Damdinsuren Bazarragchaa, Dement-Brown Jessica, Li Huifang, Tolnay Mate
Office of Biotechnology Products, Center for Drug Evaluation and Research, US Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20993, USA.
Office of Biotechnology Products, Center for Drug Evaluation and Research, US Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20993, USA.
Mol Immunol. 2016 May;73:112-21. doi: 10.1016/j.molimm.2016.04.001. Epub 2016 Apr 8.
Fc receptor-like (FCRL) proteins are novel regulators of the B cell response to antigen. Human FCRL5 binds intact IgG and modifies the strength of antigen receptor (BCR) signaling. Altering FCRL5 expression could therefore regulate the B cell response to antigen. In this study, we found that FCRL5 expression is induced specifically upon BCR stimulation and dissected the molecular mechanism. FCRL5 mRNA and cell surface protein expression required prolonged BCR stimulation and de novo protein synthesis. Using chemical inhibitors and activators, we identified roles for several signaling pathways, indicating a complex mechanism. Specifically, the PI3K/AKT, JNK, PKC and IKK2-dependent classical NF-κB pathways were involved in induced FCRL5 expression. Furthermore, induced FCRL5 expression required elevation of intracellular Ca(++) and was partially blocked by cyclosporine A, a calcineurin inhibitor. The importance of the transcription factors NF-κB, NFAT and CREB-binding protein was revealed based on sensitivity to inhibitors. Using reporter gene assays, we showed that the core FCRL5 promoter was sufficient to drive induced gene expression. Mutations of two predicted NF-κB sites or an NFAT site in the core promoter abrogated induced gene expression, suggesting direct regulation of the FCRL5 gene by NF-κB and NFAT. In support, we detected binding of NF-κB and NFAT family proteins to oligonucleotides corresponding to the predicted sites. We propose that the identified intricate mechanism serves to ensure that FCRL5 is expressed on B cells at a precise time following antigen encounter, with potential implications regarding regulation of the B cell response.
Fc受体样（FCRL）蛋白是B细胞对抗原反应的新型调节因子。人类FCRL5结合完整的IgG并改变抗原受体（BCR）信号传导的强度。因此，改变FCRL5的表达可以调节B细胞对抗原的反应。在本研究中，我们发现FCRL5的表达在BCR刺激后特异性诱导，并剖析了其分子机制。FCRL5 mRNA和细胞表面蛋白的表达需要长时间的BCR刺激和从头合成蛋白质。使用化学抑制剂和激活剂，我们确定了几种信号通路的作用，表明其机制复杂。具体而言，PI3K/AKT、JNK、PKC和IKK2依赖的经典NF-κB通路参与了FCRL5的诱导表达。此外，FCRL5的诱导表达需要细胞内Ca(++)升高，并被钙调神经磷酸酶抑制剂环孢素A部分阻断。基于对抑制剂的敏感性揭示了转录因子NF-κB、NFAT和CREB结合蛋白的重要性。使用报告基因测定，我们表明核心FCRL5启动子足以驱动诱导的基因表达。核心启动子中两个预测的NF-κB位点或一个NFAT位点的突变消除了诱导的基因表达，表明NF-κB和NFAT对FCRL5基因的直接调控。作为支持，我们检测到NF-κB和NFAT家族蛋白与对应于预测位点的寡核苷酸的结合。我们提出，所确定的复杂机制有助于确保FCRL5在抗原接触后的精确时间在B细胞上表达，这对B细胞反应的调节具有潜在意义。
