c-Rel Deficiency Increases Caspase-4 Expression and Leads to ER Stress and Necrosis in EBV-Transformed Cells

LMP1-mediated activation of nuclear factor of kappaB (NF-κB) is critical for the ligand independent proliferation and cell survival of in vitro EBV-transformed lymphoblastoid cell lines (LCLs). Previous experiments revealed that a majority of LMP1-dependent responses are regulated by NF-κB. However, the extent that individual NF-κB family members are required for these responses, in particular, c-Rel, whose expression is restricted to mature hematopoietic cells, remains unclear. Here we report that low c-Rel expression in LCLs derived from a patient with hyper-IgM syndrome (Pt1), resulted in defects in proliferation and cell survival. In contrast to studies that associated loss of NF-κB with increased apoptosis, Pt1 LCLs failed to initiate apoptosis and alternatively underwent autophagy and necrotic cell death. Whereas the proliferation defect appeared linked to a c-Rel-associated decrease in c-myc expression, identified pro-survival and pro-apoptotic targets were expressed at or near control levels consistent with the absence of apoptosis. Ultrastructural examination of Pt1 LCLs revealed a high level of cellular and ER stress that was further supported by gene expression profiling showing the upregulation of several genes involved in stress and inflammation. Apoptosis-independent cell death was accompanied by increased expression of the inflammatory marker, caspase-4. Using gene overexpression and siRNA knockdown we demonstrated that levels of c-Rel directly modulated expression of caspase-4 as well as other ER stress genes. Overall, these findings reveal the importance of c-Rel in maintaining LCL viability and that decreased expression results in ER stress and a default response leading to necrotic cell death.LMP1介导的核因子κB（nuclear factor kappa B, NF-κB）激活，对于EB病毒（Epstein-Barr virus, EBV）转化的淋巴母细胞系（lymphoblastoid cell lines, LCLs）的配体非依赖性增殖与细胞存活至关重要。既往实验研究表明，绝大多数依赖LMP1的细胞应答均受NF-κB调控。然而，这些应答过程中各NF-κB家族成员的必需作用程度，尤其是表达局限于成熟造血细胞的c-Rel，目前仍不明确。本研究发现，来自高IgM综合征（hyper-IgM syndrome）患者Pt1的LCLs中c-Rel表达水平较低，进而导致增殖与细胞存活缺陷。与既往将NF-κB缺失与细胞凋亡增加相关联的研究不同，Pt1来源的LCLs并未启动凋亡程序，而是发生了自噬与坏死性细胞死亡。尽管增殖缺陷似乎与c-Rel介导的c-myc表达下调相关，但已被鉴定的促存活与促凋亡靶基因的表达水平均处于或接近对照组水平，这与凋亡未发生的结果相符。对Pt1来源LCLs的超微结构检查显示，其存在高水平的细胞应激与内质网应激（endoplasmic reticulum stress, ER stress），而基因表达谱分析进一步佐证了这一点——该分析显示多个参与应激与炎症反应的基因均出现上调。凋亡非依赖性细胞死亡伴随炎性标志物半胱天冬酶-4（caspase-4）的表达升高。通过基因过表达与小干扰RNA（small interfering RNA, siRNA）敲低实验，我们证实c-Rel的表达水平可直接调控caspase-4以及其他内质网应激相关基因的表达。综上，本研究结果揭示了c-Rel在维持LCL存活中的重要作用，而其表达下调会引发内质网应激，并触发导致坏死性细胞死亡的默认通路。