Fig 6. Activation of MAPKs in HCS-2/8 cells treated with 5-HT or agonist of each 5-HT₂ receptor and rescue effect of inhibitors of ERK1/2 and JNK on the BW723C86-decreased CCN2 production.

(A) HCS-2/8 cells were grown until they had reached confluence. Then, the cells were treated with 5-HT, NBOH-2C-CN or BW723C86 at the indicated concentrations. After 15 min, cell lysates were prepared; and Western blot analysis was performed with the antibodies against the indicated proteins. The levels of phosphorylated ERK1/2 and JNK were increased by the treatment with BW723C86 at a concentration of 10 μM. In contrast, the levels of phosphorylated p38 MAPK were increased by the treatment with NBOH-2C-CN at the concentrations of 10 μM and 100 μM. (B) HCS-2/8 cells were grown until they had reached confluence. Then, when the cells were treated with 5-HT (10 μM) or BW723C86 (10 μM), PD98059 (MEK1 inhibitor; 50 μM) or SP600125 (JNK inhibitor; 50 μM) was applied to the cultures simultaneously. After 24 h, the cell lysates were prepared; and Western blot analysis was performed with anti-human CCN2 rabbit serum and β-actin antibody. When HCS-2/8 cells were treated with 5-HT, PD98059 or SP600125 had no effects. In contrast, when the cells were treated with BW723C86, CCN2 production was rescued by either PD98059 or SP600125. The graphs give the results of Western blotting using anti-CCN2 antibody and quantified by densitometric analysis, with normalization by the levels of β-actin. The ordinate indicates the fold change relative to untreated controls (ratio = 1.0; dotted line). The graph indicates relative densitometry (untreated control = 1.0; dotted line) from 6 independent cultures and analyzed by Bonferroni's test, and p < 0.05 (*) was considered significant.