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First published online 18 September 2007
doi: 10.1242/jcs.006916

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Cholesterol suppresses cellular TGF- responsiveness: implications in atherogenesis

¹ Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St Louis, MO 63104, USA
² Auxagen Inc., 7 Pricewoods, St Louis, MO 63132, USA
³ Departments of Ophthalmology and Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St Louis, MO 63104, USA
⁴ Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA

View larger version (45K): [in this window] [in a new window]   Fig. 1. Effects of cholesterol and LDL on Smad2 phosphorylation (A-C) and nuclear translocations (D) in Mv1Lu cells and BAECs stimulated with TGF-1. Cells were treated with increasing concentrations of cholesterol, as indicated (A,B), 50 µg protein/ml LDL (C), 5 µg protein/ml VLDL (C) or 50 µg/ml cholesterol (D) at 37°C for 1 hour and then further incubated with 50 pM TGF-1 for 30 minutes. P-Smad2 and total Smad2 in the cell lysates were analyzed by immunoblotting. The relative level of P-Smad2 (P-Smad2/Smad2) was estimated. A representative of a total of three analyses is shown (top). The quantitative analysis of the immunoblots is shown below. The relative level of P-Smad2 in cells treated with TGF-1 only was taken as 100% of TGF-1-stimulated Smad2 phosphorylation. The data are mean ± s.d. *,**Significantly lower than that in cells treated with TGF-1 only: P<0.001 and P<0.05, respectively. (D) Smad2 nuclear translocation was analyzed by indirect immunofluorescent staining. Rhodamine fluorescence represents P-Smad2 staining (a-c) whereas the nuclei were stained by DAPI staining (d-f).

View larger version (26K): [in this window] [in a new window]   Fig. 2. Effects of cholesterol, LDL, statins, -CD and nystatin on TGF-1-induced PAI-1 expression in Mv1Lu cells (A,C,E,F,G,H) and BAECs (B,D). Cells were treated with increasing concentrations of cholesterol as indicated (A,B), 50 µg/ml cholesterol (C,D,E), 50 µg/ml LDL (E), -CD (0.5%; H) or nystatin (25 µg/ml; H) at 37°C for 1 hour or with 1 µM fluvastatin or lovastatin (F,G) or with different concentrations of fluvastatin (G) at 37°C for 16 hours and then further incubated with increasing concentrations (as indicated) of TGF-1 (C,D) or 50 pM TGF-1 (A,B,E,G,H) for 2 hours. Northern blot analyses of PAI-1 and G3PDH were performed and a representative of a total of three analyses per experiment is shown (a). The relative amounts of the transcripts (PAI-1 and G3PDH) were quantified with a PhosphoImager. The ratio of the relative amounts of PAI-1 and G3PDH transcripts in cells treated without TGF-1 and cholesterol, LDL or statins on the blot was taken as 1 fold or 100% of PAI-1 expression. The quantitative data from three independent analyses was shown (b). The data are mean ± s.d. *Significantly lower than that of control P<0.001.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Effects of cholesterol and lovastatin on the TGF-1-stimulated luciferase activity (A) and TGF-1-induced growth inhibition (B) in Mv1Lu cells. (A) Cells stably expressing a luciferase reporter gene were treated with increasing concentrations (as indicated) of cholesterol at 37°C for 1 hour (a) or with 1 µM lovastatin at 37°C for 16 hours ± cholesterol (20 µg/ml) at 37°C for 1 hour (b) and then further incubated with 50 pM TGF-1 for 6 hours. The luciferase activity of the cell lysates (20 µg protein) was determined and expressed as arbitrary units (A.U.). The luciferase activity in cells treated with TGF-1 only was taken as 100% (a). The data was obtained from three or four independent analyses. *Significantly lower or higher than that in cells treated with TGF-1 only: P<0.001. (B) Cells were incubated with 0.0625 and 0.125 pM TGF-1 in the presence of increasing concentrations of cholesterol, as indicated. Cell growth was then determined by measurement of [3H-methyl]thymidine incorporation into cellular DNA. The [3H-methyl]thymidine incorporation in cells treated with vehicle only was taken as 100%. TGF-1 at 0.0625 and 0.125 pM inhibited DNA synthesis by 30% and 40%, respectively. The degree (%) of cholesterol-mediated reversal of TGF-1 growth inhibition was estimated by the equation: % reversal=[1–(T1–T2/T3–T4)]x100, where T1 is the thymidine incorporation in cells treated with cholesterol alone; T2, the thymidine incorporation in cells treated with cholesterol plus TGF-1; T3, the thymidine incorporation in cells treated with vehicle only and T4, the thymidine incorporation in cells treated with TGF-1 alone. The experiments were carried out in triplicate.

