CD70 is expressed in normal activated immune cells as well as in several types of tumors. It has been established that anti-CD70 mAb induces complement-dependent death of CD70⁺ tumor cells, but how anti-CD70 mAb affects the intrinsic signaling is poorly defined. In this report, we show that ligation of CD70 expressed on EBV-transformed B cells using anti-CD70 mAb induced production of reactive oxygen species (ROS) and subsequent apoptosis. We observed an early expression of endoplasmic reticulum (ER) stress response genes that preceded the release of apoptotic molecules from the mitochondria and the cleavage of caspases. CD70-induced apoptosis was inhibited by pretreatment with the ER stress inhibitor salubrinal, ROS quencher N-acetylcysteine, and Ca²⁺ chelator BAPTA. We supposed that ROS generation might be the first event of CD70-induced apoptosis because N-acetylcysteine blocked increases of ROS and Ca²⁺, but BAPTA did not block ROS generation. We also found that CD70 stimulation activated JNK and p38 MAPK. JNK inhibitor SP600125 and p38 inhibitor SB203580 effectively blocked upregulation of ER stress-related genes and cleavage of caspases. Inhibition of ROS generation completely blocked phosphorylation of JNK and p38 MAPK and induction of ER stress-related genes. Taken together, we concluded that cross-linking of CD70 on EBV-transformed B cells triggered ER stress-mediated apoptosis via ROS generation and JNK and p38 MAPK pathway activation. Our report reveals alternate mechanisms of direct apoptosis through CD70 signaling and provides data supporting CD70 as a viable target for an Ab-based therapy against EBV-related tumors.

Purpose.: Although Epstein-Barr virus (EBV)–associated keratitis is rare, it can cause acute corneal necrosis and neovascularization. We aimed to examine the signaling mechanism by which EBV causes epithelial–mesenchymal transition (EMT) in human corneal epithelial cells (HCECs) in vitro. Methods.: The cellular response to EBV was assessed by real-time PCR, Western blot, migration assay, invasion assay, inhibitor assay, and ELISA assay. Results.: A model of EBV-induced EMT was established in HCECs. The EBV induced morphologic changes in the cells; the loss of epithelial markers E-cadherin, ZO-1, and β-catenin; and an increase in the mesenchymal markers N-cadherin, Vimentin, Snail, and TCF8/Zeb1. The EBV infection also led to the nuclear translocation of Snail and TCF8/Zeb1; enhanced the secretion of IL-6, IL-8, VEGF, TGF-β1, TNF-α, and MCP-1; and upregulated the expression of MMP2 and MMP9. The EBV-infected HCECs exhibited increased migration and invasiveness compared to uninfected HCECs. We measured the involvement of Syk, Src, PI3K/Akt, and Erk signaling, but not Smad, in EMT by EBV-induced TGF-β1. We demonstrated that treatment with TGF-β1, TGF-β receptors, Syk, or Src inhibitor blocked TGF-β1, Syk, or Src signaling activation, and EMT development by EBV. Moreover, these inhibitors prevented PI3K/Akt and Erk activation. Conclusions.: An EBV infection in HCECs can lead to a mesenchymal fibroblast-like morphology, and cause EMT through the activation of PI3K/Akt and Erk by TGF-β1–mediated Syk and Src signaling. This phenomenon may have implications for EBV-associated keratitis and molecular approaches to treatment.

Purpose: The a-disintegrin-and-metalloprotease (ADAM) family proteins are widely expressed in the different layers of the retina throughout development. The effect of ADAM proteins on the epithelial-to-mesenchymal transition (EMT) in proliferative vitreoretinopathy (PVR) or AMD is yet to be elucidated. In this study we used Epstein-Barr virus (EBV)-transformed adult retinal pigment epithelial (ARPE) cells to investigate how sorafenib, a multikinase inhibitor, modulates ADAM proteins to control EMT. Methods: Epithelial to mesenchymal transition and related mechanisms in EBV-infected ARPE cells were determined by RT-PCR, Western blot, invasion assay, ELISA assay, and gene silencing with siRNA. Results: Mesenchymal-like ARPE/EBV cells exhibited considerably increased cellular migration and invasion compared with ARPE cells and produced EMT-related cytokines. Sorafenib significantly inhibited production of TGF-β1, VEGF, IL-6, IL-8, MCP-1, and TNF-α and blocked the activation of migration-related signaling molecules, such as HIF-1α, p-STAT3, MMP2, and Ang-1. The expression of mature ADAM10, ADAM17, and cleaved Notch 1 proteins in ARPE/EBV cells was downregulated after treatment with sorafenib through the regulatory activity of nardilysin (NRD-1). Gene silencing of NRD-1 in ARPE/EBV cells attenuated secretion of EMT-related cytokines and expression of ADAM10 and 17 and upregulated epithelial markers. Conclusions: Sorafenib controls the mesenchymal characteristics of EBV-infected ARPE cells. Nardilysin and ADAM family proteins might be new targets for the prevention or control of EMT in retinal diseases.

Song H, et al. Immune Netw. 2009 Dec;9(6):236-242. https://doi.org/10.4110/in.2009.9.6.236

Summary: B7‐H4 is a recently discovered B7 family member that has inhibitory effects on T‐cell immunity. However, the reverse signalling mechanism of the B7‐H4‐expressing cells remains unclear. Previous work has shown that B7‐H4 expression was enhanced on B cells following Epstein–Barr virus (EBV) infection, and engagement of cell‐surface‐expressed B7‐H4 induces cell death of EBV‐transformed B cells. Here we found that B7‐H4 was constitutively expressed on EBV‐positive lymphoma cells, Raji and IM‐9 cells, but was not expressed on EBV‐negative lymphoma cells (Ramos). Engagement of B7‐H4 significantly reduced cell growth of Raji and IM‐9 cells and resulted in cell cycle arrest at G0–G1 phase in a dose‐ and time‐dependent manner. To clarify the mechanism of cell cycle arrest via activation of B7‐H4, cell cycle regulatory factors were examined by reverse transcription–polymerase chain reaction and immunoblotting. We found that B7‐H4 triggered down‐regulation of CDK4/6 and up‐regulation of p21 expression at both protein and RNA levels. Furthermore, CDK2 and cyclin E/D expression was down‐regulated by B7‐H4 triggering. Additionally, the down‐regulation of phospho‐AKT and phospho‐cyclin E were clearly detected in B7‐H4‐activated Raji cells, but the phosphorylation of p53 was constitutively maintained. These results indicate that B7‐H4‐mediated signalling on EBV‐positive B‐cell lymphoma cells modulates the cell cycle through down‐regulation of the AKT pathway. Consequently, B7‐H4 may be a new potential target for use in EBV‐positive lymphoma therapy.