Molecular Mechanisms Of Human Immunoglobulin Kappa Light Chain Upregulated By Epstein-Barr Virus Encoded Latent Membrane Protein 1 In Nasopharyngeal Carcinoma Cells Via Different Signal Pathways

We previously found a transforming gene, designated as Tx, from Chinese nasopharyngeal carcinoma (NPC) cell line CNE2 genomic DNA library in 1991. Tx expressed its transforming activity when transferred into mouse epidermal JB6 cells. Sequencing and bioinformatic analysis indicated that Tx gene contains constant region and joining regions but failed to find variable region. Compared with normal human Ig kappa JC region, Tx showed 99% homogeneity. Therefore, we speculated that Tx is an aberrant human Ig kappa light chain gene.It is well known that only B lymphocytes can express immunoglobulin rather than other somatic cells including epithelial cells. However, based on the finding of Tx gene, we subsequently found that several epithelial cancer cell lines including MGC (gastric carcinoma), MCF-7 (breast carcinoma), SW480 (colorectal carcinoma), HeLa (cervical carcinoma), HNE2 (nasopharyngeal carcinoma) as well as NPC biopsies express immunoglobulin kappa light chain mRNA and protein by a series of molecular biology experiments such as immunohistochemistry, Northern Blotting. By in situ hybridization, we have also found its expression in cervical carcinoma is associated with the stages of cervical carcinogenesis. Through case-control studies, we have identified 4 single nucleotide polymorphisms (SNPs) locus in the Ig kappa gene, termed SNP3, SNP4, SNP5 and SNP6, respectively. We found the heteroduplex AT-GC genotype of the SNP4-SNP3 linkage might be a risk factor for NPC.The preliminary study of Tx expression revealed that Tx was expressed at a low level in NPC cell lines where EBV was negative, while at a significantly high level in EBV-positive B95-8 and Raji cells. In addition, Western Blotting results indicated that the levels of kappa light chain were significantly higher in EBV-encoded latent membrane protein (LMP1) positive CNE1-LMP1 NPC cells than that in LMP1-negative CNE1 NPC cells. Considering both B95-8 and Raji cells also expressed LMP1, all these implied that LMP1 might play some roles in upregulating the ectopic expression of Ig kappa light chain in NPC cells and promoted our using of NPC cell lines as model to explore the underlied mechanisms.1. LMP1 enhances kappa light chain expression in human nasopharyngeai carcinoma cellsWe use a constantly expressing full-length LMP1 cell line HNE2-LMP1 and a LMPl-inducible expression NPC cell line, Tet-on-LMP1 HNE2, in which the expression of LMP1 was tightly regulated by doxycycline addition, as the cell models, to investigate the effect of LMP1 on the expression of Ig kappa. When stimulated LMP1 expression by 0, 0.06, 0.6, 6.0μg/ml of Dox, as evidenced by immunoblotting, Dox induced a marked increase of LMP1 in a dose-dependent manner accompany a dose-dependent elevation of kappa light chain. A dose-dependent increased kappa-specific mean fluorescence intensity (MFI) by indicated concentrations of Dox in Tet-on-LMP1 HNE2 cells were also confirmed by intracellular flow cytometry analysis. RT-PCR result showed that Tet-on-LMP1 HNE2 cells under the same treatment with Dox induced a marked increase in kappa mRNA, in a dose-dependent manner.When treating HNE2 and HNE2-LMP1 cells with 2μm LMP1-specific DNAzyme DZ1, Western blotting results indicated that DNAzyme DZ1 obviously inhibited LMP1 protein expression and concomitantly downregulate kappa expression in LMPl-positive cell line HNE2-LMP 1, while has no effect on kappa expression in LMP 1-negative cell line HNE2. These results indicate that LMP1 could increase the production of kappa light chain in NPC cells at both mRNA and protein levels.2. Involvement of the NF-κB and AP-1 pathways in LMP1-augmented Ig kappa expression in human nasopharyngeal carcinoma cells Bioinformatic analysis revealed that this aberrant kappa gene contains NF-κB and AP-1 binding sites, which were located in enhancer and in J-C intronic region, respectively. It implied that the