1.LDA11 by incubation with IL-1 plus TNF-alpha. IL-6 transcription was further enhanced by 8BrcAMP co-incubation. More sensitive experiments using a luciferase reporter vector containing the GM-CSF promoter region were necessary to convincingly establish the role of TNF-alpha and 8BrcAMP on transcriptional induction of the GM-CSF gene in +/+(-)1.LDA11 stromal cells. Considering these results and an effect of 8BrcAMP on decreasing GM-CSF transcript stability in actinomycin-D (act-D) decay experiments, we conclude that the inhibitory effect of 8BrcAMP on GM-CSF expression is exerted at the posttranscriptional level. These data demonstrate that the intracellular level of cAMP has an important discriminatory role on expression of the cytokines GM-CSF and IL-6 in a model stromal cell line.
我们曾报道，细胞因子白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）以及激素环磷酸腺苷（cAMP）激动剂可调节小鼠骨髓贴壁细胞系 +/+(-)1.LDA11 中造血生长因子的产生。此前，我们报道细胞内 cAMP 水平升高会抑制由 IL-1 或 IL-1 加 TNF-α 的协同刺激所诱导的生物活性粒细胞-巨噬细胞集落刺激因子（GM-CSF）的产生。另一方面，细胞内 cAMP 水平升高会刺激 +/+(-)1.LDA11 细胞中 IL-6 的合成。此外，在诱导可溶性 IL-6 的产生方面，cAMP 与 IL-1 或 IL-1 加 TNF-α 具有相加作用。在本研究中，从信使核糖核酸（mRNA）产生水平对这些观察结果进行了机制研究。对 GM-CSF 的稳态 mRNA 进行的 Northern 印迹分析显示，用 IL-1 或 TNF-α 处理 +/+(-)1.LDA11 细胞可诱导其产生。IL-1 加 TNF-α 的联合刺激导致 +/+(-)1.LDA11 细胞中 GM-CSF 的表达出现超相加性增加。向基质细胞中添加 0.5 至 1 mM 的可溶性 cAMP 激动剂 8-溴环磷酸腺苷（8BrcAMP）可单独刺激 IL-6 mRNA 的表达，并且在刺激 IL-6 表达方面与 IL-1 或 IL-1 加 TNF-α 具有相加作用。另一方面，8BrcAMP 抑制由 IL-1 或 IL-1 加 TNF-α 刺激产生的 GM-CSF mRNA 的表达。8BrcAMP 对 GM-CSF mRNA 的抑制作用具有时间依赖性，在处理后 120 至 180 分钟开始。此外，8BrcAMP 对 +/+(-)1.LDA11 细胞中 GM-CSF 转录本表达的抑制需要一种不稳定蛋白的表达。核转录分析显示，通过与 IL-1 加 TNF-α 孵育，GM-CSF 和 IL-6 基因在 +/+(-)1.LDA11 细胞中被转录诱导。8BrcAMP 共同孵育可进一步增强 IL-6 的转录。为了令人信服地确定 TNF-α 和 8BrcAMP 对 +/+(-)1.LDA11 基质细胞中 GM-CSF 基因转录诱导的作用，需要使用含有 GM-CSF 启动子区域的荧光素酶报告载体进行更灵敏的实验。考虑到这些结果以及在放线菌素-D（act-D）降解实验中 8BrcAMP 对降低 GM-CSF 转录本稳定性的作用，我们得出结论，8BrcAMP 对 GM-CSF 表达的抑制作用是在转录后水平发挥的。这些数据表明，在一个模型基质细胞系中，细胞内 cAMP 水平对细胞因子 GM-CSF 和 IL-6 的表达具有重要的区分作用。
