Betulinic acid enhances TGF-β signaling by altering TGF-β receptors partitioning between lipid-raft/caveolae and non-caveolae membrane microdomains in mink lung epithelial cells
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TGF-β is a key modulator in the regulation of cell proliferation and migration, and is also involved in the process of cancer development and progression. Previous studies have indicated that TGF-β responsiveness is determined by TGF-β receptor partitioning between lipid raft/caveolae-mediated and clathrin-mediated endocytosis. Lipid raft/caveolae-mediated endocytosis facilitates TGF-β degradation and thus suppressing TGF-β responsiveness. By contrast, clathrin-mediated endocytosis results in Smad2/3-dependent endosomal signaling, thereby promoting TGF-β responsiveness. Because betulinic acid shares a similar chemical structure with cholesterol and has been reported to insert into the plasma membrane, we speculate that betulinic acid changes the fluidity of the plasma membrane and modulates the signaling pathway associated with membrane microdomains. We propose that betulinic acid modulates TGF-β responsiveness by changing the partitioning of TGF-β receptor between lipid-raft/caveolae and non-caveolae microdomain on plasma membrane.
We employed sucrose-density gradient ultracentrifugation and confocal microscopy to determine membrane localization of TGF-β receptors and used a luciferase assay to examine the effects of betulinic acid in TGF-β-stimulated promoter activation. In addition, we perform western blotting to test TGF-β-induced Smad2 phosphorylation and fibronectin production.
Results and conclusions
Betulinic acid induces translocation of TGF-β receptors from lipid raft/caveolae to non-caveolae microdomains without changing total level of TGF-β receptors. The betulinic acid-induced TGF-β receptors translocation is rapid and correlate with the TGF-β-induced PAI-1 reporter gene activation and growth inhibition in Mv1Lu cells.
KeywordsBetulinic acid Lipid-raft Caveolae TGF-beta
transforming growth factor-beta
betulinic acid (3b, hydroxy-lup-20(29)-en-28-oic acid)
early endosome antigen 1
type I TGF-β receptor
type II TGF-β receptor
plasminogen activator inhibitor-1
- Mv1Lu cell
mink lung epithelial cell
activin receptor-like kinase
nuclear factor kappa-light-chain-enhancer of activated B cells
peroxisome proliferator-activated receptor gamma
nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha
TGF-β is a family of 25-kDa disulfide-linked dimeric proteins. It has 3 members in mammals (TGF-β1, TGF-β2, and TGF-β3,), which share approximately 70 % of the sequence homology [11, 12]. TGF-β exhibits bifunctional growth regulation; it inhibits the growth of most cell types, including epithelial cells, endothelial cells, and lymphocytes, and stimulates the proliferation of mesenchymal cells such as fibroblasts [11, 12]. In epithelial cells, TGF-β inhibits cell proliferation, induces apoptosis, and mediates differentiation, implying that this signaling pathway engages in tumor-suppressing activities in epithelial tumors [13, 14]. However, TGF-β promotes invasive and metastatic activities in late-stage tumor progression, implying that TGF-β can paradoxically play opposing roles in human cancers, and this is seemingly dependent on the cancer stage. In addition to its growth regulatory activities, TGF-β exhibits other biological activities, including the regulation of extracellular matrix synthesis, chemotaxis, angiogenesis, and the differentiation of several cell lineages. It has been implicated in numerous pathophysiological processes including wound repair, tissue fibrosis, immunosuppression, and morphogenesis . The primary biological activities of TGF-β are mediated by specific cell surface receptors type I and type II (TβR-I and TβR-II, respectively). TGF-β exerts its effects on cells by binding to TβR-II, which induces the recruitment of TβR-I with the subsequent activation of the receptor complex. Smad2 and Smad3 are the direct substrates of the activated TGF-β receptor complex. After stimulation, the Smad complex translocates into the nucleus, where it functions as a member of different transcription factor complexes that regulate the expression of various genes [12, 13, 16].
