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First published online May 20, 2009
doi: 10.1242/10.1242/jcs.038729

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Inhibitors of clathrin-dependent endocytosis enhance TGFβ signaling and responses

¹ Departments of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Doisy Research Center, St Louis, MO 63104, USA
² Department of Pharmacology, Taipei Medical University, Taipei, Taiwan
³ Auxagen, St Louis, MO 63132, USA
⁴ Center for Biotechnology and Biomedical Engineering, National Central University, Jhongli, Taiwan
⁵ Institute of Systems Biology and Bioinformatics, National Central University, Jhongli, Taiwan

View larger version (28K): [in this window] [in a new window]   Fig. 1. Enhancement of TGFβ-stimulated Smad2 phosphorylation and nuclear localization by clathrin-dependent endocytosis inhibitors in Mv1Lu cells. (A-I,K) Cells were pretreated with 10 mg/ml β-CD (A,K), 20 µM MDC (B), 40 µM monensin (C), 20 µM TFP (D) and 0.45 M sucrose (K) or several concentrations (as indicated) of β-CD (E), TFP (F), monensin (G), chloroquine (H) and dynasore (I), at 37°C for 1 hour. The cells were then stimulated with vehicle only or 100 pM TGFβ1 (A-I) or several concentrations (as indicated) of TGFβ1 (K). At various time-points, as indicated, at 37°C, cell lysates were analysed by 7.5% SDS PAGE followed by western blot analysis using antibodies against P-Smad2 and Smad2 and chemiluminescence development (a or top) and quantitation by densitometry (b or bottom). The data shown (a or top) are representative of three independent experiments. The relative level of P-Smad2 was expressed as arbitrary units (A.U.) (A,B,C,D,K) (b). The relative level of P-Smad2 in cells treated with TGFβ alone was taken as 100% of control (E-I, bottom). Experiments were performed in triplicate. The data are means ± s.d. Asterisks (*) indicate results significantly higher than that in cells treated with TGFβ alone; P<0.001. (J) Cells were pretreated with 10 mg/ml β-CD (c,d) at 37°C for 1 hour. The cells were then stimulated with vehicle only (a) or with 100 pM TGFβ (b,d). After 30 minutes at 37°C, the cells were fixed and subjected to indirect immunofluorescence staining using an antibody against P-Smad2. The data shown are representative of three independent experiments. Scale bar: 5 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 2. Enhancement of colocalization and accumulation of SARA and TβR-I at the plasma membrane by inhibitors of clathrin-dependent endocytosis in Mv1Lu cells. Cells were pretreated with vehicle only or 20 µM TFP, 10 mg/ml β-CD, 40 µM dynasore (Dyn) and 200 µM chloroquine (CQ) at 37°C for 1 hour. Treated cells were then stimulated with and without 100 pM TGFβ1. After 30 minutes at 37°C, cells were fixed and analyzed by indirect immunofluorescence staining using antibody against TβR-I (panels 1-10) and SARA (panels 11-20); DAPI (nuclear) staining was also performed (panels 21-30). Merged staining is also shown (panels 31-40). Insets indicate the colocalization and accumulation of SARA and TβR-I at the plasma membrane.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Enhancement of TGFβ-stimulated expression of PAI-1 by inhibitors of clathrin-dependent endocytosis in Mv1Lu cells. (A-G) Mv1Lu cells were pretreated with vehicle alone or with several concentrations (as indicated) of β-CD (A,C), thioridazine (B), TFP (D), chloroquine (E), monensin (F) and MDC (G) at 37°C for 1 hour. Treated cells were then stimulated with 50 pM TGFβ1. After 2 hours at 37°C, the mRNAs encoding PAI-1 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH, as control) in the cell lysates were analyzed by northern blot (top) and quantified using a PhosphorImager (bottom) (A,B) or real-time RT-PCR (C-G). The TGFβ-stimulated expression of PAI-1 in cells treated with vehicle only was taken as 100% (A,B) or one fold (C-G) of control. Experiments were carried out in triplicate. The data are means ± s.d. Asterisks indicate result significantly higher than control treated without the inhibitor (*, P<0.001; **, P<0.05). (H) Mv1Lu cells were pretreated with different concentrations (as indicated) of dynasore at 37°C for 1 hour and then stimulated with 50 pM TGFβ1. After 2 hours at 37°C, the mRNAs for PAI-1 and β-actin were quantitated by real-time RT-PCR. The relative mRNA level of PAI-1:β-actin in cells treated with TGFβ1 alone was taken as 1.0 (100%). Experiments were carried out in triplicate. The data are means ± s.d. Asterisk (*) indicates result significantly higher than control treated without the inhibitor, P<0.001. (Ia) Mv1Lu cells were pretreated with SB 431542 (10 µM) at 37°C for 1 hour and dynasore (50 µM) at 37°C for 0.5 hours, alone or together, and then stimulated with and without 20 pM TGFβ1. After 2 hours at 37°C, the mRNAs for PAI-1 and β-actin were quantitated by real-time RT-PCR. The relative mRNA level of PAI-1:β-actin in cells treated with vehicle only (control) was taken as 1.0. Experiments were carried out in triplicate. The data are means ± s.d. Asterisks (*) indicates result significantly lower than cells treated with the same agent(s) but without SB 431542; P<0.001. (Ib) Mv1Lu cells stably expressing the luciferase reporter gene driven by the PAI-1 promoter (MLECs-clone 32) were pretreated with SB 431542 (10 µM) at 37°C for 1 hour and several concentrations (as indicated) of dynasore at 37°C for 0.5 hours, alone or together, and then stimulated with and without 20 pM TGFβ1 at 37°C for 6 hours. The stimulated cells were lysed in 100 µl of lysis buffer. The cell lysates were assayed using a luciferase kit (Promega). The luciferase activity (A.U.) in treated and stimulated cells was determined. Experiments were carried out in triplicate. The data are means ± s.d.

View larger version (76K): [in this window] [in a new window]   Fig. 4. Histological analysis (A,B,C) and TβR-II and P-Smad2 immunofluorescent stainings (D,E) of the coronary arteries of wild-type and ApoE-null mice. Wild-type (A,D,E) and ApoE-null mice (A-E) were treated with DMSO only (control) (a,c) or 1 mg/kg dynasore (b,d) every two days for 8 weeks. The coronary arteries (A,D,E) and hearts (B,C) were removed from the animals and subjected to histological analysis by haematoxylin and eosin (H&E) staining (A-C) and indirect immunofluorescent staining for TβR-II (D) and P-Smad2 (E). The asterisk (*) in A, B, D and E indicates the location of the lumen in the coronary artery and descending aorta, and the small asterisks in B indicates the atherosclerotic lesions. A quantification of atherosclerotic lesion areas in descending aortas right above the aortic valves of ApoE-null mice (six mice per experimental group) treated with and without dynasore was performed (C). Areas of atherosclerotic lesions and blood vessel media were determined using the NIH image J program. The magnitude of the atherosclerotic lesion is presented as the percentage of lesion area/media area. The asterisk indicates a result significantly lower than the control; P<0.05 (C). The arrows and arrowheads indicate the immunofluorescent stainings of TβR-II and P-Smad2 in the aortic endothelium and smooth muscle, respectively (D,E).

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