View larger version (45K): [in this window] [in a new window]   Fig. 4. Sucrose density gradient analysis of TR-II in the plasma membrane of Mv1Lu cells treated with or without cholesterol and stimulated with and without TGF-1. Cells were treated with or without 50 µg/ml cholesterol at 37°C for 1 hour and further incubated with and without 50 pM TGF-1 for 2 hours. 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-TR-I, anti-TR-II, anti-TfR-1 and anti-caveolin-1 antibodies. The arrow indicates the locations of TR-I, TR-II, caveolin-1 and TfR-1. Fractions 4 and 5 contained lipid rafts/caveolae whereas fractions 7 and 8 are non-lipid raft fractions. Treatment with cholesterol alone did not affect the total amounts of TGF- receptor proteins and cell proteins. Open arrowheads indicate the increased amount of TR-I or TR-II in the fraction as compared with that of untreated control. *The decreased amount of TR-II in the fraction as compared with that of untreated control. #The decreased amount of TR-II in the fraction as compared with that of treatment with cholesterol or TGF-1 alone.

View larger version (55K): [in this window] [in a new window]   Fig. 5. Immunofluorescent localization of TR-I and caveolin-1 in Mv1Lu cells treated with and without cholesterol and TGF-1. Cells were treated with or without 50 µg/ml cholesterol at 37°C for 1 hour and incubated with and without 100 pM TGF-1 at 37°C for 30 minutes. The cells were then fixed with cold methanol and incubated with a goat antibody to TR-I (e-h) and rabbit antibody to caveolin-1 (a-d) followed by incubation with Rhodamine-conjugated donkey anti-goat antibody or FITC-conjugated mouse anti-rabbit antibody. The fluorescence in cells was examined using a fluorescent confocal microscope. Bar, 20 µm. The arrows indicate colocalization of TR-I and caveolin-1 at the cell surface (j).

View larger version (29K): [in this window] [in a new window]   Fig. 6. Concentration dependence of cholesterol (A) or LDL (B) in enhancing TGF-1-induced degradation of TR-II in Mv1Lu cells. Cells were treated with several concentrations of cholesterol (A) or LDL (B), as indicated, at 37°C for 1 hour, then incubated with and without 1% -CD at 37°C for 1 hour and further incubated with 50 pM TGF- for 2 hours. The cell lysates were then subjected to western blot analysis using anti-TR-II and anti--actin antibodies (a) and quantification by densitometry (b). The ratio of the relative amounts of TR-II and -actin in cells treated without TGF-1 was taken as the 100% level of TR-II. The data are representative of a total of three independent analyses; values are mean ± s.d. *Significantly lower than control cells: P<0.001.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Effects of the treatments with lovastatin, fluvastatin and nystatin on the plasma-membrane microdomain localization (A) and TGF-1-induced degradation of TR-II (B) in Mv1Lu cells. Cells were treated with or without lovastatin (1 µM), fluvastatin (1 µM) or nystatin (25 µg/ml) at 37°C for 16 hours or 1 hour, respectively. The treated cells were directly analyzed by sucrose density gradient ultracentrifugation analysis (A) or further incubated with 50 pM TGF- at 37°C for several time periods as indicated (B). Western blot analyses of the sucrose density gradient fractions (A) and of TGF-1-treated cell lysates (B) were performed using anti-TR-II, anti-caveolin-1, anti-TfR-1 and anti--actin antibodies. The open arrowheads indicate the increased amount of TR-II in the fraction as compared with that of the untreated control. The data are representative of a total of three independent analyses; values are mean ± s.d. *Significantly higher than that in cells treated without fluvastatin: P<0.05.

