PMC:2783367 / 35436-38700 JSONTXT

{"project":"2_test","denotations":[{"id":"19736547-15817814-66178371","span":{"begin":594,"end":596},"obj":"15817814"},{"id":"19736547-15817814-66178371","span":{"begin":594,"end":596},"obj":"15817814"},{"id":"19736547-15817814-66178372","span":{"begin":707,"end":709},"obj":"15817814"},{"id":"19736547-15817814-66178372","span":{"begin":707,"end":709},"obj":"15817814"},{"id":"19736547-18177486-66178373","span":{"begin":1273,"end":1275},"obj":"18177486"},{"id":"19736547-18177486-66178373","span":{"begin":1273,"end":1275},"obj":"18177486"},{"id":"19736547-18177486-66178374","span":{"begin":1874,"end":1876},"obj":"18177486"},{"id":"19736547-18177486-66178374","span":{"begin":1874,"end":1876},"obj":"18177486"},{"id":"19736547-18160255-66178375","span":{"begin":1878,"end":1880},"obj":"18160255"},{"id":"19736547-18160255-66178375","span":{"begin":1878,"end":1880},"obj":"18160255"},{"id":"T22352","span":{"begin":594,"end":596},"obj":"15817814"},{"id":"T21050","span":{"begin":594,"end":596},"obj":"15817814"},{"id":"T65912","span":{"begin":707,"end":709},"obj":"15817814"},{"id":"T33215","span":{"begin":707,"end":709},"obj":"15817814"},{"id":"T58596","span":{"begin":1273,"end":1275},"obj":"18177486"},{"id":"T57697","span":{"begin":1273,"end":1275},"obj":"18177486"},{"id":"T51487","span":{"begin":1874,"end":1876},"obj":"18177486"},{"id":"T55603","span":{"begin":1874,"end":1876},"obj":"18177486"},{"id":"T45850","span":{"begin":1878,"end":1880},"obj":"18160255"},{"id":"T56336","span":{"begin":1878,"end":1880},"obj":"18160255"}],"text":"Curcumin interrupts canonical Wnt signaling in activated HSCs in vitro\nTo explore the mechanisms by which curcumin eliminated the stimulatory effect of Wnt signaling on the induction of lox-1 expression, we assumed that the activation of PPARγ by curcumin antagonistically interacted with the Wnt signaling pathway and led to the interruption of canonical Wnt signaling in activated HSCs. To test the assumption, passaged HSCs were transfected with the plasmid TOPflash, or FOPflash. TOPflash was a canonical Wnt signaling luciferase reporter, which contained 8 copies of TCF/LEF binding sites 33. FOPflash was used as a control luciferase reporter, which contained 8 copies of mutant TCF/LEF binding sites 33. After recovery, cells were treated with curcumin at various concentrations (0-30 μM) in DMEM with FBS (10%) for 24 hr. Luciferase activity assays in Fig. 9A demonstrated that curcumin caused a dose-dependent reduction in luciferase activities in cells transfected with TOPflash. However, curcumin had no impact on luciferase activities in cells transfected with FOPflash. These results suggested that curcumin interrupted canonical Wnt signaling in HSCs.\nCanonical Wnt signaling is activated only when dephosphorylated β-catenin is translocated into the nucleus 42. To verify the effect of curcumin on interrupting the Wnt signaling pathway, HSCs were treated with curcumin at various concentrations (0-30 μM) for 24 hr. Total nuclear proteins were prepared and the abundance of nuclear β-catenin was evaluated by Western blotting analyses. It was revealed in Fig. 9B that curcumin dose-dependently reduced the abundance of nuclear β-catenin. The nuclear protein Histone H1 was used as an invariant control.\nNuclear β-catenin forms a complex with the transcription factor TCF/LEF to bind to the promoter of target genes and to stimulate target gene transcription 42, 43. EMSA were conducted to further verify the effect of curcumin on interrupting canonical Wnt signaling. Nuclear protein extracts from HSCs treated with curcumin at 0-30 μM for 24 hr were incubated with biotin-labeled double-stranded oligonucleotide probe P(TCFwt). The probe P(TCFwt) contained the consensus TCF/LEF binding site found in the lox-1 promoter. As shown in Fig. 9C, curcumin dose-dependently reduced the DNA binding activity of the transcription factor TCF/LEF to the probe. To examine the DNA binding specificity of TCF/LEF to the probe, competition assays were performed using a 10-, 50-, or 100-fold excess of the unlabeled probe P(TCFwt) (lanes 3-5 in Fig. 9D), or the unlabeled probe P(TCFmut) (lanes 6-8 in Fig. 9D). The probe P(TCFmut) contained the TCF/LEF binding site found in the lox-1 promoter with site-directed mutations. It was found that the amount of TCF/LEF binding to the biotin-labeled probe P(TCFwt)) was competitively reduced by the unlabeled probe P(TCFwt), but not by the mutant probe P(TCFwt). These results indicated that curcumin dose-dependently reduced the DNA binding activity of TCF/LEF to the promoter of LOX-1 gene in activated HSCs. Taken together, these results indicated that curcumin interrupted canonical Wnt signaling in activated HSCs by reducing the abundance of nuclear β-catenin and reducing the DNA binding activity of TCF/LEF."}