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

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.

Conclusions: Vitamin D3 may have utility as a novel long-term therapeutic and/or preventive option for uterine fibroids.

Figure 1.

UFs having Med12 somatic mutations showed an up-regulation of β-catenin, Wnt4, and Wisp1 as compared with Med12-negative adjacent normal myometrium. A, Protein lysates were prepared from paired myometrium (M) and uterine fibroid (F; n = 5) from five individual subjects. Equal amounts of each protein lysates (30 μg) were analyzed by Western blots using anti-β-catenin, anti-Wnt4, and anti-Wisp1 antibodies. B, The intensity of each protein band was quantified using image-analyzing software, normalized to corresponding β-actin, and relative values were used to generate data graphs. Each underline shows UFs and the adjacent myometrium from the same patient. P1, P2, P3, P4, and P5 indicate fibroid subjects. Med12 mutation status in fibroids from subjects P1, P2, P3, P4, and P5 are 130G>A, 107T>C, 105A>T, 131 G>A, and 105A>T, respectively.

Figure 2.

Effect of vitamin D3 on β-catenin protein expression in cultured HuLM cells. A, HuLM cells were serum starved and treated with increasing concentrations of vitamin D3 (0, 10, 100, and 1000 nM) for 48 hours. Equal amounts of each cell lysate were analyzed by Western blots using anti-β-catenin antibody. β-Actin Western blot was used as loading control. The intensity of each protein band was quantified and normalized to corresponding β-actin. *, P < .05 when compared with control. B, Immunofluorescence analyses were performed using HuLM cells cultured on glass coverslips and treated with increasing concentrations of vitamin D3 (0, 10, 100, and 1000 nM) for 48 hours. Cells were fixed, permeabilized, and stained with monoclonal anti-β-catenin antibody followed by incubating with carbocyanine 3-conjugated antimouse secondary antibody. β-Catenin staining (red) was monitored by a fluorescence microscopy. Nuclei of cells were stained with 4′,6-diamino-2-phenylindole. Pictures were taken at ×200 magnification. These data are representative of at least two independent experiments, each performed in duplicate.

Figure 3.

Effect of vitamin D3 on protein expression of Wnt4, Wisp1, and FEN1 in cultured human uterine fibroid cells. A, HuLM cells were serum starved and treated with increasing concentrations of vitamin D3 (0, 1, 10, 100, and 1000 nM) for 48 hours. Equal amounts of each cell lysate were analyzed by Western blots using anti-Wnt4, anti-Wisp1, and anti-FEN1 antibodies. β-Actin Western blot was used as loading control. B and C, Immunofluorescence analyses were performed using both HuLM cells (B) and human PUF cells (C) cultured on glass coverslips and treated with increasing concentrations of vitamin D3 (0, 10, 100, and 1000 nM) for 48 hours. Cells were fixed, permeabilized, and stained with anti-Wnt4 antibody (1:50 dilution) followed by incubating with carbocyanine 3-conjugated antirabbit secondary antibody. Wnt4 staining (red) was monitored by fluorescence microscopy. Nuclei of cells were stained with 4′, 6-diamino-2-phenylindole. Pictures were taken at ×200 magnification.

Figure 4.

Effect of vitamin D3 on protein expression of DDIT4, TSC1, and TSC2 in cultured human uterine fibroid cells. A, HuLM cells were serum starved and treated with increasing concentrations of vitamin D3 (0, 1, 10, 100, and 1000 nM) for 48 hours, as described above. Equal amounts of each cell lysate were analyzed by Western blots using anti-DDIT4, anti-TSC1, and anti-TSC2 antibodies. β-Actin Western blot was used as loading control. B and C, Immunofluorescence analyses were performed using both HuLM cells (B) and human PUF cells (C) cultured on glass coverslips and treated with increasing concentrations of vitamin D3 (0, 10, 100, and 1000 nM) for 48 hours. Cells were fixed, permeabilized, and stained with anti-DDIT4 antibody (1:50 dilution) followed by incubating with carbocyanine 3-conjugated antirabbit secondary antibody. DDIT4 staining (red) was monitored by fluorescence microscopy. Nuclei of cells were stained with 4′,6-diamino-2-phenylindole. Pictures were taken at ×200 magnification.

Figure 5.

Effect of vitamin D3 on activation of mTOR signaling in cultured HuLM and human PUF cells. HuLM cells (A) and human PUF cells (B) were serum starved and treated with increasing concentrations of vitamin D3 (0, 1, 10, 100, and 1000 nM) for 48 hours. Equal amounts of each cell lysate were analyzed by Western blots using anti-mTOR, anti-p-mTOR, anti-p-p70S6 kinase, and anti-p70S6 kinase antibodies, as indicated. β-Actin Western blot was used as loading control.

Figure 6.

Knockdown VDR induces Wnt4/β-catenin and mTOR signaling and induces proliferation of UtSMC cells. A, VDR was targeted for knockdown in UtSMC cells by infecting with lentiviruses expressing VDR-specific shRNA or scrambled control. Cell lysates were analyzed by Western blotting using anti-VDR, anti-β-catenin, anti-Wnt4, anti-p-mTOR, and anti-mTOR antibodies. β-Actin was used as loading control. B, Quantification of protein expression of above proteins were normalized to β-actin and shown. *, P < .05 as compared with control. C, Cell lysates were also analyzed by Western blotting using antifibronectin, anticollagen type 1, and anti-PCNA antibodies. D, Normalized protein levels are shown. *, P < .05 as compared with control. E, Both scrambled control and VDR knockdown cells were seeded into 12-well plates and cultured in phenol-free DMEM/F12 medium containing 10% charcoal stripped fetal bovine serum. Cultures were replenished every other day with fresh conditioned media. Cell proliferation MTT assay was performed at days 2, 3, 4, and 5. Each data point is the mean SD of triplicate wells (n = 3). *, P < .05 as compared with the corresponding control.