3509Research Article

IntroductionTransforming growth factor-� (TGF-�) is a family of 25-kDadimeric growth factors or cytokines and has three members(TGF-�1-3) in mammalian species (Massague, 1990; Roberts,1998). TGF-� is a bifunctional growth regulator: it inhibits cellgrowth of most cell types, but stimulates growth ofmesenchymal cells. The growth inhibitory activity of TGF-�has been implicated in its immune-suppressing and tumorsuppressor activities. TGF-� is a potent stimulator ofextracellular matrix synthesis and is involved in the processesof wound healing and tissue fibrosis (Massague, 1990; Roberts,1998). TGF-� is also an anti-inflammatory factor whichinhibits migration and differentiation of inflammatory cells(Shull et al., 1992; Kulkarni et al., 1993; Li, M. et al., 2006).Because of its anti-inflammatory, immunomodulatory andfibrogenic activities, TGF-� is hypothesized to be a protectivecytokine in blood for atherosclerosis (Metcalfe and Grainger,1995). This hypothesis has been supported by several lines ofevidence: (1) TGF-� antagonizes many events involved inatherosclerosis (Owens et al., 1988; Gamble et al., 1993;Mallat et al., 2001); (2) TGF-� is an anti-inflammatorycytokine (Shull et al., 1992; Kulkarni et al., 1993; Li, M. et al.,2006), and inflammation is pivotal in the initiation andpromotion of the late stages of atherosclerosis (Libby, 2002);and (3) patients with high plasma levels of TGF-� tend not todevelop atherosclerotic cardiovascular disease (Grainger et al.,1995). A causal link between atherosclerosis and low TGF-�responsiveness in vascular cells and/or low TGF-� levels in

plasma has been demonstrated in several relevant in vivomodels (McCaffrey et al., 1997; Grainger et al., 2000; Mallatet al., 2001; Reckless et al., 2001; Robertson et al., 2003; Li,D. et al., 2006). This implies that physiological factors that arecapable of suppressing TGF-� responsiveness in vascular cellsare potentially atherogenic.

Cholesterol is an essential structural component of lipid raftsand caveolae which are cholesterol- and sphingolipid-enrichedmicrodomains in plasma membranes (Galbiati et al., 2001;Simons and Ehehalt, 2002). Lipid rafts and caveolae are alsoenriched in signaling proteins, including Src-family kinases,heterotrimeric G protein subunits, and growth factor receptortyrosine kinases (Galbiati et al., 2001). Lipid rafts and caveolaehave been shown to support signaling by functioning asplatforms for recruitment and organization of signaltransduction molecules and to suppress signaling bysequestering signaling proteins (Simons and Toomre, 2001;Gomez-Mouton et al., 2004). The signaling induced by insulin(Bickel, 2002), NGF (Encinas et al., 2001) and PDGF-BB (Liuet al., 1996) is reduced following cholesterol depletion;conversely, the signaling induced by EGF is enhancedfollowing disruption of lipid rafts and/or caveolae (lipidrafts/caveolae) (Ringerike et al., 2002). More recently, lipidraft/caveolae-mediated endocytosis has been shown tofacilitate TGF-� degradation and suppress TGF-�responsiveness (Di Guglielmo et al., 2003; Mitchell et al.,2004; Le Roy and Wrana, 2005; Huang and Huang, 2005;Chen et al., 2006). Among the growth factors and hormones

Hypercholesterolemia is a major causative factor foratherosclerotic cardiovascular disease. The molecularmechanisms by which cholesterol initiates and facilitatesthe process of atherosclerosis are not well understood.Here, we demonstrate that cholesterol treatmentsuppresses or attenuates TGF-� responsiveness in all celltypes studied as determined by measuring TGF-�-inducedSmad2 phosphorylation and nuclear translocation, TGF-�-induced PAI-1 expression, TGF-�-induced luciferasereporter gene expression and TGF-�-induced growthinhibition. Cholesterol, alone or complexed in lipoproteins(LDL, VLDL), suppresses TGF-� responsiveness byincreasing lipid raft and/or caveolae accumulation ofTGF-� receptors and facilitating rapid degradation of

