The Epstein-Barr virus latent membrane protein 1 regulates gene expression by engaging multiple signaling pathways

Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that is associated with a variety of human malignancies of lymphoid and epithelial origins, such as post-transplant lymphoproliferative disorder (PTLD), Burkitt's lymphoma (BL), Hodgkin disease (HL), nasopharyngeal carcinoma, and others. Latent membrane protein 1 (LMP1), which is expressed in latency type II and III, is considered the EBV oncoprotein due to its ability to transform rodent fibroblasts and its critical roles in EBV-mediated B cell transformation. Two major signaling domains of LMP1, carboxyl-terminal activating region (CTAR) 1 and 2, are responsible for the majority of LMP1-mediated signaling pathways. However, CTAR1 and CTAR2 induce distinctive signal transductions and only CTAR1 is essential for LMP1-mediated cell transformation. CTAR1 recruits TNFR-associated factors (TRAFs) to induce signaling pathways including PI3K/Akt, MEK/ERK, and complex NF-kappaB signaling. CTAR1 is also responsible for LMP1-regulated expression of cellular genes, such as epidermal growth factor receptor (EGFR).;In this study, multiple signaling pathways engaged by LMP1 through CTAR1 to mediate EGFR upregulation were evaluated. LMP1 has been shown to induce EGFR expression by activating transcriptional active complex p50-homodimer/Bcl-3. This study shows CTAR1 also induces the expression of Bcl-3 through activating transcriptional regulator of Bcl-3, STAT3, and the STAT3 activation is required for CTAR1's complete activity to induce EGFR expression. Using mouse embryonic fibroblasts (MEF) defective for different NF-kappaB effectors and TRAFs, we identified that CTAR1-mediated EGFR upregulation is dependent on NIK but not canonical or noncanonical NF-kappaB pathways. However NIK alone is not sufficient to induce EGFR expression, indicating that additional pathways are also needed. Consistent with previous studies, TRAF2 and TRAF3 but not TRAF6 are required for CTAR1-mediated EGFR induction. TRAF2 potentially contributes to EGFR induction by regulating p50 activation, and this activation is not mediated through the canonical or noncanonical NF-kappaB pathways.;Both EGFR and STAT3 are activated by CTAR1 in a serum-independent manner, however, the CTAR1-activated EGFR can also activate STAT3 in response to EGF treatment and induce the expression of STAT3 target genes. CTAR1 was found to activate STAT3 through PKCdelta, as inhibition of PKCdelta by a chemical inhibitor reduced CTAR1-induced serine phosphorylation of STAT3 and Bcl-3 expression. These findings indicate that CTAR1 manipulates STAT3 signaling through multiple mechanisms. PKCdelta was also shown to mediate CTAR1-induced ERK activation, and inhibition of PKCdelta block CTAR1-mediated transformation of rodent fibroblast cells. These results suggest that CTAR1 induce EGFR expression by engaging multiple downstream signaling pathways that contribute to LMP1-mediated gene regulation.