Zearalenone exposure affects the Wnt/β-catenin signaling pathway and related genes of porcine endometrial epithelial cells in vitro

Abstract

Objective: ZEA has estrogen-like effects. Our previous study has shown that ZEA (0.5-1.5 mg/kg) could induce abnormal uterine proliferation through TGF signaling pathway. In order to further study the other regulatory networks of uterine hypertrophy caused by ZEA, the potential mechanism of ZEA on porcine endometrial epithelial cells was explored by the Illumina Hiseq 2000 sequencing system in this study. Methods: The PECs were treated with ZEA at 0 (ZEA0), 5 (ZEA5), 20 (ZEA20), and 80 (ZEA80) μmol/L for 24 h. The collected cells were subjected to cell cycle, RNA-seq, qRT-PCR, immunofluorescence, and western blot analysis. Results: The proportion of cells in the S and G2 phases decreased (p < 0.05), but the proportion of cells in the G1 phase increased (p < 0.05) in the ZEA80 treatment. Data analysis revealed that the expression of Wnt pathway-related genes, estrogen-related genes, and MAPK pathway-related genes increased (p < 0.05), but the expression of genetic stability genes decreased (p < 0.05) with increasing ZEA concentrations. The relative mRNA and protein expression of WNT1, β-catenin, glycogen synthase kinase 3β (GSK-3β) were increased (p < 0.05) with ZEA increasing, while the relative mRNA and protein expression of cyclin D1 (CCND1) was decreased (p < 0.05). Moreover, our immunofluorescence results indicate that β-catenin accumulated A c c e p t e d A r t i c l e around the nucleus from the cell membrane and cytoplasm with increasing ZEA concentrations. Conclusion: In summary, ZEA can activate the Wnt/β-catenin signaling pathway by up-regulating WNT1 and β-catenin expression, so as to promote the proliferation and development of porcine endometrial epithelial cells. At the same time, the up-regulation of GSK-3β and down-regulation of CCND1, as well as the mRNA expression of other pathway related genes indicated that other potential effects of ZEA on the uterine development need further study.

Zearalenone and the Immune Response

“…In mouse ovarian granulosa cells Chen et al [ 96 ] and Zhang et al [ 97 ] demonstrated by MTT, EdU and flow cytometry that ZEA suppressed in vitro cell viability at 30–150 μM and increased apoptosis at 15–60 μM after 24 or 72h of exposure. Close to the results recorded by Song et al [ 95 ] in pig (a decreased of cell proportion in the S and G2 phase at ZEA 80 µmol/L), Zhang et al [ 97 ], found that mouse granulosa cells were arrested in G1 phase of cell cycle and the cells proportion decreased in phase S and G2 after 30 μM ZEA treatment. However, in another study this author [ 98 ] found species specific ZEA effect, pig being more sensitive than mouse.…”

“…In weaned piglets, ZEA (0.5, 1.0 and 1.5 mg/kg) induces ovarian development by accelerating ovarian follicles proliferation through the activation of ERs/GSK-3β-dependent Wnt-1/β-catenin signaling pathway [ 94 ]. Also, ZEA (0.5–1.5 mg/kg) determined an abnormal uterine proliferation through TGF signaling pathway [ 95 ]. Investigating other regulatory pathways involved by ZEA in uterine hypertrophy, these authors exposed porcine endometrial epithelial cells to ZEA 0, 5, 20 and 80 μmol/L for 24 h and cell cycle was analyzed.…”

“…Investigating other regulatory pathways involved by ZEA in uterine hypertrophy, these authors exposed porcine endometrial epithelial cells to ZEA 0, 5, 20 and 80 μmol/L for 24 h and cell cycle was analyzed. A significant lower proportion of cells in S and G2 phases and an increase in the phase of G1 was found at ZEA 80 μmol/L [ 95 ]. The related mechanism involved also the activation of Wnt/β-catenin signaling pathway.…”

Impact of Fusarium-Derived Mycoestrogens on Female Reproduction: A Systematic Review

“…In pig endometrial stromal cells, β-ZOL altered transcription of cell cycle-dependent kinases and reduced phosphorylation of MAPK and AKT (PKB) kinases [ 119 ]. Additionally, recent studies in porcine endometrial cells indicate that ZEN can activate the WNT/β-catenin signaling pathway to promote proliferation [ 123 ]. Overall, the evidence from murine models points toward strong apoptotic effects of mycoestrogens in the uterus and decreased cell viability in endometrial stromal and granulosa porcine-derived cells [ 101 , 118 , 120 ].…”

Quantitative Proteomic Analysis of Zearalenone Exposure on Uterine Development in Weaned Gilts

“…In addition, the relative mRNA and protein expressions of proliferating cell nuclear antigen (PCNA), BCL-2 associated X protein (BAX), and B-cell lymphoma/leukemia-2 (BCL-2) were up-regulated through the TGF-β1/Smad3 signaling pathway in uteri of gilts fed 1.5 mg/kg of a ZEA-contaminated diet [ 12 ]. Changes of relative mRNA and protein expressions of GSK-3 and Cyclin D1 of the Wnt/β-catenin signaling pathway in ZEA-treated porcine endometrial epithelial cells were also observed [ 13 ]. However, the potential physiological differences and certain molecular mechanisms of ZEA-induced uterine hypertrophy are unclear.…”