View larger version (29K): [in this window] [in a new window]   Fig. 8. A lower ratio of 125I-TGF-1 binding to TR-II and TR-I (A) and suppressed TGF- responsiveness (B) in the aortic endothelium of ApoE-null mice fed a high-cholesterol diet and in cultured BAECs treated with cholesterol. (A) 125I-TGF- affinity labeling. (a) The aortic endothelium from wild-type and ApoE-null (ApoE–/–) mice fed a high-cholesterol diet (lanes 1 and 2, respectively) and BAECs treated with and without 50 µg/ml cholesterol at 37°C for 1 hour, were affinity-labeled with 125I-TGF-1, extracted with 1% Triton X-100, analyzed by 7.5% SDS-PAGE and autoradiography (top), and quantified using a PhosphoImager (bottom). A representative of a total of five animals each analyzed or of three independent BAEC analyses is shown. The number on the top of the bar charts is the estimated ratio of 125I-TGF-1 binding to TR-II and TR-I. (B) Western blot analysis. The aortic endothelium from wild-type (top, lanes 1 and 2) and ApoE-null mice (ApoE–/–) (top, lanes 3 and 4) mice fed a high-cholesterol diet were extracted with 1% Triton X-100. Equal protein amounts (100 µg) of the Triton X-100 extracts were then subjected to western blot analysis using antibodies to Smad2, P-Smad2, VCAM-1 and -actin (top). Two representatives (lanes 1 and 2, and 3 and 4) of a total of five animals each analyzed are shown (top). The relative levels of P-Smad2 (P-Smad2/Smad2) and VCAM-1 (VCAM-1/-actin) were estimated (bottom). Statistical comparisons between groups were made by use of the Mann-Whitney test (bottom). Data represent median (interquartile). *P<0.001 versus wild-type mice.

View larger version (71K): [in this window] [in a new window]   Fig. 9. Immunofluorescent localization of P-Smad2 in the coronary artery from wild-type and ApoE-null mice fed a high cholesterol diet. (A,B) Representative photographs of the coronary artery from wild-type (A) mice exhibited a plaque-free section; that from ApoE-null mice fed a high cholesterol diet (B) showed an advanced plaque. (C,D) Immunofluorescent confocal microscopic analysis of the tissue cross sections revealed that P-Smad2 is present in wild-type mice (C) whereas no P-Smad2 was detected in the endothelium of the coronary artery from ApoE-null mice fed a high cholesterol diet (D). *The location of the artery lumen. The magnification is 200x (A and B); bar, 20 µm (C,D). The arrows in C indicate the localization of P-Smad2 in the artery endothelium.

View larger version (33K): [in this window] [in a new window]   Fig. 10. A model for the cholesterol effect on TGF- partitioning between lipid rafts/caveolae- and clathrin-mediated endocytosis. In cells, there are two major TR-I–TR-II complexes (Complex I and Complex II) present on the cell face. Complex I and Complex II are mainly localized in the non-lipid raft and lipid raft/caveolae microdomains of the plasma membrane, respectively. The numbers of TR-I and TR-II molecules (blue rectangles) in Complex I and Complex II shown in the model are arbitrary and intended to indicate that Complex I and Complex II contain TR-II>TR-I and TR-I>TR-II, respectively. The ratio of TR-II to TR-I can be determined by 125I-TGF-1 affinity labeling (Chen et al., 2006). Cholesterol increases the formation and/or stabilization of lipid rafts/caveolae by integration into the plasma membrane, thereby increasing the localization of TR-I and TR-II in lipid rafts/caveolae (as Complex II), facilitating rapid degradation of TGF- and attenuating TGF- responsiveness (Smad dependent). Complex II may also be capable of mediating Smad2/3-indepentent signaling which leads to different cellular responsiveness such as fibrogenesis in fibroblasts (Pannu et al., 2007). Depletion of cholesterol in the plasma membrane, by treating cells with cholesterol-lowering agents (e.g. statins) or cholesterol-depleting agents (e.g. -CD), facilitates the localization of TR-I and TR-II in non-lipid raft microdomains. In the presence of ligand, Complex I undergoes clathrin-mediated endocytosis, promoting Smad2/3-dependent endosomal signaling and TGF- responsiveness. In hypercholesterolemic mice, cell-surface TGF- receptor complexes in the aortic endothelium contain more Complex II than Complex I. In normal mice, cell-surface TGF- receptor complexes contain more Complex I than Complex II in the aortic endothelium.