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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: BACKGROUND: Expression of kappa gene is under the control of distinct cis-regulatory elements, including the kappa intron enhancer (iE kappa) and the kappa 3' enhancer (3'E kappa). 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-kappaB and AP-1 signaling pathways, we thus use NPC cells as model to further explore the molecular mechanisms of nonlymphoid cells expressing Ig kappa. RESULTS: In this study, luciferase reporter plasmid containing human wild-type iE kappa, and its derivative plasmids containing mutant binding sites for transcription factor NF-kappaB or AP-1 were constructed. Luciferase reporter assays demonstrate iE kappa is active in Ig kappa-expressing NPC cells and LMP1 expression can upregulate the activity of iE kappa in NPC cells. Mutation of the NF-kappaB or AP-1 site within and downstream the iE kappa, inhibition of the NF-kappaB 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 I kappaB alpha (DNMI kappaB alpha) or of c-Jun (TAM67) indicate that both sites are functional and LMP1-enhanced iE kappa activity is partly regulated by these two sites. Gel shift assays show that LMP1 promotes NF-kappaB subunits p52 and p65 as well as AP-1 family members c-Jun and c-Fos binding to the kappa NF-kappaB and the kappa AP-1 motifs in vitro, respectively. Both chemical inhibitors and dominant negative mutants targeting for NF-kappaB 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 kappaB motif as well as c-Jun and c-Fos binding to the AP-1 motif of Ig kappa gene in vivo. CONCLUSION: These results suggest that human iE kappa is active in Ig kappa-expressing NPC cells and LMP1-stimulated NF-kappaB and AP-1 activation results in an augmenting activation of the iE kappa. LMP1 promotes the interactions of heterodimeric NF-kappaB (p52/p65) and heterodimeric AP-1 (c-Jun/c-Fos) transcription factors with the human iE kappa enhancer region are important for the upregulation of kappa light chain in LMP1-positive nasopharyngeal carcinoma cells.

SUBMITTER: Liu H

PROVIDER: S-EPMC2774294 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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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.

Liu HaiDan H Zheng Hui H Duan Zhi Z Hu DuoSha D Li Ming M Liu SuFang S Li ZiJian Z Deng XiYun X Wang ZhenLian Z Tang Min M Shi Ying Y Yi Wei W Cao Ya Y

Molecular cancer 20091027

<h4>Background</h4>Expression of kappa gene is under the control of distinct cis-regulatory elements, including the kappa intron enhancer (iE kappa) and the kappa 3' enhancer (3'E kappa). 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 ...[more]

PMID: 19860880

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Project description:Accumulating evidence indicates that epithelial cancer cells, including nasopharyngeal carcinoma (NPC) cells, express immunoglobulins (Igs). We previously found that the expression of the kappa light chain protein in NPC cells can be upregulated by the EBV-encoded latent membrane protein 1 (LMP1). In the present study, we used NPC cell lines as models and found that LMP1-augmented kappa production corresponds with elevations in ERKs phosphorylation. PD98059 attenuates LMP1-induced ERKs phosphorylation resulting in decreased expression of the kappa light chain. ERK-specific small interfering RNA blunts LMP1-induced kappa light chain gene expression. Luciferase reporter assays demonstrate that immunoglobulin κ 3' enhancer (3'E(κ)) is active in Igκ-expressing NPC cells and LMP1 upregulates the activity of 3'E(κ) in NPC cells. Moreover, mutation analysis of the PU binding site in 3'E(κ) and inhibition of the MEK/ERKs pathway by PD98059 indicate that the PU site is functional and LMP1-enhanced 3'E(κ) activity is partly regulated by this site. PD98059 treatment also leads to a concentration-dependent inhibition of LMP1-induced Ets-1 expression and phosphorylation, which corresponds with a dose-dependent attenuation of LMP1-induced ERK phosphorylation and kappa light chain expression. Suppression of endogenous Ets-1 by small interfering RNA is accompanied by a decrease of Ig kappa light chain expression. Gel shift assays using nuclear extracts of NPC cells indicate that the transcription factor Ets-1 is recruited by LMP1 to the PU motif within 3'E(κ)in vitro. ChIP assays further demonstrate Ets-1 binding to the PU motif of 3'E(κ) in cells. These results suggest that LMP1 upregulates 3'E(κ) activity and kappa gene expression by activating the Ets-1 transcription factor through the ERKs signaling pathway. Our studies provide evidence for a novel regulatory mechanism of kappa expression, by which virus-encoded proteins activate the kappa 3' enhancer through activating transcription factors in non-B epithelial cancer cells.

Project description:Latent membrane protein 1 (LMP1), which is encoded by the Epstein‑Barr virus (EBV), has been considered as an oncogene, although the detailed mechanism behind its function remains unclear. It has been previously reported that LMP1 promotes tumorigenesis by upregulation of peroxisome proliferator‑activated receptor‑γ coactivator‑1β (PGC1β). The present study aimed to investigate the potential mechanism for transient EBV/LMP1 exposure‑mediated persistent PGC1β expression and subsequent tumorigenesis through modification of mitochondrial function. Luciferase reporter assay, chromatin immunoprecipitation and DNA mutation techniques were used to evaluate the PGC1β‑mediated expression of dynamin‑related protein 1 (DRP1). Tumorigenesis was evaluated by gene expression, oxidative stress, mitochondrial function and in vitro cellular proliferation assays. The potential effects of EBV, LMP1 and PGC1β on tumor growth were evaluated in an in vivo xenograft mouse model. The present in vitro experiments showed that LMP1 knockdown did not affect PGC1β expression or subsequent cell proliferation in EBV‑positive tumor cells. PGC1β regulated DRP1 expression by coactivation of GA‑binding protein α and nuclear respiratory factor 1 located on the DRP1 promoter, subsequently modulating mitochondrial fission. Transient exposure of either EBV or LMP1 in human hematopoietic stem cells caused persistent epigenetic changes and PGC1β upregulation after long‑term cell culture even in the absence of EBV/LMP1, which decreased oxidative stress, and potentiated mitochondrial function and cell proliferation in vitro. Enhanced tumor growth and shortened survival were subsequently observed in vivo. It was concluded that PGC1β expression and subsequent cell proliferation were independent from LMP1 in EBV‑positive tumor cells. PGC1β modulated mitochondria fission by regulation of DRP1 expression. Transient EBV/LMP1 exposure caused persistent PGC1β expression, triggering tumor growth in the absence of LMP1. The present study proposes a novel mechanism for transient EBV/LMP1 exposure‑mediated tumorigenesis through persistent epigenetic changes and PGC1β upregulation, uncovering the reason why numerous forms of lymphoma exhibit upregulated PGC1β expression, but are devoid of EBV/LMP1.

Dataset Information

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.

Publications

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.

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