We have previously reported that suppressed TGF-β responsiveness in the aortic endothelium plays a critical role in the pathogenesis of atherosclerosis in hypercholesterolemic animals [17, 18]. A high concentration of cholesterol in the culture medium suppresses TGF-β responsiveness in cultured cells, including in endothelial cells, by inducing the accumulation of cell-surface TGF-β-TGF-β receptor complexes in the lipid rafts/caveolae of the plasma membrane, facilitating the rapid degradation of these complexes, thereby attenuating TGF-β-stimulated signaling and related responses [17, 18, 19]. This effect of cholesterol is believed to be mediated by the increasing formation or stabilization of the lipid rafts/caveolae, presumably through the direct insertion of cholesterol into the plasma membranes of target cells [17, 18]. Lipid raft/caveolae are thought to form platforms collecting assemblies of proteins involved in many key cellular functions, including signal transduction, membrane fusion, cytoskeleton organization, lipid sorting, protein trafficking, and the localization and activities of specific membrane channels [20, 21, 22]. Because BetA shares a similar chemical structure with cholesterol, and was reported to insert into the plasma membrane , it is rational to speculate that BetA changes the fluidity of the plasma membrane and modulates the signaling pathway associated with membrane microdomains. We speculate that BetA modulates TGF-β responsiveness by changing the partitioning of the TGF-β receptor between the microdomains of the lipid raft/caveolae and non-caveolae on the plasma membrane.
Because the TGF-β signaling pathway is a key mediator that controls proliferation and inflammation, and BetA has antitumor and anti-inflammation properties, this study investigates the effect of BetA on TGF-β signaling, and we attempt to elucidate the mechanisms involved. We found that BetA activates TGF-β receptors by moving them from lipid raft to non-raft membrane microdomains, as perceived by the enhancement in TGF-β-specific reporter gene activity, sucrose gradient fractionation of the plasma membrane, fibronectin protein levels, Smad2/3 phosphorylation, nuclear translocation, and TGF-β-induced growth inhibition.
Dulbecco’s modified Eagle's medium (DMEM), phenylmethanesulfonyl fluoride (PMSF), Betulinic acid [(3b)-3-hydroxy-lup-20(29)-en-28-oic acid], bovine serum albumin (BSA), and peroxidase-conjugated anti-rabbit IgG were obtained from Sigma (St. Louis, MO). The pre-stained protein ladder (64, 49, 37, 26, and 20 kDa) and fetal calf serum (FCS) was obtained from Invitrogen (Carlsbad, CA). TGF-β1 was purchased from Austral Biologicals (San Ramon, CA). The polyclonal antibodies against early endosome antigen 1 (EEA1), transferrin receptor (TfR), Smad2/3, Caveolin-1, flotillin-2, epidermal growth factor receptor (EGFR), TβR-I, TβR-II, and HA-probe were obtained from Santa Cruz (Santa Cruz, CA). The rabbit polyclonal antibody to phospho-Smad2 was purchased from Cell Signaling (Boston, MA).
Mink lung epithelial (Mv1Lu) cells and Mv1Lu cell stably express plasminogen activator inhibitor-1 (PAI-1) luciferase promoter were kindly provided by Dr. Jung San Huang (Saint Louis University, Saint Louis, MO). The Mv1Lu cells were cultured in DMEM supplemented with 10 % FCS, 1 % penicillin, and streptomycin (pH 7.4). The cells were seeded in tissue culture plates (Falcon, Bedford, MA, USA) and incubated at 37 °C in a humidified atmosphere of 5 % CO2. The Mv1Lu cells were subcultured twice per week through trypsinization in a 0.25 % trypsin-EDTA solution after washing with Ca2+-Mg2+-free saline.
Treatment of Mv1Lu cells with TGF-β after preincubation with BetA
The Mv1Lu cells were grown in 12-well plastic plates (4 × 105 cells/mL with 1 mL/well 10 % FCS-DMEM) for 24 h in a humidified CO2 incubator at 37 °C. Afterward, the medium was replaced with fresh 1 % FCS-DMEM. The cells were subsequently preincubated with BetA for 1 h at 37 °C. Next, TGF-β was added to the medium, and incubation continued for an additional 48 h; the cultures were then washed twice with cold PBS, and harvested for Western blot analysis. To observe Smad2/3 phosphorylation, the cells in the 12-well plastic plates were preincubated with BetA for 1 h at 37 °C. Afterward, TGF-β1 was added to the medium, and incubation continued for 30 min.
