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.
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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LMP1 增强的 kappa 内含子增强子活性有助于通过 NF-kappaB 和 AP-1 通路上调鼻咽癌细胞中 Ig kappa 轻链的表达
DOI：
10.1186/1476-4598-8-92
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发表时间：
2009-10-27
期刊：
影响因子：
37.3
通讯作者：
Cao Y
中科院分区：
文献类型：
--
作者：
Liu H;Zheng H;Duan Z;Hu D;Li M;Liu S;Li Z;Deng X;Wang Z;Tang M;Shi Y;Yi W;Cao Y
kappa基因的表达受不同的顺式调控元件控制，包括kappa内含子增强子（iEκ）和kappa 3'增强子（3' E κ）。免疫球蛋白的活性增强子和表达通常被认为仅限于B淋巴细胞。然而，越来越多的证据表明，上皮癌细胞，包括鼻咽癌（NPC）细胞系，表达免疫球蛋白。免疫球蛋白在非淋巴细胞中表达的机制尚不清楚。我们在前期研究的基础上，发现EB病毒编码的潜伏膜蛋白1（LMP 1）可通过激活NF-κB和AP-1信号通路上调鼻咽癌细胞κ轻链的表达。因此，我们以鼻咽癌细胞为模型，进一步探讨非淋巴细胞表达IG κ的分子机制。本研究构建了含人野生型iEκ的荧光素酶报告质粒及其衍生质粒，这些质粒含有转录因子NF-κB或AP-1的突变结合位点。荧光素酶报告基因分析表明iEκ在表达IGκ的NPC细胞中是有活性的，LMP 1的表达可上调iEκ的活性。iEκ内和下游的NF-κB或AP-1位点突变，NF-κB和AP-1通路被其各自的化学抑制剂Bay 11 -7082和SP 600125抑制，以及IκBα（DNMIκBα）或c-Jun（TAM 67）的显性阴性突变体的稳定或瞬时表达，表明这两个位点都有功能，LMP 1增强的iEκ活性部分受这两个位点调节。凝胶位移分析表明，LMP 1促进NF-κB亚基p52和p65以及AP-1家族成员c-Jun和c-Fos在体外分别与κNF-κB和κAP-1基序结合。针对NF-κB和AP-1通路的化学抑制剂和显性失活突变体都可以减弱LMP 1增强的结合。使用来自HNE 2-LMP 1细胞的核提取物的Co-IP测定揭示了p52和p65、c-Jun和c-Fos蛋白在内源性水平上彼此相互作用。ChIP分析进一步证实了p52和p65与κB基序的结合以及c-Jun和c-Fos与IG κ基因的AP-1基序的结合。这些结果表明，人iEκ在表达IGκ的NPC细胞中是有活性的，并且LMP 1刺激的NF-κB和AP-1活化导致iEκ的增强活化。LMP 1促进异源二聚体NF-κB（p52/p65）和异源二聚体AP-1（c-Jun/c-Fos）转录因子与人iEκ增强子区域的相互作用，对于LMP 1阳性鼻咽癌细胞中κ轻链的上调是重要的。
Expression 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. In 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. These 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.
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