transcription factors binding to corresponding site might exert effects on the enhancer and finally modulate kappa gene expression.It has been shown that aκB biding site within the J_κ-C_κregion of Ig kappa gene played a key role in modulating Ig kappa light chain gene expression and an AP-1 site downstream the rd3 site also played a role in kappa gene regulation in B cell lines. Among the specific signal transduction pathways activated by LMP1, the NF-κB and AP-1 signaling pathways are the most important, since their activation results in the overexpression of most LMP1 target genes. We therefore investigated whether LMP 1-augmented Ig kappa expression had deal with the aberrant activation of these two pathways by LMP1 in human nasopharyngeal carcinoma cells.Two inhibitors, Bayll-7082 and SP600125, were used to block NF-κB and AP-1 pathways, respectively. Treatment of HNE2-LMP 1 cells with both inhibitors resulted in a dose-dependent suppression of kappa chain induction by the LMP 1, which is correlated with a dose-dependent attenuation of JNK and IκBαphosphorylation induced by LMP1. Flow cytometry analysis also indicated that both compounds inhibited LMP1-induced Ig kappa expression.Our previous studies showed that dominant-negative mutant of IκBα(DNMIκBα) and c-Jun (TAM67) could efficiently inhibit the activations of NF-κB and AP-1 pathways induced by LMP1, respectively. By Westem blot and Flow cytometric analysis, we found that HNE2-LMP1 cells, when stably expressing DNMIκBαor TAM67, exhibited a significant decrease of Ig kappa production compared with their parental cells HNE2-LMP1. Overall, these results confirm the concept that upregulation of kappa light chain by LMP1 occurs via activation of NF-κB and AP-1 pathways.3. Upregulated the activity of human lg kappa light chain intronie enhancer in nasopharyngeal carcinoma cells by LMP1 via NF-κB and AP-1 pathwaysThe regulation of gene expression is under the control of cis-regulatory elements and trans-acting factors interaction. On the basis of our previously findings that both NF-κB and AP-1 pathways are involved in LMP1-augmented Ig kappa expression in human NPC cells, we try to confirm the exact element which transcription factors NF-κB and AP-1 target for. The activation of enhancers is required for Ig kappa gene expression in B cell lines. The human Ig kappa gene contains aκB site within the iEκand a perfect consensus AP-1 site downstream the iEκ. In B cell lines, both enhancers can be activated by nuclear factor NF-κB and AP-1 at different degrees. Thus raises an issue whether LMP1 upregulated Ig kappa by upregulating the iEκactivity via NF-κB and AP-1 pathways.To confirm the point, a 575bp genomic fragment containing the intact iEκwas subcloned into enhancerless pGL3-promoter vector. This construct, which contains wild typeκB site within the iEκand wild type AP-1 site downstream the 3' flank of the iEκ(designated pwt575), was introduced into HNE2 and HNE2-LMP1 cells to test the activity of iEκ. Transfection of pwt575 can increase the luciferase activity whatever in LMPl-negative and in LMP 1-positive NPC cells. These results indicated that the iE_κis active in NPC cells which expressed immunoglobulin kappa light chain. Moreover, the activity of iE_κin HNE2-LMP1 cells (～35-fold) was significantly higher than that in LMP 1-negative (-5-fold), which was consistent with our antecedent report that the levels of kappa light chain were significantly higher in HNE2-LMP1 cells than in HNE2 cells. We infer that the upregulation of kappa light chain by LMP 1 is likely due to increase the activity of iE_κby LMP 1.To determine whether the NF-κB or AP-1 sequence is required for LMPl-enhanced iEk activation, site-directed mutagenesis by Overlap Extension PCR was used to introduce mutations into each and both of these sequences. From the pwt575 construct, which contains wild typeκB site within the iEκand wild type AP-1 site downstream the 3' flank of the iEκ, we created constructs termed pmt575.κB, pmt575.AP-1, pmt575.