| 摘要: | TNF-α為pro-inflammatory cytokine。TNF-α不只參與發炎及感染反應，也與抑制紅血球分化有關。Activin A為transforming growth factor (TGF)-β superfamily其中的一個成員，會促進紅血球的分化。過去本實驗室利用PDTC (NF-κB抑制劑) 證明在CML病人建立的K562細胞中TNF-α可透過NF-κB路徑增加c-Jun表現而抑制Activin A誘導的紅血球分化。在本論文中，利用transient transfection或穩定細胞株大量表現NF-κB p50或NF-κB p65更進一步證明於K562細胞中NF-κB於TNF-α誘導c-Jun表現所扮演的角色。實驗結果顯示在K562細胞中，NF-κB p65會活化c-Jun promoter activity，NF-κB p50會抑制c-Jun promoter activity。c-Jun或NF-κB p65透過AP-1 binding site調控其c-Jun promoter，因此推測NF-κB p65透過c-Jun作用於AP-1 binding site，這可能為TNF-α抑制紅血球分化的原因之ㄧ。JNK抑制劑SP600125可抑制因TNF-α活化的c-Jun promoter活性，此結果表示TNF-α也可以透過JNK路徑調控c-Jun的表現。此外，NF-κB p65活化c-Jun promoter的活性部分可被JNK抑制劑所抑制。所以推測TNF-α是分別透過JNK以及NF-κB兩個個別路徑調控c-Jun的表現。RT-PCT的結果顯示Activin A可誘導K562細胞中erythroid genes(α-globin, ζ-globin, NF-E2p45 and GATA-1)的表現；TNF-α則抑制這些genes的表現。當NF-E2大量表現時會增加Activin A誘導ζ-globin reporter的活性，而c-Jun大量表現時，會抑制此現象。由這些實驗結果顯示TNF-α透過NF-κB增加的c-Jun表現可抑制NF-E2增加Activin A誘導紅血球的分化。未來，將利用ChIP assay研究NF-κB p65是否結合於c-Jun promoter 上AP-1 binding site的c-Jun而增加c-Jun promoter活性。
The pro-inflammatory cytokine, tumor necrosis factor (TNF)-α, is linked to erythropoietic inhibition and may contribute to different forms of anemia. Activin A, a member of the transforming growth factor (TGF)-β superfamily, is an erythroid differentiation factor. In our previous study, we demonstrated that TNF-α up-regulated c-Jun expression through the NF-κB pathway to inhibit Activin A-mediated erythropoiesis with NF-κB inhibitor PDTC in chronic myeloid leukemia (CML)-derived K562 cells. In this study, we carry out the over-expression of different NF-κB family members, p50 and p65, by transient transfection or stable expression in K562 cells to further explore the role of NF-κB on TNF-α-induced c-Jun expression. Our data show that p65 activates c-Jun promoter; however, p50 represses c-Jun promoter. The c-Jun or NF-κB p65 activates c-Jun promoter through AP-1 binding site, suggesting NF-κB p65 act on AP-1 binding site through c-Jun which may lead to erythropoietic inhibition in TNF-α signaling. The JNK inhibitor SP600125 represses the TNF-α-activated c-Jun promoter, indicating that TNF-α?n also exerts its effect through JNK activation. Furthermore, p65-activated c-Jun promoter is partly inhibited by JNK inhibitor, suggesting TNF-α ?nregulates the c-Jun promoter through both NF-κB and JNK pathways. The results of RT-PCR show that activin A up-regulates the expression of erythroid genes (α-globin, ζ-globin, NF-E2p45 and GATA-1) and TNF-α?n down-regulates these genes. The over-expression of NF-E2 enhances Activin A-induced ζ-globin reporter activity, co-expression of c-Jun and NF-E2 can inhibit these effects. These results indicate that TNF-α up-regulates c-Jun through NF-κB pathway to antagonize the NF-E2 transcriptional activity by Activin A in erythroid differentiation. In the future work, whether NF-κB p65 binding to c-Jun on the AP-1 binding site of c-Jun promoter to enhance the c-Jun promoter activity will be investigated by ChIP assay. |
