IL-4诱导THP-1细胞表达DC—SIGN的信号通路调节研究

IL-4 induced signal pathways regulating DC-SIGN expression in THP-1 cells

目的研究IL-4诱导THP-1细胞表达DC—SIGN的信号调节通路，探索DC．SIGN表达的信号调控网络。方法以佛波脂（PMA）刺激THP—l细胞24h后加入IL4作用48h诱导DC．SIGN的表达，并设ERK阻断剂、NF—KB阻断剂、JAK-STAT阻断剂和MAPK阻断剂处理组。用RT—PCR检测DC—SIGN的mRNA表达，Westernblot检测胞质内DC—SIGN蛋白的表达，流式细胞术检测细胞表面DC—SIGN的表达。另外，提取IL-4诱导0、10、20、30、60和120min的THP一1细胞胞质和胞核蛋白，Westernblot检测不同信号通路的信号蛋白及其磷酸化蛋白的变化。结果IL．4可以大幅提高DC—SIGN在THP-1细胞上的表达，包括mRNA和胞膜蛋白水平。在mRNA、胞质和细胞表面蛋白表达3个水平上，ERK通路阻断剂阻断效果最好，几乎完全阻断了IL4的诱导效果，JAK—STAT和NF—KB通路阻断剂具有部分阻断效果，而p38通路阻断剂无阻断效果。信号蛋白检测结果显示，IL4诱导0～120min内，胞质磷酸化ERKl／2、磷酸化STAT6以及NF—KBp65、NF—KBpS0、磷酸化IKB和磷酸化AKT随时间推移浓度逐渐升高，而p38MAPK及其磷酸化蛋白浓度无明显改变。胞核内胞质磷酸化ERKl／2、磷酸化STAT6以及NF．KBp65和NF．KBpSO随时间推移浓度逐渐升高。结论ERK、JAK-STAT和NF．KB通路参与了DC．SIGN启动子的活化，其中以ERK通路为主。

Objective To detect the signal pathways through which IL-4 regulates expression of DC-SIGN in THP-1 cells. Methods We used phorbol 12-myristate 13-acetate（PMA） differentiated THP-1 cells as the in vitro model of monoeyte/macrophage cells to study the signal pathways involved in IL-4 regula- ted expression of DC-SIGN. DC-SIGN mRNA expression was detected by RT-PCR. Cytoplasmic DC-SIGN protein was tested by Western blot. Flow eytometry was used to detect eel1 surface expression of DC-SIGN. Cytoplasm and nuclear protein of PMA stimulated THP-1 cells induced by IL-4 for 0, 10, 20, 30, 60 and 120 min was extracted and detected by Western blot for signal pathway signaling protein and phosphoprotein. Results We found that a high expression of DC-SIGN could be induced by IL-4 at the levels of mRNA and cell surface protein. Up-regulated expression of DC-SIGN was almost completely blocked by the specific in- hibitor of ERK pathway, and partly reduced by the specific inhibitors of JAK-STAT and NF-KB pathways. The activation of the three signaling pathways was directly confirmed by testing the phosphorylation of protein kinase within the cytoplasm and nucleus over time. Conclusion Multiple signaling pathways are involved in IL-4 induced high expression of DC-SIGN on THP-1 cells, in which ERK pathway is the main signal pathway.