Western blot analysis
The cell lysates of Mv1Lu cells (approximately 50 μg protein) were subjected to 10 % SDS-PAGE under reducing conditions, and then electrotransferred to PVDF membranes. After being incubated with 5 % nonfat milk in Tris-buffered saline plus Tween 20 (TBST) (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 0.05 % Tween 20) for 1 h at room temperature, the membranes were incubated further with specific polyclonal antibodies to TβR-I and TβR-II in TBST/non-fat milk at 4 °C for 18 h, and washed 3 times with TBST for 10 min each. The bound antibodies were detected using peroxidase-conjugated anti-rabbit IgG and visualized using the ECL system (ImageQuant).
Luciferase activity assay
The Mv1Lu cells were transiently transfected with fibronectin  and collagen  luciferase promoter plasmids through electroporation. The cell suspension was mixed with 14 μg/mL of plasmid DNA (and renilla control plasmid) before it was transferred into an electroporation cuvette (0.4 cm gap, Bio-Rad, Hercules, CA) and pulsed (950 μF, 250 V, Gene Pulser II, Bio-Rad). After electroporation, the cells were seeded in 24-well cluster plates (Corning), and growth continued for an additional 24 h. The Mv1Lu cells stably express luciferase reporter gene driven by the PAI-1 promoter (MLECs – Clone 32), grown to near confluence on 24-well plates, were treated with different concentrations of BetA, with or without varying concentrations of cholesterol at 37 °C for 1 h. The treated cells were further incubated with 50 pM TGF-β1 at 37 °C for 6 h, and lysed in 100 μL of a lysis buffer (Promega). The cell lysates (approximately 20 μg of protein) were then mixed with a D-luciferin (Gold Biotechnology, St. Louis, MO) assay buffer and assayed using the luminometer (Titertek-Berthold, Pforzheim, Germany). The luciferase count was corrected for renilla activity, and a relative increase in corrected luciferase count was calculated versus the controls. A constitutively active ALK-5 (caALK-5) construct was cotransfected at a concentration of 1.2 μg/mL in the indicated experiments.
Immunofluorescent detection of Smad2/3
Cells were grown on 24-mm round coverglass (Paul Marienfeld). After 1 h of serum starvation and 1 h of pretreatment with 5 μg/mL of BetA or the vehicle, cells were stimulated with TGF-β1 (20 pM) for 30 min. They were then washed with phosphate buffered saline (PBS) and fixed in cold methanol for 15 min. Afterward, the cells were blocked with 5 % goat serum (Dako) in 1 % BSA/PBS. After incubation with mouse-anti-Smad2/3 (H-2; Santa Cruz Biotechnology) at 1:100 dilution in 1 % BSA/PBS for 18 h at 4 °C, the cells were incubated with donkey anti-mouse-FITC (Gene Tex) at room temperature for 1 h. The coverglass was mounted with a slow-fade gold antifade reagent and DAPI (Invitrogen). Photomicrographs were taken using a Zeiss Axio Observer Z1 microscope equipped with a Photometrics HQ2 camera.
Immunofluorescent confocal microscopy
The Mv1Lu cells were placed in a 24-mm coverglass and transiently transfected with TβR-II-HA plasmid (0.4 μg) by using lipofectamin 2000 (Invitrogen) in accordance to the manufacturer protocol. The transfected cells were pretreated with 5 μg/mL of BetA at 37 °C for 1 h, and then incubated with 100 pM of TGF-β1 for 30 min. After TGF-β1 stimulation, the cells were fixed in methanol at −20 °C for 15 min, washed with PBS, and then blocked using 0.2 % gelatin in PBS for 1 h. Cells were incubated overnight at 4 °C in a humidified chamber with a goat antibody against HA-probe (F-7, Santa Cruz Biotechnology) and a rabbit antibody against caveolin-1 (N-20, Santa Cruz Biotechnology) at 1:100 dilutions. After extensive washing, the cells were incubated with Rhodamine-conjugated donkey anti-goat antibody and FITC-conjugated mouse anti-rabbit antibody at a 1:50 dilution for 1 h. Images were acquired using a Leica TCS SP confocal microscope (Leica Microsystems Ltd., Heidelberg, Germany). The measurements of the colocalization rate were analyzed using a Leica Application Suite.
