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Regulation and Biological Functions of DUSP6 and DUSP8 in Epstein-Barr viral infection
|Advisor:||蔡錦華(Ching- Hwa Tsai)|
Epstein-Barr virus,MAPKs,Dual specificity phosphatase (DUSPs),LMP1,
|Publication Year :||2014|
|Abstract:||EB病毒(Epstein-Barr virus)屬於疱疹病毒家族的一員，是第一個由WHO組織認證為與腫瘤高度相關的DNA病毒。在體外實驗，具有能夠將B細胞不朽化(Immortalize)，使其成為不斷增生的淋巴母芽細胞株(Lymphoblastic cell line, LCLs)。文獻指出EB病毒與許多人類惡性腫瘤有高度相關，如巴氏淋巴瘤(Burkitt’s lymphoma)、霍金氏淋巴瘤(Hodgkin’s lymphoma)、移植後淋巴增生疾病(Post-transplant lymphoproliferative disorder, PTLD)、鼻咽癌(nasopharyngeal carcinoma, NPC)等。
EB病毒為了長期存活於宿主細胞內，會抑制細胞凋亡因子，並且持續性活化利於生長之訊息傳遞路徑，例如NF-kB、ERK、JNK、p38、Akt。最重要地，EB病毒藉由調控宿主免疫反應，使得EB病毒得以逃避免疫攻擊，穩定存活於細胞內。本實驗室先前利用cDNA微陣列實驗，分析CD19+初級B細胞(primary B cells)及LCLs，探查兩者細胞內基因表現之變化情形。發現EB病毒感染B細胞後，影響B細胞內多種雙特異性磷酸酶(Dual specificity phosphatase, DUSPs)之基因表現，如DUSP1、DUSP2、DUSP6、DUSP8。DUSP於細胞內主要使特定目標之MAPK去磷酸化，使MAPK活性降低，扮演負向調控角色。其中，DUSP6及DUSP8分別可調控ERK或JNK/p38之磷酸化程度。因此，我們想了解MAPK之表現與調控對EB病毒在不朽化B細胞之過程有何影響。根據DUSPs對於下游目標MAPK專一性，我們選擇其中13種DUSPs，利用RT-PCR分析CD19+初級B細胞及相配對之LCLs細胞，發現當B細胞受到EB病毒感染時DUSP1、DUSP2、DUSP6、DUSP8表現量明顯受到抑制。同樣地利用西方墨點法也觀察到DUSP6及DUSP8蛋白質表現量下降。進一步確認上述現象，以四個來源之初級B細胞及相對應LCLs觀察到DUSP6及DUSP8蛋白質表現量有相同趨勢。
Epstein-Barr virus (EBV) belongs to human Herpesviridae family. This virus is first virus defined by WHO as a human oncogenic virus. In vitro, EBV can immortalize primary B cells into lymphoblastoid cell lines (LCLs). Many studies evidence that EBV is associated with many human malignancies, such as Burkitt’s lymphoma, Hodgkin’s lymphoma, Post-transplant lymphoproliferative disorder, nasopharyngeal carcinoma etc and so on.
In order to persistent in host cells, EBV activate downstream signaling transduction pathway, such as NF-kB, ERK, JNK, p38, Akt, to induce host cell constitutive proliferation, and to prevent host cell from program cell death. Most importantly, EBV manipulates the host immune response so that it can escape the immune surveillance and persist in host cells. Previously, we utilized cDNA microarray to compare the whole panel of cellular gene expression between CD19+ primary B cells and LCLs which immortalized with B95.8 strain EBV. Preliminarily, we found that EBV influences the gene expression of many kinds of dual specificity phosphatase (DUSP), such as DUSP1, DUSP2, DUSP6 and DUSP8. One of the major functions for DUSP is to negatively regulate MAPK activities by dephosphorylaion of MAPK and inactivate their kinase activities. So, we wonder the expression and regulation of MAPK during the EBV immortalization process. According to their substract specificity, we selected 13 DUSPs to test their kinetic mRNA expression by RT-PCR. Among them, we saw the DUSP1, 2, 6, and 8 are obviously down-regulated when primary B cells become LCLs. Among them, we chose the DUSP6 and 8 as our study targets. In further Western blotting assay, the protein expression of both DUSP6 and 8 are also decreased. To further confirm these phenomena, four pairs of primary B cells and LCLs are detected for their protein expression of DUSP6 and 8. Next, we would like to know which viral gene is involved in these down-regulation effects. According to the results of lentiviral infection, the EBV latent membrane protein 1(LMP1) is responsible for this down-regulation. These results were both seen in Akata and BJAB cell lines. Furthermore, we show that LMP1 is necessary for DUSP 6 and 8 downregulation by shRNA approach. C-terminal activation domain (CTAR)1 and CTAR2 of LMP1 were required for this downregulation. In signaling, LMP1-mediated ERK pathway was involved in both DUSP-downregulation. In addition, ERK-downstream Elk1 was required for DUSP6 but not DUSP8 down-regulation. Based on the results of luciferase reporter assay, we demonstrated that LMP1 downregualtes DUSP6 and DUSP8 through inhibiting their promoter activity, but probably with different transcriptional factors.
Of note, we desired to address the influence of this down-regulation. LCL clumping morphology was shrinking upon the DUSP 6-expressive lentivirus were transducted into LCLs. Consistently, the cell number were decreased during lentivirus infection. To clarify the question what is fate of DUSP6-overexpressive LCLs, we checked the cell cycle by flowcytometry. The sub-G1 population was obviously increased after over-expression. Meanwhile, we examined the expression of p-ERK, ERK, caspase and PARP. Obviously, Western-blotting data indicated that the amounts of p-ERK are decreased, while cleaved caspase 3 and cleaved PARP are increased. All the results hinted that over-expression DUSP 6 can cause cell apoptosis. Of note, the kinase mutated DUSP 6 would not trigger the above phenomena. In the case of DUSP8, the LCL clumping was also shrinking and the sub-G1 population was also increased. However, their apoptotic mechanism is different form over-expression DUSP6. During over-expression of DUSP8, p38 was decreased but caspase 3 kept the same. On the other hand, cleaved Bcl-2 and PUMA were increased. However, the kinase dead DUSP 8 did not cause any change for cell proliferation.
So, we conclude that DUSP6 and DUSP8 are downregulated during EBV immortalization since LMP1 should prevent their effect from apoptosis.
|Appears in Collections:||微生物學科所|
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