Vitamin D3 Inhibits Wnt/β-Catenin and mTOR Signaling Pathways in Human Uterine Fibroid Cells

Abstract

Abstract Context: Somatic mutations in the Med12 gene are known to activate Wnt/β-catenin signaling in human uterine fibroids (UFs). Objective: The objective of the study was to examine the role of vitamin D3 in the modulation of Wnt/β-catenin and mammalian target of rapamycin (mTOR) signaling in human UF cells. Design: Immortalized human UF cells (HuLM) and human primary UF (PUF) cells were treated with increasing concentrations of vitamin D3 and thereafter analyzed using Western blots and immunocytochemistry. Main Outcome Measures: Wnt/β-catenin and mTOR signaling proteins in cultured HuLM and PUF cells were measured. Results: UF tumors with Med12 somatic mutations showed an up-regulation of Wnt4 and β-catenin as compared with adjacent myometrium. Vitamin D3 administration reduced the levels of Wnt4 and β-catenin in both HuLM and PUF cells. Vitamin D3 also reduced the expression/activation of mTOR signaling in both cell types. In contrast, vitamin D3 induced the expression of DNA damaged-induced transcription 4 (an inhibitor of mTOR) and tuberous sclerosis genes (TSC1/2) in a concentration-dependent manner in HuLM cells. Furthermore, we observed a concentration-dependent reduction of Wisp1 (Wnt induced signaling protein 1) and flap endonuclease 1 proteins in HuLM cells. Additionally, abrogation of vitamin D receptor expression (by silencing) in normal myometrial cells induces Wnt4/β-catenin as well as prompts a fibrotic process including an increase in cell proliferation and increased extracellular matrix production. Together these results suggest that vitamin D3 functions as an inhibitor of Wnt4/β-catenin and mTOR signaling pathways, which may play major roles in fibroid pathogenesis. Conclusion: Vitamin D3 may have utility as a novel long-term therapeutic and/or preventive option for uterine fibroids.