Abstract
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′Eκ) is active in Igκ-expressing NPC cells and LMP1 upregulates the activity of 3′Eκ in NPC cells. Moreover, mutation analysis of the PU binding site in 3′Eκ and inhibition of the MEK/ERKs pathway by PD98059 indicate that the PU site is functional and LMP1-enhanced 3′Eκ 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′Eκ in vitro. ChIP assays further demonstrate Ets-1 binding to the PU motif of 3′Eκ in cells. These results suggest that LMP1 upregulates 3′Eκ 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.
Highlights
The restriction of immunoglobulin (Ig) expression to cells of the B-cell lineage is well established
latent membrane protein 1 (LMP1) can activate the Ras/ERK/MAPK signaling pathway [23] and our previous study indicated that LMP1 upregulates kappa light chain expression in nasopharyngeal carcinoma (NPC) cell lines [2]
The level of ERK phosphorylation was higher in HNE2-LMP1 cells than in HNE2 cells, which demonstrated that LMP1 activates the ERK pathway in NPC cells (Figure 1A)
Summary
The restriction of immunoglobulin (Ig) expression to cells of the B-cell lineage is well established. Two important kappa enhancers: the intronic enhancer (iEk), which lies between the Jk-Ck region, and the 39 enhancer (39Ek), which is located downstream of the Ck region, have been identified [8,9,10] Both enhancers are inactive at the pro-B and pre-B cell stages and active at the Igk-expressing mature B cell and plasma cell stages [10,11]. The activity of these enhancers is generally transcriptionally silent in other cells that cannot produce the kappa chain, such as T-lymphoid cells (Jurkat) [10], epithelial cells (HeLa) [10] and NIH3T3 fibroblasts [12] Based on these observations, the activation of these regulatory elements is generally believed to be required for immunoglobulin kappa gene expression and is a B cell lineage-restricted event [10]. Whether the other kappa enhancer, 39Ek, is functional in Igk-expressing epithelial cancer cells remains unknown
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