Transforming growth factor-β signaling pathway cross-talking with ERα signaling pathway on regulating the growth of uterine leiomyoma activated by phenolic environmental estrogens in vitro

The aim of this paper is to study the participation of transforming growth factor-beta (TGF-beta) signaling pathway in mediating the growth of human uterine leiomyoma (UL) activated by phenolic environmental estrogens (EEs), via the interaction between TGF-beta and ER signaling pathways. The UL cells were prepared by primary culture and subculture methods. To validate the role of TGF-beta 3 (5 ng/ml) for the viability of human uterine leiomyoma cells, CCK-8 assay was performed in each of five treatment groups including E-2 group (E-2 10(9) mol/l), BPA group (bisphenol A 10 mu mol/l), NP group (nonylphenol 32 mu mol/l), OP group (octylphenol 8 mu mol/l), or control group (DMSO only). Subsequently, qRT-PCR was applied to detect mRNA expressions of ER alpha and c-fos, while western blot assay was used to test the expressions of p-Smad3, SnoN, and c-fos proteins in all settings mentioned above; the expressions were compared among different groups, and also in settings with and without synchronous treatment of ICI 182,780. Primarily cultured UL cells were successfully established. Compared with the control group, there were statistically significant increases in the proliferation rate of the UL cells in all EE groups or treated with TGF-beta 3 only (p < 0.05). Nevertheless, a slight decrease in proliferation rate of UL was detected in coexistence with TGF-beta 3 in all EE groups (p > 0.05). Interestingly, mRNA expressions of ER alpha and c-fos reduced in the setting of coexistence of TGF-beta 3 and EEs compared to isolated EE treatment (p < 0.05). Compared with the control group, the expression of p-Smad3 and c-fos proteins significantly decreased (p < 0.05) in each of E-2, BPA, NP, and OP group, and the expression of SnoN protein also significantly reduced only in BPA and NP groups (p < 0.05), followed by TGF-beta 3 treatment. When adding ICI 182,780, the expression of p-Smad3 protein significantly increased in OP group (p < 0.05), but slightly increased in E-2, BPA, NP, and OP groups (p > 0.05). However, compared with the control group, the expressions of SnoN and c-fos proteins significantly decreased (p < 0.05) after adding ICI182,780. Moreover, there was a significant statistical difference in the expression of p-Smad3, SnoN, and c-fos proteins between pre- and post-treatment of ICI 182,780 in all groups (p < 0.05). The ER alpha signaling pathway and TGF-beta signaling pathway have different roles in the control of UL cell proliferation. The phenolic EEs can be a promoter of UL cell proliferation, which is mediated by ER alpha signaling pathway and its cross-talking with TGF-beta signaling pathway. Both less exposure to EEs and blockade of TGF signaling pathway are necessary strategies to prevent UL.