κB.AP-1, respectively. These four constructs were individually introduced into HNE2 and HNE2-LMP1 cells. Comparing HNE2 with HNE2-LMP1 cells, the transfection of pwt575 into respective cells increased the iEκluciferase activity from～6-fold to -160-fold. Mutation of the NF-κB (pmt575.κB), the AP1motif (pmt575.AP-1) alone in the context of the iEκwas shown to decrease LMP 1-increased activation of the iEκto～70-fold and～90-fold, respectively. Combined mutation of r,B and AP-1 motif (pmt575.κB.AP-1) decreased LMP1-increased iEκactivity to the similar extent as mutation of the AP-1 site alone. This suggested that the two pathways through which LMP1 activated the iEκshowed no evidence of synergy. The magnitude of the reduction for pmt575.AP-1 is less than that for pmt575.κB, implying that of two signaling pathways, NF-κB pathway may play a leading role in LMP 1-augmented iEκactivity in NPC cells.To further confirm the functional activity of the NF-κB and the AP1 site require for LMP1-augmented activation of the iEκ. We used various specific inhibitors and dominant negative mutants for AP-1 and NF-κB signaling pathways to block the LMP1-mediated iEκactivation. We found the increased activity of iEκby LMP1 was abolished by 20μm Bay11-7082 (～30-fold) and 20μm SP600125 (～110-fold) but not by the DMSO vehicle control and was greatly attenuated when DNMIκBαand TAM67 were stably tranfected into HNE2-LMP1 cells. Together, the results indicated that both NF-rJ3 and AP-1 binding sites are involved in human kappa light chain intronic enhancer activation by LMP1 in NPC cells.Since we have demonstrated that both transcription factors NF-κB and AP-1 were involved in the cis-acting element iEκactivation in NPC cells, we further investigate whether the transcription factors directly bind to the DNA fragments covering the NF-κB and AP-1 binding motif by EMSA. The biotin-labeledκNFκB andκAP-1 oligonucleotides used were derived from the sequence of the r,B site within the human kappa intron enhancer and the sequence of AP-1 site 330 bp 3' of theκB site within human Ig kappa gene Jκ-Cκregion, respectively. We also synthesized biotin-labeled mutantκNFκB andκAP-1 oligonucleotides in which nucleotide mutations introduced were the same as reporter assay used. Equal amounts of nuclear extracts from HNE2, HNE2-LMP1, HNE2-LMP1-DMNIκB, HNE2-LMP1-TAM67, Bay11-7082-treated HNE2-LMP1 and SP600125-treated HNE2-LMP1 cells were used.We found LMP1 caused a much stronger NF-κB DNA-binding activity in HNE2-LMP1 cells than that in HNE2 cells. The induction of NF-κB DNA binding activity by LMP1 was clearly inhibited by 201aM Bay 11-7082. We also found that the nuclear lysates isolated from HNE2-LMP 1-DMNIκB cells induced a weaker electromobility shift band than that from their parental cells HNE2-LMP1. The nuclear lysates isolated from these cell lines did not induce an electromobility shift when biotin-labeledκNF-κB mutant-type oligonucleotide was introduced. Supershift analysis with antibodies specific for NF-κB family members showed that NF-κB DNA/protein complex composed of p65 and p52 subunits in nuclear extracts of HNE2-LMP1 cells, which suggested that p65/p52 heterodimers directly bind to the human kappa intronic enhancer.Similarly, LMP1 increased the formation of AP-1 DNA binding complex. The nuclear lysates isolated from HNE2-LMP1-TAM67 cells induced a weaker electromobility shift band than that from their parent cells HNE2-LMP1. The induction of AP-1 DNA binding activity by LMP1 was clearly inhibited by 20μM SP600125. The nuclear lysates isolated from these cells did not induce an electromobility shift when biotin-labeledκAP-1 mutant-type oligonucleotide was introduced. Supershift analysis confirmed that c-Jun and c-Fos are components of the complex bound to the human kappa AP-1 site. Our results clearly demonstrated that c-Jun/c-Fos heterodimers directly bind to the intronic enhancer-containing Jκ-Cκregion of human kappa gene sequence. 