Objective: To explore the molecular mechanism of the synergetic effect of mitogen-activated protein kinase (MAPK) pathway on expression of tumor necrosis factor alpha (TFG-alpha) gene in murine monocyte (RAW264.7 cells) induced by lipopolysaccharide (LPS).
Methods: RAW264.7 cells were cultured, transfected with vectors with mutants of different constitutively active MAPK kinases, i. e., ERK, JNK, and p38, vectors with inactivated mutants of MAPK kinase, and murine TNF-alpha driven report gene pGL2TNFLuc, and then stimulated by LPS. Protein kinase assay was used to analyze the activity of MAPKs. Western blotting was used to detect the amount of target proteins. RT-PCR was used to detect the influence of the MEK/REK inhibitor PD98059, JNK inhibitor SP600125, and p38 inhibitor SB203580 on the expression of TNF-alpha gene induced by LPS.
Results: ERK1, JNK1, and p38 MAPKs were transiently activated by LPS. After the vectors with activated MAPK mutants were co-transfected with murine TNF-alpha driven report gene pGL2TNFLuc into RAW264.7 cells, the transcription activity of TNF-alpha promoter was induced to different degrees. After the vectors with inactivated MAPK mutants were co-transfected with murine TNF-alpha driven report gene pGL2TNFLuc into RAW264.7 cells, the transcription activity of TNF-alpha promoter was inhibited.
Conclusion: The transactivity of TNF-alpha promoter stimulated by LPS involves ERK, JNK, and p38 pathways. These three major MAPK pathways exert synergetic effects on the gene regulation of TNF-alpha expression.