TGF-� and thus suppressing TGF-�-induced signaling.Conversely, cholesterol-lowering agents (fluvastatin andlovastatin) and cholesterol-depleting agents (�-cyclodextrin and nystatin) enhance TGF-� responsivenessby increasing non-lipid raft microdomain accumulationof TGF-� receptors and facilitating TGF-�-inducedsignaling. Furthermore, the effects of cholesterol on thecultured cells are also found in the aortic endothelium ofApoE-null mice fed a high-cholesterol diet. These resultssuggest that high cholesterol contributes to atherogenesis,at least in part, by suppressing TGF-� responsiveness invascular cells.

Key words: Cholesterol, TGF-�, TGF-� receptors

Summary

Cholesterol suppresses cellular TGF-�responsiveness: implications in atherogenesisChun-Lin Chen1, I-Hua Liu2, Steven J. Fliesler3, Xianlin Han4, Shuan Shian Huang2 and Jung San Huang1,*1Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St Louis, MO 63104, USA2Auxagen Inc., 7 Pricewoods, St Louis, MO 63132, USA3Departments of Ophthalmology and Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 S. Grand Blvd.,St Louis, MO 63104, USA4Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA*Author for correspondence (e-mail: huangjs@slu.edu)

Accepted 22 July 2007Journal of Cell Science 120, 3509-3621 Published by The Company of Biologists 2007doi:10.1242/jcs.006916

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whose receptors are known to be associated with lipidrafts/caveolae, only TGF-� is present at high levels in normalblood vessel walls (Grainger, 2004).

Since cholesterol is atherogenic (Steinberg, 2005) and sincelipid rafts/caveolae play a role in the modulation of TGF-�responsiveness (Di Guglielmo et al., 2003; Chen et al., 2006)and the development of atherosclerosis (Lee et al., 2004), wehypothesize that high cholesterol initiates and/or facilitatesatherogenesis by suppressing TGF-� responsiveness (Smad2and/or Smad3 dependent) in vessel-wall cells via promotingformation of or stabilizing lipid rafts/caveolae and facilitatingTGF-� degradation. To test this hypothesis, we determined theeffects of cholesterol and/or lipoproteins and cholesterol-lowering agents or cholesterol-depleting agents on TGF-�responsiveness and on lipid raft/caveolae localization anddegradation of TGF-� receptors in several cell types. We alsoexamined the indicators for TGF-� responsiveness, includingthe ratio of TGF-� binding to the type II and type I TGF-�receptors (T�R-II and T�R-I; also known as TGF�R2 andTGF�R1 – Mouse Genome Informatics) and the level ofphosphorylated Smad2 (P-Smad2), in the aortic endotheliumof ApoE-null mice fed a high-cholesterol diet (atheroscleroticmice). In this study, we demonstrate that cholesterol, both freeand complex forms (e.g. low density lipoprotein; LDL),suppresses TGF-� responsiveness in all cell types studiedwhereas cholesterol-lowering agents (lovastatin andfluvastatin) and cholesterol-depleting agents [�-cyclodextrin(�-CD) and nystatin] enhance TGF-� responsiveness in thesecells. We show that cholesterol increases and statins, �-CD andnystatin decrease lipid raft/caveolae localization and TGF-�-induced degradation of the TGF-� receptors. Furthermore, weshow that suppressed TGF-� responsiveness occurs in theaortic endothelium of ApoE-null mice fed a high-cholesteroldiet, similar to that previously observed in vascular cellsderived from atherosclerotic patients with hyper -cholesterolemia (McCaffrey et al., 1997).

