前列腺素E1活化轉錄因子C/EBPβ 與促使介白素-6表現之機轉研究

Abstract

C/EBPβ 是CCAAT/Enhancer Binding Protein 家族其中的一員，在許多生理作用上扮演著重要的角色，例如細胞的分化與增生、免疫反應、癌化與其他許多細胞內之反應。在實驗室之前的研究中發現，前列腺素E1 (PGE1) 在人類腎臟上皮細胞 (HEK 293) 中，可以促使帶有C/EBPβ 可辨識區域報導基因的表現，這一活化的過程，被證實是經由PKA的路徑。在我們現今的研究中，我們發現在前列腺素E1的處理下，C/EBPβ 會從細胞質轉進到細胞核中，而這一現象需要經由PKA的活化才能完成。此外，C/EBPβ 和DNA的結合力，在前列腺素E1的處理下會增加。C/EBPβ 下游的基因介白素-6，在前列腺素E1的刺激下，mRNA的表現量會增加，而這樣的現象，以PKA抑制劑H89處理之下，介白素-6 mRNA的表現量有明顯被抑制的結果。綜合上述結果，我們發現介白素-6 mRNA的表現量，確實是經由PKA活化的C/EBPβ 所調控。 在許多文獻中指出，C/EBPβ 會與其他蛋白質結合，進而影響下游基因的調控，目前已知會和C/EBPβ 結合的蛋白質有TIF-1, CUGBP1, Mediator complexes, Egr1, glucocorticoid receptor, NF-kappa B, C/ATF, 以及C/EBP 家族中的其他成員。為了找出在前列腺素E1的刺激下，是否有其他蛋白質會和C/EBPβ 形成複合物，而影響C/EBPβ 的活化情形，我們利用共同免疫沉澱法□(co-immunoprecipitation)與介質輔助雷射脫附游離□飛行時間式質譜儀 (MALDI-TOF)來分析與鑑定蛋白質的身分。在共同免疫沉澱法的結果中，我們發現有許多蛋白質會和C/EBPβ 形成複合物，目前我們已經證實poly (ADP-ribose) polymerase-1 (PARP-1)確實會和C/EBPβ 結合，另一方面，我們利用PARP酵素活性的抑制劑3-aminobenzamide，發現由PARP家族所造成的poly(ADP-ribosyl)ation，在前列腺素E1引發C/EBPβ 的活化中，扮演著抑制C/EBPβ 活性的角色；此外，PARP-1與C/EBPβ 的親合力在前列腺素E1的刺激下會有所變化，此一變化與PARP-1在細胞核中的分佈情形相似。藉由找出與C/EBPβ 結合的蛋白質，我們可以更清楚的闡明前列腺素E1活化C/EBPβ 與促使介白素-6基因表現的機制。

C/EBPβ, a CCAAT/Enhancer Binding Protein β, was found to play an essential role in the differentiation, inflammatory response, proliferation, tumorigenesis, and other numerous cellular responses. Recently, we found that E-type prostaglandin (PGE1) could activate C/EBPβ responding element (C/EBPβRE)-based reporter gene expression in HEK 293 cells. This process is mediated by a PKA-dependent manner. In this study, we found that the localization of C/EBPβ could be changed by PGE1 from cytoplasm into nucleus which was also dependent on the activity of PKA. Furthermore, DNA binding of C/EBPβ was also increased in response to the treatment of PGE1. The upregulation of C/EBPβ downstream gene, IL-6, was increased upon PGE1 treatment. This PGE1-induced IL-6 upregulation could be suppressed by H89, an inhibitor of PKA. This result suggests that the PGE1-mediated increase of IL-6 mRNA level is possibly via the PKA-dependent activation of C/EBPβ. It has been shown that C/EBPβ can be regulated by co-activator, such as TIF-1, CUGBP1, Mediator complexes, Egr1, glucocorticoid receptor, NF-kappa B, C/ATF and other C/EBP family. Interestingly, we found that in the nucleus C/EBPβ also associated with several proteins in responses to the treatment of PGE1 by co-immunoprecipitation. One of associated protein was further identified as poly (ADP-ribose) polymerase-1 (PARP-1) using matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF). This observation was confirmed by Western blotting. Further studies showed that poly(ADP-ribosyl)ation played a repressive role in PGE1-induced C/EBPβ activation. In conclusion, we find that PGE1 can induce the activity of C/EBPβ via a PKA-dependent pathway. In the nucleus, the activity of C/EBPβ may be further regulated by a co-regulator, PARP-1.