4. LMP1 induced transcription factor Ets-1 phosphorylation to target for 3'Eκenhancer via ERK signal pathway, thus upregulating Ig kappa light chain expressionBesides iEk, 3'Eκis another enhancer which also plays an important role in regulating Ig kappa gene expression. Several positive regulatory elements in the core region of human 3'Eκwere characterized, including a consensus binding site for the B-cell and macrophage-specific transcription factor PU.1 and the family of Ets-related proteins. According to our previous findings that LMP 1 could upregulate the Ets-1 phosphorylation level, we supposed that a LMP1-ERK/MAPK-Ets pathway was likely to exist through which LMP1 upregulated Ig kappa induction.We used PD98059, a specific ERK inhibitor, to block the LMPl-induced ERK activation. Treatment of HNE2-LMP1 cells with various concentrations of PD98059 resulted in a dose-dependent suppression of LMP1-induced ERK phosphorylation, which is correlated with a dose-dependent attenuation of Ets-1 phosphorylation and Ig kappa expression induced by LMP 1.The results indicated LMP1 upregulated Ig kappa induction in an ERK-dependent Ets-1 phosphorylation manner.To elucidate that LMPl-induced transcription factor Ets-1 phosphorylation targets for 3'Eκenhancer, we synthesized biotin-labeled κPU oligonucleotides according to PU binding site DNA sequences within human Ig kappa gene 3' enhancer as well as biotin-labeled mutant r, PU oligonucleotides. Equal amounts of nuclear extracts from HNE2, HNE2-LMP1, and PD98059-treated HNE2-LMP1 cells were subject to EMSA analysis.Supershift-EMSA analysis proved that, in NPC cells, it was transcription factor Ets-1 but not PU.1 that binds toκPU consensus sequences within 3' enhancer, thus suggesting that, to some extent, the mechanisms by which NPC cell lines expressing Ig differ from B cell lines. We found LMP1 caused a much stronger Ets-1 DNA-binding activity in HNE2-LMP1 cells than that in HNE2 cells. 50μM PD98059 completely inhibited LMPl-induced Ets-1 DNA binding activity. The nuclear lysates isolated from these cells did not induce Ets-1/DNA complex shift bands when biotin-labeledκPU mutant-type oligonucleotides were introduced. We therefore concluded that LMP1 induced transcription factor Ets-1 phosphorylation to target for 3'Eκenhancer via ERK signal pathway, thus upregulating Ig kappa light chain expression.With our advantages in the research field of signal transduction, we start our study from abnormally modulated signal pathways by EBV-LMP1. We used the NPC cell lines as model in order to explore the mechanisms by which human cancer cells of epithelial origin produced immunoglobulins. We confirmed that the activation of transcription factors by EBV-LMP 1 played an important role in upregulating Ig kappa light chain expression in NPC cells and found that, for the first time, in Ig-expressing NPC cells, the kappa gene intronic enhancer has functional activity, which is modulated by LMP1. We identified three main signal pathways, three key transcription factors and two kappa light gene enhancers (iEκand 3'Eκ), all of which were modulated by LMP1. We proved that LMP1 stimulated transcription factors NF-κB, AP-1, Ets-1 binding to the corresponding site in Ig kappa gene via NF-κB,JNK/MAPK and ERK/MAPK signal pathways and finally upregulated Ig kappa light chain induction. In addition, we found the mechanism by which LMP1 induced transcription factor Ets-1 targeting for 3'Eκenhancer via ERK signal pathway thus upregulating Ig kappa light chain expression in NPC cells was different from that by B cells.The present study presented novel experimental proofs on the mechanisms upregulating the ectopic expression of kappa light chain by LMP1 in NPC cells. Since other virus-encoded oncoproteins, such as HBX, E6, E7, can also activate many signal pathways including NF-κB,AP-1 and ERK pathways, these oncoproteins might induce immunoglobulin gene expression through the mechanism similar to EBV-LMP 1. The innovative findings might recruit the classical theory of immunology and give us a new insight into this abnormal phenomenon. Our study might provide some hints of possibility mechanisms by which other human cancer cell lines of epithelial origin produced immunoglobulins and lay foundations for further studies, as well as new strategies for cancer therapy.