| Summary: | 碩士 === 國立中興大學 === 生物醫學研究所 === 93 === The process of differentiation from monocytes to macrophages plays a pivotal role in inflammation and immune responses, but the molecular mechanism underlying this process remains elusive. THP-1, a human acute monocytic leukemia cell line, can be induced to differentiate into macrophage-like cells by phorbol ester PMA and thus represents an excellent in vitro model to study this process. In this study, the molecular basis of the monocytes-to-macrophages differentiation was explored by molecular profiling of gene expression in THP-1 differentiation induced by PMA. A cDNA microarray containing 9,600 human cDNAs was used to analyze the gene expression profile of macrophage differentiation. After statistical analysis, we found there were 127 genes whose expression was up-regulated at least two times during differentiation, while the expression level of 123 genes was two times lower. We further performed real-time quantitative PCR and western blotting analysis to confirm the change of expression for certain genes at the transcriptional and translational levels, respectively. In particular, our data indicated that the expression of PEA-15 mRNA and protein was greatly increased after PMA treatment in a time-dependent manner, implicating its important role in macrophage differentiation. Furthermore, we investigated the role of MAPK kinase pathway in the differentiation of monocytic THP-1 cells into macrophages. We found that ERK phosphorylation was increased after PMA induction with maximal level reached at 3 hours, while the differentiation was completely abolished with the treatment with U0126, a potent MAPK inhibitor. Significantly, PEA-15 expression began to emerge at the time point when ERK phosphorylation started to decline, and the expression was completely lost in the presence of U0126. Taken together, our data demonstrated the increased expression of PEA-15 in the process of differentiation is dependent on MAPK kinase activity. Furthermore, the reciprocal change of ERK phosphorylation and PEA-15 expression during differentiation suggests the possible cross-talk between ERK activation and PEA-15 expression. Lastly, we investigated the role of PEA-15 in THP-1 differentiation by overexpression of PEA-15 in THP-1 cells after transfection of pIRES2- EGFP-PEA/15, which encodes both PEA-15 and EGFP. Although the transfection efficiency was extremely low, we found that all the cells with successful pIRES2-EGFP-PEA/15 transfection did not show cell attachment to their cultured dishes, the first step of macrophage differentiation. Based on our results, we speculate that PEA-15 may be dispensible for initiating differentiation, but rather required for the stages of macrophage maturation.
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