Smad signal transduction pathway involved in supression of VEGF on TGF-β1 induced EMT of HK-2 cells

Objective To examine the relationship between the inhibitory effect of VEGF on EMT of HK-2 cells and the influence of changes in the expressions of SnoN and Smad pathway on the effect of VEGF mentioned above. Methods The cultured HK-2 cells were divided into four groups:①without treatment group, ②treated with TGF-β1 (5 μg/L) alone as positive control group, ③ treated with VEGF165 (100 μg/L) alone group, ④treated with TGF-β1 (5 μg/L) and VEGF165 (100 μg/L) as the co-treatment group. The α-SMA, p-Smad 2/3 and Smad 2/3, Smad 7, SnoN expressions were assessed with western blot and RT-PCR, respectively. Results By western blot and RT-PCR analysis, it's found α-SMA expression was significantly increased in HK-2 cells treated with TGF-β1 (5 μg/L) compared with no treatment (P 0.05), and the ratio of p-Smad 2/3 and Smad 2/3 were significantly increased in both 30 min and 60 min (P 0.05). These phosphorylation marker mentioned above decreased in HK-2 cells co-treatment group compared with the positive controls, especially at 30 min (P 0.05). But Smad 7 expression was exactly opposite to that of these phosphorylation marker mentioned above. VEGF165 dramatically abrogated TGF-β1 induced α-SMA expression and phosphorylation of Smad 2/3 and restored Smad 7 expression in HK-2 cells (P 0.05). However, VEGF165 itself failed to induce phosphorylation of Smad 2/3 and the expression of Smad 7. SnoN protein expression significantly increased in HK-2 cells treated with TGF-β1 (5 μg/L) compared with no treatment (P 0.05) patients, but was not different in HK-2 cells co-treatment patients compared with positive controls(P ﹥0.05). SnoN mRNA expression was not significantly different in all groups (P ﹥0.05). Conclusions The results documented that VEGF165 may partially inhibit TGF-β1-induced EMT in HK-2 cells in vitro, and this effect is related to suppress of phosphorylation of Smad 2/3 directly, and up-regulates Smad 7 expression. However, the effect of VEGF165 on EMT may be not related to SnoN expression.