Fig 1.
Chemical structures of (A) cholesterol and (B) euphol.
Fig 2.
Inhibition of TGF-β-induced transcriptional activation by euphol.
(A) Mv1Lu cells with stable expression of the PAI-1 luciferase promoter were treated with increasing concentration of euphol. The gray bars in (A) represent the cells without TGF-β treatment. The black bars represent the cells treated with 100 pM TGF-β. (B,C,D) Mv1Lu cells were transfected with CAGA12-Luc, collagen, or a fibronectin luciferase plasmid, and AGS (E) and MKN45 (F) gastric cancer cells were transfected with CAGA12-Luc, Mv1Lu, AGS, and MKN45 cells were treated with TGF-β (100 pM, +β), euphol (Eu), or MβCD (CD). Luciferase activity was measured as described in the methods section. Columns show mean of three independent experiments performed in triplicated; bars indicate s.d.; *P<0.05 (compare with TGF-β treatment), **P<0.01.
Fig 3.
(A) Euphol suppresses TGF-β-induced Smad2/3 phosphorylation. Mv1Lu cells were pretreated with 10 μg/ml euphol for 1 h and stimulated with different concentration of TGF-β for 30 min. (B) Treatment with high concentrations of euphol (40 μg/ml) shows stronger inhibition in Smad2/3 phosphorylation than cells treated with 10 μg/ml euphol (Fig 3A). Results from Fig 3A and 3B were quantified by densitometry showing in upper right panel (C) AGS cells and (D) MKN45 cells pretreated with euphol (40 μg/ml) were stimulated with increasing concentration of TGF-β (0, 5, 10, 20, 50, and 200 pM) and then whole-cell lysates were blotted with antibodies as indicated. (E) Mv1Lu cells and (F) AGS cells pretreated with increasing concentration of euphol (0, 5, 10, 20, 40, and 60 μg/ml) were stimulated with 100 pM and then whole-cell lysates were blotted with the antibodies as indicated. The band intensity was quantitated and the statistical analysis of three independent experiments was provided (*P<0.05). (G) Euphol blocks TGF-β-induced nuclear localization of Samd2/3. TGF-β induces nuclear translocation of Smad2/3 after 30 min (Gb), and this effect is blocked by euphol (Gc). Nystatin reverses euphol induced inhibition of Smad nuclear localization (Gd), the distribution pattern of Smad2/3 was detected by immunofluorescence staining with an anti-Smad2/3 antibody. Bar, 10 μm (Right). The nuclear/cytoplasmic ratio of the Smad2/3 signal was quantified (H). Data are means ± SEM for ≥10 fields. s.d.; **P<0.01 versus TGF-β-induced. (I) Mv1Lu cells and (J) AGS cells treated with euphol were exposed to TGF-β for 45 min before nuclear protein extraction. Nuclear translocation of pSmad2 was observed after SDS-PAGE (10%) followed by Western blot analysis. Lamin B was used to check nuclear isolation and loading.
Fig 4.
Sucrose density gradient analysis of TGF-β receptors in the plasma membranes of Mv1Lu (A), AGS (B), and MKN45 (C) cells treated with or without euphol and nystatin.
Cells were treated with or without 10 μg/ml euphol at 37°C for 1 hour, and the cell lysates from these treated cells were subjected to sucrose density gradient ultracentrifugation. The sucrose gradient fractions were then analyzed by Western blot analysis using anti-TβR-I, anti-TβR-II, anti-TfR-1, anti-EGFR, anti-flotillin, anti-CD-55 and anti-caveolin-1 antibodies. Fractions 4 and 5 contained lipid rafts/caveolae whereas fractions 7–10 were non-lipid raft fractions. Treatment with euphol and nystatin did not affect the abundance of TGF-β receptor proteins and other cell proteins (S2 Fig). Open arrowheads (ρ) indicate increased abundance of TβR-I or TβR-II in the fraction in comparison with that of the untreated control cells. The stars (*) indicate decreased abundance of TβR-I or TβR-II in the fraction in comparison with that of the untreated control cells. In Fig 4C, due to the poor expression level of TfR in MKN45 cells, we show the blot of early endosome antigen-1 (EEA-1), which is an alternative marker for non-lipid raft fractions.
Fig 5.
Immunofluorescent localization of TβR-II and caveolin-1(or flotillin-2) in Mv1Lu cells treated with and without euphol and TGF-β.
Mv1Lu cells that transiently expressed TβR-II-HA were treated with or without 10 μg/ml euphol at 37°C for 1 hour, after which they were incubated with or without 100 pM TGF-β at 37°C for 30 minutes. The cells were then fixed with cold methanol and incubated with mouse anti-HA antibodies (Fig 5A and 5B, a-d), rabbit anti-caveolin-1 antibodies (Fig 5A, e-h), or rabbit anti-flotillin-2 antibodies (Fig 5B, e-h) followed by incubation with rhodamine-conjugated donkey anti-mouse antibodies or FITC-conjugated goat anti-rabbit antibodies. The fluorescence in the cells was measured using confocal fluorescence microscopy. Bar, 20 μm. The white arrows indicate colocalization of TβR-II-HA and caveolin-1 (or flotillin-2) at the cell surface (j) and in endocytic vesicles (l).
Fig 6.
Euphol decreased the abundance of TβR-I and TβR-II in Mv1Lu (A) and MKN45 (B) cells after.
Mv1Lu and MKN45 cells were treated with several concentrations of euphol at 37°C for 48 hours, after which the cell lysates were subjected to Western blot analysis using anti-TβR-I, anti-TβR-II, TfR, EEA-1, caveolin-1, and anti-β-actin antibodies (A and C), followed by quantification by densitometry (B and D). The ratio of the relative amounts of TβR-I, TβR-II, and β-actin in cells treated without euphol was taken as 100% TGF-β receptor expression. The data are representative of a total of four independent analyses; values are mean ± s.d. significantly lower than control cells: *P<0.05 versus control.
Fig 7.
Euphol inhibits TGF-β-induced fibronectin expression.
(A) AGS cells were treated with TGF-β (100 pM) ± euphol (5–40μg/ml) for 24 hours. Total RNA were isolated and the expressions of fibronectin were determined by RT-PCR. GAPDH was used as a loading control. (B) AGS cells were treated with TGF-β (100 pM) ± euphol for 48 hours. Total protein extracts from treated cells were Western blotted with anti-fibronectin or anti-β-actin monoclonal antibody. β-actin was used as a loading control; results were quantified by densitometry showing in lower panel. Data represent the means ± s.d. of three independent experiments *P<0.01 versus TGF-β-induced expression.
Fig 8.
A model for the effect of euphol on TGF-β receptor partitioning between lipid rafts/caveolae- and clathrin-mediated endocytosis.
In mammalian cells, there are 2 major TβR-I-TβR-II complexes (complex I and complex II) present on the cell surface. Complex I and complex II are mainly localized in the non-lipid raft and lipid raft/caveolae microdomains of the plasma membrane, respectively. Euphol increases the formation and/or stabilization of lipid rafts/caveolae by integration into the plasma membrane, thereby increasing the localization of TβR-I and TβR-II in lipid rafts/caveolae (as complex II), facilitating rapid degradation of TGF-β and attenuating Smad dependent-TGF-β signaling.