ResultsTGF-�-induced signaling is modulated by cholesteroland statinsSince cholesterol is an important structural component of lipidrafts and caveolae (Pike, 2003; Lee et al., 2004), the treatmentof cells with cholesterol may suppress TGF-�-inducedsignaling, and thus TGF-� responsiveness by promotingformation of or stabilization of lipid rafts and caveolae. To testthe effect of cholesterol on TGF-�-induced signaling, wedetermined the effect of cholesterol treatment on TGF-�-induced Smad2 phosphorylation and nuclear translocation,both of which are key signaling events, leading to TGF-�responsiveness (Heldin et al., 1997; Massague, 1998;Moustakas et al., 2001). Mink lung epithelial (Mv1Lu) cells,which are a standard model system for investigating TGF-�responsiveness, and bovine aorta endothelial cells (BAECs)were treated with increasing concentrations of cholesterol at37°C for 1 hour and then incubated with 50 pM TGF-�1 at37°C for 30 minutes. P-Smad2 and Smad2 in the cell lysateswere determined by 7.5% SDS-PAGE followed by western blotanalysis using anti-P-Smad2 and anti-Smad2 antibodies andthe enhanced chemiluminescence (ECL) system, andquantified by densitometry. As shown in Fig. 1A,B, cholesteroleffectively suppressed Smad2 phosphorylation stimulated by

TGF-�1 in a concentration-dependent manner in both Mv1Lucells and BAECs. Cholesterol treatment appreciablysuppressed Smad2 phosphorylation at concentrations of 6-100 �g/ml. At 25 �g/ml, cholesterol suppressed Smad2phosphorylation by ~55% and ~90% in Mv1Lu cells andBAECs, respectively. Cholesterol also suppressed Smad2phosphorylation in a concentration-dependent manner in NRKcells. At 25 �g/ml, cholesterol suppressed Smad2phosphorylation by ~40% in these cells (data not shown). Sincecholesterol is mainly present as lipoprotein complexes (e.g.LDL and VLDL) in plasma, we determined the effects of lowdensity lipoprotein (LDL) and very low density lipoprotein(VLDL) on Smad2 phosphorylation in Mv1Lu cells. As shownin Fig. 1C, LDL (50 �g protein/ml) treatment suppressedSmad2 phosphorylation by ~60% in Mv1Lu cells and VLDL(5 �g/ml) slightly suppressed Smad2 phosphorylation in thesecells. At 50 �g protein/ml, VLDL suppressed Smad2phosphorylation by ~55±5% (n=4) in Mv1Lu cells. Theconcentration (50 �g/ml) of LDL used in the experiment waschosen because it caused inhibition of Smad2 phosphorylationby ~60%, which was similar to that induced by 25 �g/mlcholesterol (Fig. 1A). To determine the effect of cholesterol onSmad2 nuclear translocation, Mv1Lu cells were treated with50 �g/ml cholesterol at 37°C for 1 hour and then furtherincubated with and without 50 pM TGF-�1 at 37°C for 30minutes. These cells were subjected to immunofluorescentstaining using anti-P-Smad2 antibody and nuclear 4�,6-diamidine-2-phenylindole (DAPI) staining. As shown in Fig.1D, cholesterol suppressed Smad2 nuclear translocation (Fig.1D,c versus b). Counting cells with Smad2 nuclear localizationfrom four separate experiments indicated that TGF-�1 inducedSmad2 nuclear translocation in all of the treated cells, whereascholesterol suppressed Smad2 nuclear translocation in 60±5%of these cells. Taken together, these results suggest thatcholesterol treatment suppresses TGF-�1-induced signaling.

TGF-�-induced gene expression is modulated bycholesterol and statinsOne important biological activity of TGF-� is thetranscriptional activation of genes coding for extracellularmatrix (ECM) proteins and their regulatory proteins such asplasminogen activator inhibitor-1 (PAI-1) (Massague, 1990;Heldin et al., 1997; Roberts, 1998; Moustakas et al., 2001).This transcriptional activation is mediated by the Smad2/3signaling pathway. To define the effect of cholesterol on TGF-� responsiveness, we determined the effect of cholesterol onPAI-1 expression in cells stimulated with TGF-�1 by northernblot analysis using a PhosphoImager. PAI-1 expression iscommonly used to determine TGF-� responsiveness (Smad2/3dependent) in many cell types (Lund et al., 1987; Massague,1990; Roberts, 1998). Mv1Lu cells and BAECs were treatedwith increasing concentrations of cholesterol at 37°C for 1 hourand then further incubated with 50 pM TGF-�1 at 37°C for 2hours. Northern blot analysis of PAI-1 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) transcripts in the celllysates was performed; G3PDH expression was used as aninternal control. As shown in Fig. 2A,B, treatment of Mv1Lucells and BAECs with cholesterol attenuated PAI-1 expressionin a concentration-dependent manner: at 2 �g/ml (~5 �M),cholesterol attenuated PAI-1 expression by ~50% in both celltypes, and at 50 �g/ml (~125 �M) by ~70-80% in these cells.

