Abstract:

Objective To investigate the effects of endothelin-1 （ET-1） and endothelin-3 （ET-3） on the expression of transformation growth factor-beta 1 （TGF-β1） and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group （no stimulation）, ET-1 group （stimulated with ET-1）, ET-1 + BQ123 group （treated with ET-1 and BQ123）, ET-1 + BQ788 group （treated with ET-1 and BQ788）, ET-3 group （stimulated with ET-3）, to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 （P-Smad 3）. Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells （both P < 0.05）. BQ123 significantly blocked the up-regulatory effect of ET-1 on the expression TGF-β1 protein（P < 0.05）, but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1 significantly elevated the expression of P-Smad 3 in A375 cells （P < 0.05）, and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells （P < 0.05）. Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.