LMP1-augmented kappa intron enhancer activity contributes to upregulation expression of Ig kappa light chain via NF-kappaB and AP-1 pathways in nasopharyngeal carcinoma cells

Abstract

BackgroundExpression of kappa gene is under the control of distinct cis-regulatory elements, including the kappa intron enhancer (iEκ) and the kappa 3' enhancer (3'Eκ). The active enhancers and expression of immunoglobulin is generally considered to be restricted to B lymphocytes. However, accumulating evidence indicated that epithelial cancer cells, including nasopharyngeal carcinoma (NPC) cell lines, express immunoglobulins. The mechanisms underlying the expression of Igs in nonlymphoid cells remain unknown. On the basis of our previous finding that expression of kappa light chain in NPC cells can be upregulated by EBV-encoded latent membrane protein 1(LMP1) through the activation of NF-κB and AP-1 signaling pathways, we thus use NPC cells as model to further explore the molecular mechanisms of nonlymphoid cells expressing Ig kappa.ResultsIn this study, luciferase reporter plasmid containing human wild-type iEκ, and its derivative plasmids containing mutant binding sites for transcription factor NF-κB or AP-1 were constructed. Luciferase reporter assays demonstrate iEκ is active in Igκ-expressing NPC cells and LMP1 expression can upregulate the activity of iEκ in NPC cells. Mutation of the NF-κB or AP-1 site within and downstream the iEκ, inhibition of the NF-κB and AP-1 pathways by their respective chemical inhibitor Bay11-7082 and SP600125 as well as stable or transient expression of dominant-negative mutant of IκBα (DNMIκBα) or of c-Jun (TAM67) indicate that both sites are functional and LMP1-enhanced iEκ activity is partly regulated by these two sites. Gel shift assays show that LMP1 promotes NF-κB subunits p52 and p65 as well as AP-1 family members c-Jun and c-Fos binding to the κNF-κB and the κAP-1 motifs in vitro, respectively. Both chemical inhibitors and dominant negative mutants targeting for NF-κB and AP-1 pathways can attenuate the LMP1-enhanced bindings. Co-IP assays using nuclear extracts from HNE2-LMP1 cells reveal that p52 and p65, c-Jun and c-Fos proteins interact with each other at endogenous levels. ChIP assays further demonstrate p52 and p65 binding to the κB motif as well as c-Jun and c-Fos binding to the AP-1 motif of Ig kappa gene in vivo.ConclusionThese results suggest that human iEκ is active in Igκ-expressing NPC cells and LMP1-stimulated NF-κB and AP-1 activation results in an augmenting activation of the iEκ. LMP1 promotes the interactions of heterodimeric NF-κB (p52/p65) and heterodimeric AP-1 (c-Jun/c-Fos) transcription factors with the human iEκ enhancer region are important for the upregulation of kappa light chain in LMP1-positive nasopharyngeal carcinoma cells.

Heterogeneity of aberrant immunoglobulin expression in cancer cells

“…Our recent studies showed that the level of the cancerous Ig k light chain could be upregulated by the exogenous oncoprotein, latent membrane protein 1 (LMP1), which is encoded by the Epstein-Barr virus in nasopharyngeal carcinoma (NPC) and triggers several signal pathways mediated through nuclear factor-kappa B (NF-KB) and activator protein-1 (AP-1). 19,20 Our findings indicated that cancerous Ig plays a potential role in the process of cell transformation. Overall, cancerous Ig is different from normal Ig.…”

Cancerous Immunoglobulins and Ca215: Implications in Cancer Immunology

“…Besides demonstrating the gene expression of immunoglobulins, the expression of essential genes necessary for the assembly of immunoglobulin genes, such as Recombinant Activating Gene (RAG1), Recombinant Activating Gene 2 (RAG2) and Activation-Induced cytidine Deaminase (AID) gene, were also documented in each case (Chen and Gu, 2007;Huang et al, 2008;2009;Niu et al, 2011a;2011b;Zhang et al, 2010;Zhao et al, 2011). However, in general, the mechanisms of action behind the expressions of these atypical immunoglobulins in normal human tissues were discussed with limited experimental evidence (Babbage et al, 2006;Chen and Gu, 2007;Geng et al, 2007;Hu et al, 2003;Huang et al, 2008;2009;Li et al, 2001;Liu et al, 2009;Niu et al, 2011a;2011b;Okabe et al, 2001;Yang et al, 2002;Zhang et al, 2010;Zhao et al, 2011;Zheng et al, 2007b;Zhu et al, 2008). In most cases, the expressions of the atypical immunoglobulins in the immunologically privileged sites of normal human tissues were found to only implement the possible functions of immune protection (Chen and Gu, 2007;Huang et al, 2008;2009;Niu et al, 2011a;2011b;Zhang et al, 2010;Zhao et al, 2011).…”

Rearrangement and expression of the immunoglobulin μ-chain gene in human myeloid cells

“…[7][8][9] Moreover, non-hematopoietic cell-derived Ig shares some interesting features, including the restricted or biased usage of certain sequences, 9,10 a unique glycosylation profile 11,12 and novel regulatory mechanisms of gene expression. 13,14 Furthermore, non-hematopoietic cellderived Ig has a role in cell growth and survival. 1,3,15,16 These findings suggest that Ig derived from non-hematopoietic cells might be a novel molecule with a unique structure and function and might play a role in carcinogenesis and/or tumor progression.…”