Journal of Cell Science 120 (20)

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3511Cholesterol and TGF-� activity

To further define the effect of cholesterol on TGF-�responsiveness, we determined the effect of cholesterol (50�g/ml) on PAI-1 expression in cells treated with severalconcentrations of TGF-�1. As shown in Fig. 2C,D, treatmentwith 50 �g/ml cholesterol effectively suppressed PAI-1expression in Mv1Lu cells (Fig. 2C) and BAECs (Fig. 2D)stimulated with several concentrations of TGF-�1, rangingfrom 2 to 100 pM. Cholesterol suppressed TGF-�1-stimulatedPAI-1 expression by ~65-70% and ~50-80% in Mv1Lu cellsand BAECs, respectively. In NRK cells, cholesterol (50 �g/ml)suppressed TGF-�1-stimulated PAI-1 expression by ~60%(data not shown).

The above results suggest that cholesterol suppresses TGF-�1-stimulated PAI-1 expression in these various cell types. Todefine the physiological relevance of the cholesterol effect, weexamined the effect of LDL (a major cholesterol vehicle inblood), or fluvastatin or lovastatin (potent HMG-CoAreductase inhibitors commonly employed as cholesterol-lowering agents) (Alberts, 1988; Yuan et al., 1991) on PAI-1expression in Mv1Lu cells stimulated by TGF-�1. Mv1Lucells were pretreated with LDL (50 �g protein/ml) orcholesterol (50 �g/ml) at 37°C for 1 hour, or with fluvastatin

or lovastatin (1 �M) at 37°C for 16 hours and then incubatedwith 50 pM TGF-�1 at 37°C for 2 hours. The PAI-1 expressionin these TGF-�1-stimulated cells was determined by northernblot analysis using a PhosphoImager. At 1 �M, fluvastatin orlovastatin inhibited cholesterol synthesis by >90% asdescribed previously (Negre-Aminou et al., 1997). As shownin Fig. 2E, LDL and cholesterol suppressed TGF-�1-inducedPAI-1 expression by ~50-60% in these cells (Fig. 2Ea,b). TheLDL and cholesterol effects were abolished in the presence of25 �g/ml nystatin (a cholesterol-sequestering compound; datanot shown). Conversely, both fluvastatin and lovastatinenhanced PAI-1 expression stimulated with 50 pM TGF-�1 inMv1Lu cells (Fig. 2Fa). Fluvastatin appeared to be morepotent than lovastatin on a molar basis. Fluvastatin (1 �M)enhanced PAI-1 expression stimulated with 10 pM TGF-�1 bythreefold whereas lovastatin (1 �M) had no effect (Fig. 2Fb).However, lovastatin was capable of enhancing PAI-1expression stimulated with 50 pM TGF-�1 by approx.threefold in these cells (Fig. 2Fb). Fluvastatin enhanced PAI-1 expression stimulated by 50 pM TGF-�1 in a concentration-dependent manner (Fig. 2Ga) with an EC50 of ~0.5 �M (Fig.2Gb).

