Objective: To investigate the role of ERK/JNK in the alteration of activator protein-1(AP-1) signaling pathway in human embryonic lung fibroblasts(HELFs) induced by carbon black.
Methods: HELFs were cultured in RPMI 1640 medium containing 0, 15, 30, 60, 120 or 240 μg/mL carbon black for 24 h, and the appropriate dose of carbon black was determined by MTT assay result. HELFs were divided into three groups: HELFs, HELFs transfected with ERK dominant negative mutant plasmid(DN-ERK) and HELFs transfected with JNK dominant negative mutant plasmid(DN-JNK). 100 μg/mL carbon black was used to treat HELFs(CB), DN-ERK HELFs(CB-DN-ERK), DN-JNK HELFs(CB-DN-JNK), and HELFs without any treatment were considered as control group. At 0, 1, 2, 4, 8, 12, 24 and 36 h of CB and control groups HELFs, the western blot was used to detect ERK, p-ERK, JNK, p-JNK, p38, p-p38, c-Jun, p-c-Jun, c-Fos, p-c-Fos protein expression levels, and AP-1 activity was detected by luciferase method. Whereas CB-DN-ERK and CB-DN-JNK HELFs were detected only at 24 h.
Results: Compared with the protein expression levels at 0 h, CB group HELFs ERK and p-ERK protein expression increased at each time point, whereas p38 protein expression decreased. AP-1 activity of CB group HELFs was declined to the lowest at 8 h(0.72±0.12), and upregulated to the peak at 36 h(1.38±0.11). CB group HELFs c-Fos, p-c-Fos and c-Jun protein expression levels at each time point from 1 h to 24 h were greater than those of 0 h, and p-c-Jun protein expression levels at 1 h, 2 h, 4 h, 8 h, 36 h were also greater than those of 0 h. CB group HELFs AP-1 activity, ERK, p-ERK, JNK, p-JNK, p38, p-p38, c-Jun, p-c-Jun, c-Fos, p-c-Fos protein expression levels changes followed biphasic patterns. There were no statistically significant differences in AP-1 activity between CB group HELFs(1.03±0.10) and CB-DN-ERK group(1.02±0.04) or CB-DN-JNK group(1.09±0.10) HELFs(t=0.16, P=0.88; t=0.73, P=0.50). However, compared with CB group HELFs, c-Fos(t=5.31, P=0.01), p-c-Fos(t=4.33, P=0.01), p-c-Jun(t=10.95, P& lt; 0.01)in CB-DN-JNK group, and c-Fos protein expression levels in CB-DN-ERK group(t=42.72, P& lt; 0.01)were significantly decreased.
Conclusion: While carbon black induces HELFs increased protein expression levels of ERK, p-ERK, c-Jun, p-c-Jun, c-Fos and p-c-Fos, JNK may upregulate c-Fos, p-c-Fos, p-c-Jun protein expression levels, and ERK may upregulate c-Fos protein expression level.
Keywords: activator protein-1; c-Jun N-terminal kinases; carbon black; extracellular signal-regulated kinases; human embryonic lung fibroblasts.