Offermann M K, Zimring J, Mellits K H, Hagan M K, Shaw R, Medford R M, Mathews M B, Goodbourn S, Jagus R
Division of Hematology/Oncology, Emory University, Atlanta, USA.
Eur J Biochem. 1995 Aug 15;232(1):28-36. doi: 10.1111/j.1432-1033.1995.tb20777.x.
Double-stranded RNA (dsRNA) induces the vascular cell adhesion molecule VCAM-1 to high levels of expression in human umbilical vein endothelial (HUVE) cells. Although VCAM-1 is also induced by the cytokine interleukin 1 beta (IL-1 beta), activation of the dsRNA-activated protein kinase (PKR) occurs only in response to incubation with dsRNA but not with IL-1 beta. Incubation of HUVE cells with the synthetic dsRNA, poly (I).poly (C), activates PKR with increased autophosphorylation, increased phosphorylation of the translation factor eIF2 alpha, and increased activation of the transcription factor NF-kappa B. Promoter analysis in HUVE cells using a VCAM-1 promoter linked to CAT reporter gene demonstrates that poly (I).poly (C) responsiveness resides in the minimal VCAM-1 promoter that contains two NF-kappa B sites, and deletion of the NF-kappa B sites eliminates basal and poly (I).poly (C)-induced CAT activity, supporting the importance of NF-kappa B in the poly (I).poly (C)-mediated induction of VCAM-1. In vitro studies using purified reagents demonstrate that PKR is capable of phosphorylating I kappa B alpha (the inhibitory subunit of NF-kappa B) in a dsRNA-dependent manner. This suggests that phosphorylation of I kappa B alpha by PKR could be an initial step in the activation of NF-kappa B by dsRNA. NF-kappa B is also activated by IL-1 beta in HUVE cells, but this activation occurs without increased PKR autophosphorylation or eIF2 alpha phosphorylation. Poly (I).poly (C) induces VCAM-1 mRNA levels that are dramatically higher and sustained longer than levels induced by IL-1 beta. Although phosphorylation of eIF2 alpha interferes with protein translation, sufficient VCAM-1 mRNA translation occurs in response to poly (I).poly (C) to yield VCAM-1 protein levels that are similar to levels that are induced by IL-1 beta. This suggests that the higher, sustained VCAM-1 mRNA levels that occur in response to incubation with poly (I).poly (C) compensate for the partial translational block resulting from increased eIF2 alpha phosphorylation. These studies indicate that transcriptional and translational regulatory events that occur in response to activation of PKR by dsRNA are important in the regulation of VCAM-1 gene expression in HUVE cells.
双链RNA（dsRNA）可诱导人脐静脉内皮（HUVE）细胞中血管细胞黏附分子VCAM-1高水平表达。尽管细胞因子白细胞介素1β（IL-1β）也可诱导VCAM-1表达，但双链RNA激活蛋白激酶（PKR）仅在与dsRNA孵育时被激活，而与IL-1β孵育时则不会激活。用合成的dsRNA聚（I）·聚（C）孵育HUVE细胞，可通过增加自身磷酸化、增加翻译因子eIF2α的磷酸化以及增加转录因子NF-κB的激活来激活PKR。在HUVE细胞中使用与CAT报告基因相连的VCAM-1启动子进行启动子分析表明，聚（I）·聚（C）反应性存在于包含两个NF-κB位点的最小VCAM-1启动子中，删除NF-κB位点可消除基础和聚（I）·聚（C）诱导的CAT活性，这支持了NF-κB在聚（I）·聚（C）介导的VCAM-1诱导中的重要性。使用纯化试剂的体外研究表明，PKR能够以dsRNA依赖的方式磷酸化IκBα（NF-κB的抑制亚基）。这表明PKR对IκBα的磷酸化可能是dsRNA激活NF-κB的初始步骤。在HUVE细胞中，IL-1β也可激活NF-κB，但这种激活在PKR自身磷酸化或eIF2α磷酸化未增加的情况下发生。聚（I）·聚（C）诱导的VCAM-1 mRNA水平比IL-1β诱导的水平显著更高且持续时间更长。尽管eIF2α的磷酸化会干扰蛋白质翻译，但响应聚（I）·聚（C）仍会发生足够的VCAM-1 mRNA翻译，以产生与IL-1β诱导水平相似的VCAM-1蛋白水平。这表明，响应与聚（I）·聚（C）孵育而出现的更高、持续的VCAM-1 mRNA水平可补偿因eIF2α磷酸化增加导致的部分翻译阻滞。这些研究表明，dsRNA激活PKR后发生的转录和翻译调控事件在HUVE细胞中VCAM-1基因表达的调控中很重要。
