Agrawal Alok, Samols David, Kushner Irving
Department of Pharmacology, East Tennessee State University, Johnson City, TN 37614, USA.
Mol Immunol. 2003 Oct;40(6):373-80. doi: 10.1016/s0161-5890(03)00148-2.
Induction of C-reactive protein (CRP) synthesis in hepatocytes by cytokines occurs at the transcriptional level. In Hep3B cells, the transcription factors C/EBPbeta, STAT3, and Rel p50 have been shown to participate in this process. A C/EBP binding site centered at -53 and an overlapping nonconsensus kappaB site on the promoter are critical for CRP expression. We have previously found that an oligonucleotide containing a kappaB site diminished binding of C/EBPbeta to the C/EBP site, suggesting that unidentified Rel proteins present in Hep3B nuclei facilitate the formation of C/EBPbeta-complexes. The current studies were undertaken to determine which of the five Rel proteins, p50/p65/p52/c-Rel/RelB, play such a role. Mutation of the nonconsensus kappaB site did not abolish binding of C/EBPbeta to its binding site, indicating that this site was not necessary for the formation of C/EBPbeta-complexes. Depletion of Rel proteins from Hep3B nuclei led to decreased formation of C/EBPbeta-complexes on a CRP promoter-derived oligonucleotide that contained only the intact C/EBP binding site but not the nonconsensus kappaB site. This finding indicates that Rel proteins are involved in the binding of C/EBPbeta to its binding site by a kappaB site-independent mechanism. Electrophoretic mobility shift assays (EMSAs) revealed that it was c-Rel that facilitated formation of C/EBPbeta-complexes and that c-Rel bound directly to C/EBPbeta-complexes formed on the C/EBP site. Cotransfection of c-Rel enhanced the induction of CRP promoter-driven luciferase activity and enhanced endogenous CRP expression in cells transfected with C/EBPbeta. We conclude that c-Rel regulates CRP expression without the requirement of binding to a kappaB site, and binds directly to C/EBPbeta to facilitate the binding of C/EBPbeta to the CRP promoter.
细胞因子诱导肝细胞合成C反应蛋白（CRP）发生在转录水平。在Hep3B细胞中，转录因子C/EBPβ、STAT3和Rel p50已被证明参与此过程。启动子上以-53为中心的C/EBP结合位点和一个重叠的非典型κB位点对CRP表达至关重要。我们先前发现，含有κB位点的寡核苷酸会减少C/EBPβ与C/EBP位点的结合，这表明Hep3B细胞核中存在的未鉴定Rel蛋白促进了C/EBPβ复合物的形成。当前的研究旨在确定五种Rel蛋白（p50/p65/p52/c-Rel/RelB）中哪一种发挥这样的作用。非典型κB位点的突变并未消除C/EBPβ与其结合位点的结合，表明该位点对于C/EBPβ复合物的形成并非必需。从Hep3B细胞核中耗尽Rel蛋白导致在仅包含完整C/EBP结合位点而不包含非典型κB位点的CRP启动子衍生寡核苷酸上C/EBPβ复合物的形成减少。这一发现表明Rel蛋白通过一种不依赖κB位点的机制参与C/EBPβ与其结合位点的结合。电泳迁移率变动分析（EMSA）显示，促进C/EBPβ复合物形成的是c-Rel，并且c-Rel直接结合到在C/EBP位点形成的C/EBPβ复合物上。共转染c-Rel增强了CRP启动子驱动的荧光素酶活性的诱导，并增强了用C/EBPβ转染的细胞中内源性CRP的表达。我们得出结论，c-Rel调节CRP表达而无需与κB位点结合，并直接与C/EBPβ结合以促进C/EBPβ与CRP启动子的结合。