Separation of lipid-raft and non-lipid raft microdomains of plasma membranes by sucrose density gradient ultracentrifugation
Sucrose density gradient analysis was performed at 4 °C as described previously . In brief, cells were grown to near-confluence in 100-mm dishes (5–10 × 106 cells per dish). Cells were incubated with BetA (5 μg/mL) or cholesterol (25 μg/mL) at 37 °C for 4 h. After 2 washes with ice-cold PBS, the cells were scraped in 0.85 mL of 500 mM sodium carbonate (pH 11). Homogenization was performed with 10 strokes of a tightfitting Dounce homogenizer, followed by three 15-s bursts of an ultrasonic disintegrator (Qsonica, Newtown, CT, USA) to disrupt the cell membranes. The homogenates were adjusted to 45 % sucrose by adding 0.85 mL of 90 % sucrose in 25 mM 2-(N-morpholino) ethanesulfonic acid (pH 6.5) and 0.15 M NaCl (MBS), and placed at the bottom of an ultracentrifuge tube. A discontinuous sucrose gradient was generated by overlaying 1.7 mL of 35 % sucrose and of 5 % sucrose in MBS on top of the 45 % sucrose solution, and it was subsequently centrifuged at 40 000 rpm for 16–20 h in a P50S2 rotor (Himac, Tokyo, Japan). Ten 0.5-mL fractions were collected from the top of the tube, and a portion of each fraction was analyzed through SDS-PAGE, followed by Western blot analysis using antibodies to TβRI (ALK-5), TβRII, TfR-1, EEA-1, flotillin-2, EGFR, and caveolin-1. The relative amounts of TβRI, TβRII, TfR-1, and caveolin-1 on the blot were quantified through densitometry. The protein recovery and localization of caveolin-1, flotillin-2, and TfR-1 (fractions 4 to 5 and 8 to 10, respectively) did not change significantly with any of the treatment protocols (Fig. 8). An HA-tagged TβRII (TβRII-HA) construct was transfected at a concentration of 0.5 μg/mL in the indicated experiments.
MTT viability assay
The effect of BetA on TGF-β-induced growth inhibition was determined using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) assay. In brief, the cells were plated at 1× 104 cells per well in 150 μL of a culture medium containing 1 % FCS and the desired concentrations of BetA, before they were diluted with the culture media to achieve final concentrations ranging from 0.5 to 5 μg/mL. The EtOH concentration remained within the maximum permissible concentration of 0.01 % in both the control and treated samples. After pretreatment with BetA, the cells were treated with TGF-β, and incubation continued for 30 h at 37 °C in a humidified incubator, after which cell viability was determined. Afterward, 50 μL of MTT (4 μg/mL in PBS stock, diluted to a working strength of 1 μg/mL with the media) was added to each well and incubated for 2 h. After the medium was carefully removed, 0.1 mL of DMSO was added to each well, and the plates were shaken. Absorbance was recorded on a microplate reader, at a wavelength of 570 nm. The effect of BetA on growth inhibition was assessed as the percentage of cell viability, where the vehicle-treated cells were considered 100 % viable.
The data represent the means ± standard deviation (SD). Group means were compared by one-way ANOVA (analysis of variance), followed by Tukey’s procedure for multiple comparisons if necessary, using statistical software program SPSS ver. 17.0 for Windows (SPSS, Chicago, IL, USA). In all comparisons, a value of p < 0.01 was considered to indicate a statistically significant difference.
BetA enhances TGF-β-induced transcriptional responsive
BetA enhances TGF-β-induced Smad2 phosphorylation and nuclear translocation
TGF-β1-induced fibronectin expression is promoted by BetA
TGF-β-induced Mv1Lu cell growth inhibition is enhanced by BetA
BetA increases TGF-β receptor accumulation in the non-caveolae microdomain
Discussion and conclusion
This work is supported by the National Science Council of Taiwan (101-2320-B-110-003, 102-2320-B-110-007, 103-2314-B-037-064, and 103-2320-B-037-014), KMU Center for Stem Cell Research (KMU-TP103G01, KMU-TP103G00, KMU-TP103G03, KMU-TP103G04 & KMU-TP103G05), VGH-NSYSU Joint Research Project (VGHNSU103-004), and NSYSU-KMU Joint Research Project (NSYSUKMU2013-I006).
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