A phytoestrogen diarylheptanoid mediates estrogen receptor/Akt/glycogen synthase kinase 3β protein-dependent activation of the Wnt/β-catenin signaling pathway

Abstract

Estrogen promotes growth in many tissues by activating Wnt/β-catenin signaling. Recently, ASPP 049, a diarylheptanoid isolated from Curcuma comosa Roxb., has been identified as a phytoestrogen. This investigation determined the involvement of Wnt/β-catenin signaling in the estrogenic activity of this diarylheptanoid in transfected HEK 293T and in mouse preosteoblastic (MC3T3-E1) cells using a TOPflash luciferase assay and immunofluorescence. ASPP 049 rapidly activated T-cell-specific transcription factor/lymphoid enhancer binding factor-mediated transcription activity and induced β-catenin accumulation in the nucleus. Interestingly, the effects of ASPP 049 on the transcriptional activity and induction and accumulation of β-catenin protein in the nucleus of MC3T3-E1 cells were greater compared with estradiol. Activation of β-catenin in MC3T3-E1 cells was inhibited by ICI 182,780, suggesting that an estrogen receptor is required. In addition, ASPP 049 induced phosphorylations at serine 473 of Akt and serine 9 of GSK-3β. Moreover, ASPP 049 also induced proliferation and expressions of Wnt target genes Axin2 and Runx2 in MC3T3-E1 cells. In addition, ASPP 049 increased alkaline phosphatase expression, and activity that was abolished by DKK-1, a blocker of the Wnt/β-catenin receptor. Taken together, these results suggest that ASPP 049 from C. comosa induced osteoblastic cell proliferation and differentiation through ERα-, Akt-, and GSK-3β-dependent activation of β-catenin signaling. Our findings provide a scientific rationale for using C. comosa as a dietary supplement to prevent bone loss in postmenopausal women.

FIGURE 1.

ASPP 049 and E2 activate TCF/LEF reporter activity. A, time-dependent activation of TCF/LEF reporter activity by ASPP 049. HEK 293T cells were transiently cotransfected with TOPflash, β-catenin-FLAG, ERα, and Renilla luciferase vectors. After 48 h, cells were treated with ASPP 049 for the indicated time. Activity of the β-catenin signaling was quantified by measuring the relative firefly luciferase activity units normalized to Renilla luciferase activity. Data are expressed as the fold change compared with the cells transfected with pcDNA3.1 empty vector. B, concentration-dependent activation of TCF/LEF reporter activity by ASPP 049. HEK 293T cells were transiently cotransfected with TOPflash, β-catenin-FLAG, ERα, and Renilla luciferase vectors. After 48 h, cells were treated with the indicated concentration of ASPP 049 for 60 min and harvested for luciferase-based TOPflash assay for Wnt signaling. C, ASPP 049 and E2 activate TCF/LEF-driven luciferase activity. HEK 293T cells were transfected with the indicated plasmids. Treatment with ASPP 049 or E2 (60 min, 0.1 μm) selectively increased transcription from the TOPflash reporter plasmid compared with FOPflash. Data are represented as corrected luciferase; means ± S.E. (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with β-catenin-FLAG-transfected cells and treatment with vehicle (ANOVA). ###, p < 0.001 compared with β-catenin-FLAG-transfected cells and treatment with E2 (Student's t test).

FIGURE 2.

ASPP 049 and E2 increase β-catenin protein expression and transactivation in mouse preosteoblastic (MC3T3-E1) cells. A, representative blot showing time-dependent effects of ASPP 049 and E2 on β-catenin protein expression. MC3T3-E1 cells were treated with ASPP 049 (50 μm) or E2 (0.1 μm) for different times (30–120 min) and harvested for immunoblotting using anti-β-catenin and anti-β-actin antibodies. Maximum increase in β-catenin protein occurred by 60 min and then markedly decreased by 120 min after treatment with ASPP 049, whereas E2 induced a gradual increment of β-catenin up to 120 min. B, representative blot showing concentration-dependent effects of ASPP 049 and E2 on β-catenin protein expression. MC3T3-E1 cells were treated with various concentrations of ASPP 049 (0.01–50 μm) or E2 (0.001–1 μm) for 60 min and harvested for immunoblotting using anti-β-catenin and anti-β-actin antibodies. C, ASPP 049 and E2 augment TCF/LEF-dependent transcription in MC3T3-E1 cells. After 24 h of transfection with the indicated reporter plasmids, cells were treated with 0.1 μm ASPP 049 or 0.1 μm E2 for 60 min. Activity of the β-catenin signaling pathway was quantified by measuring the relative firefly luciferase activity units normalized to Renilla luciferase activity. ASPP 049 and E2 selectively increased transcription only from the TOPflash reporter plasmid. Data are represented as mean ± S.E. (n = 4). ***, p < 0.001 compared with vehicle control treatments (ANOVA); #, p < 0.05 compared with E2-treated cells (Student's t test).

FIGURE 3.

ASPP 049 and E2 increase nuclear localization of β-catenin. MC3T3-E1 cells were treated with 0.1 μm ASPP 049 or 0.1 μm E2 for 60 min. Cells were fixed and immunostained for β-catenin using anti-β-catenin (A) and anti-dephosphorylated β-catenin (anti-ABC) (B) antibodies (red). TO-PRO3 was used as a nuclear marker (blue). Samples were visualized with confocal laser microscopy. β-catenin immunostaining was predominantly localized in the nucleus after treatments with ASPP 049 and E2. Scale bar = 20 μm. DMSO, dimethyl sulfoxide.

FIGURE 4.

Activation of Wnt/β-catenin signaling by ASPP 049 and E2 is GSK-3β- and Akt-dependent. A, GSK-3β-dependent activation of Wnt/β-catenin signaling. The activation of Wnt/β-catenin signaling by ASPP 049 or E2 was measured in the presence of constitutively active β-catenin mutant S33Y, which is insensitive to GSK-3β-mediated phosphorylation. HEK 293T cells were transiently cotransfected with TOPflash, Renilla luciferase vector, and the indicated plasmids. After 24 h, cells were incubated with 0.1 μm ASPP 049 or 0.1 μm E2 for 60 min. Activity of the β-catenin signaling pathway was quantified by measuring the relative firefly luciferase activity units normalized to Renilla luciferase activity. Data is expressed as fold change compared with pcDNA3.1-transfected cells and represented as mean ± S.E. (n = 4). **, p < 0.01; ***, p < 0.001 compared with β-catenin-FLAG-transfected cells treated with vehicle (ANOVA); #, p < 0.05 compared with β-catenin-FLAG-transfected cells and treatment with E2 (Student's t test). B, ASPP 049 and E2 induce GSK-3β-dependent β-catenin expression. MC3T3-E1 cells were pretreated with 40 mm LiCl or 40 mm NaCl for 16 h and then incubated in medium containing 0.1 μm ASPP 049 or 0.1 μm E2 for 60 min. Cells were harvested and analyzed by Western blotting using anti-β-catenin and anti-β-actin antibodies. ASPP 049 and E2 failed to increase β-catenin expression after LiCl pretreatment. C, ASPP 049 and E2 increase GSK-3β and Akt phosphorylation in MC3T3-E1 cells. MC3T3-E1 cells were treated with 0.1 μm ASPP 049 or 0.1 μm E2 for different times (30–120 min) and then harvested for Western blotting using anti-phospho-GSK-3β (Ser-9), anti-GSK-3β, anti-phospho-Akt (Ser-473), and anti-Αkt antibodies. DMSO, dimethyl sulfoxide. D, LY 294002 blocks ASPP 049- and E2-induced GSK-3β and Akt phosphorylation. MC3T3-E1 cells were treated with 0.1 μm ASPP 049 or 0.1 μm E2 for 60 min with or without pretreatment with LY 294002, a specific PI3 Kinase inhibitor (10 μm for 3 h), and then harvested for Western blotting using the indicated antibodies.

FIGURE 5.

Induction of β-catenin protein expression by ASPP 049 and E2 requires estrogen receptors. MC3T3-E1 cells were preincubated with ER antagonist ICI 182,780 (1 μm) for 2 h prior to treatment with 0.1 μm ASPP 049 or 0.1 μm E2 for 60 min. Cells were harvested and immunoblotted for β-catenin protein expression using anti-β-catenin and anti-β-actin antibodies. Bar graphs show the mean normalized densitometry values and the corresponding standard deviations from four independent experiments. Data are represented as means ± S.E. (n = 4). *, p < 0.05 and ***, p < 0.001 compared with vehicle control treatments (ANOVA); ##, p < 0.01; ###, p < 0.001 compared with E2 and ASPP 049 treatments (Student's t test). DMSO, dimethyl sulfoxide.

FIGURE 6.

ASPP 049 and E2 enhance cell proliferation and Wnt target gene expression. A, ASPP 049 increases MC3T3-E1 cell proliferation. MC3T3-E1 cells were treated with 0.1 μm ASPP 049, 0.1 μm E2 or 10 mm LiCl for the indicated times. The proliferation of MC3T3-E1 cells was observed by MTT assay. Cell proliferation is expressed as % of control group (dimethyl sulfoxide, DMSO). B and C, ASPP 049 and E2 increase Wnt target gene expression. MC3T3-E1 cells were treated with 0.1 μm ASPP 049 or 0.1 μm E2 for the indicated times (1, 6, 24, and 48 h). Total RNAs were extracted for quantitative PCR of the Wnt target genes: Axin2 (B) and RUNX2 (C). Data are represented as mean ± S.E. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with vehicle control treatments (ANOVA).

FIGURE 7.

ASPP 049-induced preosteoblastic cell differentiation is mediated by Wnt/β-catenin signaling. A, ASPP 049 induces ALP expression. MC3T3-E1 cells were cultured in differentiation medium and treated with various concentrations of ASPP 049 or E2 for 21 days. The ALP expression was stained with Naphthol AS-MX phosphate. DMSO, dimethyl sulfoxide. B, ASPP 049 induces ALP activity. MC3T3-E1 cells were cultured in differentiation medium and treated with indicated concentrations of ASPP 049 or E2 for 7 days. Cells were harvested, and ALP activity was measured at 405 nm and normalized with total protein. C, DKK-1 blocks ASPP 049-induced ALP activity. MC3T3-E1 cells were cultured in differentiation medium and treated with 1 μm ASPP 049 or 0.1 μm E2 for 7 days with or without pretreatment with DKK-1 (0.2 μg/ml) for 3 h, as indicated. Cells were harvested, and ALP activity was measured at 405 nm and normalized with total protein. Data are expressed as fold change compared with the cells treated with vehicle control and are mean ± S.E. (n = 4). **, p < 0.01; ***, p < 0.001 compared with treatments (ANOVA). ##, p < 0.01; ###, p < 0.001 compared with the presence and absence of DKK-1 (Student's t test).