Fig. 1. Effects of cholesterol and LDL on Smad2 phosphorylation (A-C) and nuclear translocations (D) in Mv1Lu cells and BAECs stimulatedwith TGF-�1. Cells were treated with increasing concentrations of cholesterol, as indicated (A,B), 50 �g protein/ml LDL (C), 5 �g protein/mlVLDL (C) or 50 �g/ml cholesterol (D) at 37°C for 1 hour and then further incubated with 50 pM TGF-�1 for 30 minutes. P-Smad2 and totalSmad2 in the cell lysates were analyzed by immunoblotting. The relative level of P-Smad2 (P-Smad2/Smad2) was estimated. A representative ofa total of three analyses is shown (top). The quantitative analysis of the immunoblots is shown below. The relative level of P-Smad2 in cellstreated with TGF-�1 only was taken as 100% of TGF-�1-stimulated Smad2 phosphorylation. The data are mean ± s.d. *,**Significantly lowerthan that in cells treated with TGF-�1 only: P<0.001 and P<0.05, respectively. (D) Smad2 nuclear translocation was analyzed by indirectimmunofluorescent staining. Rhodamine fluorescence represents P-Smad2 staining (a-c) whereas the nuclei were stained by DAPI staining (d-f).

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To further define the role of endogenouscholesterol in the modulation of TGF-�responsiveness, we determined the effects ofcholesterol-depleting agents �-CD andnystatin (Wang et al., 1998; Subtil et al.,1999) on TGF-�-induced PAI-1 expressionin Mv1Lu cells. As shown in Fig. 2Ha,b,nystatin (25 �g/ml) and �-CD (0.5%)enhanced TGF-�-induced PAI-1 expressionby 1.8 fold and fourfold, respectively. Theeffect of cholesterol or lovastatin on TGF-�responsiveness was further characterizedusing Mv1Lu cells stably expressing aluciferase reporter driven by the PAI-1 genepromoter. As shown in Fig. 3, treatment ofcells with 50 �g/ml cholesterol suppressedluciferase activity stimulated with 50 pMTGF-�1 by ~40% (Fig. 3Aa), whereas 1 �Mlovastatin enhanced the activity by ~1.7 fold(Fig. 3Ab). The stimulatory effect oflovastatin was abolished by treatment ofMv1Lu cells with cholesterol (20 �g/ml) for1 hour prior to TGF-�1 stimulation (Fig.3Ab). Cholesterol (20 �g/ml) alone did notaffect TGF-�1-stimulated luciferase activityunder the experimental conditions used (Fig.3Aa). These results suggest that thelovastatin effect is mainly mediated by itsability to inhibit cholesterol synthesis.

TGF-�-induced growth inhibition isreversed by cholesterolAnother prominent biological activity ofTGF-� is growth inhibition of many differentcell types (Massague, 1990; Heldin et al.,

Journal of Cell Science 120 (20)

Fig. 2. Effects of cholesterol, LDL, statins, �-CDand nystatin on TGF-�1-induced PAI-1expression in Mv1Lu cells (A,C,E,F,G,H) andBAECs (B,D). Cells were treated with increasingconcentrations of cholesterol as indicated (A,B),50 �g/ml cholesterol (C,D,E), 50 �g/ml LDL(E), �-CD (0.5%; H) or nystatin (25 �g/ml; H) at37°C for 1 hour or with 1 �M fluvastatin orlovastatin (F,G) or with different concentrationsof fluvastatin (G) at 37°C for 16 hours and thenfurther 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 blotanalyses of PAI-1 and G3PDH were performedand a representative of a total of three analysesper experiment is shown (a). The relativeamounts of the transcripts (PAI-1 and G3PDH)were quantified with a PhosphoImager. The ratioof the relative amounts of PAI-1 and G3PDHtranscripts in cells treated without TGF-�1 andcholesterol, LDL or statins on the blot was takenas 1 fold or 100% of PAI-1 expression. Thequantitative data from three independentanalyses was shown (b). The data are mean ±s.d. *Significantly lower than that of controlP<0.001.

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3513Cholesterol and TGF-� activity

1997; Hocevor and Howe, 1998; Roberts, 1998; Moustakas etal., 2001; Huang and Huang, 2005). If cholesterol suppressesTGF-� responsiveness, it should antagonize TGF-�1 growthinhibitory activity which is also mediated by the Smad2/3signaling pathway. To test this, Mv1Lu cells were treated withseveral concentrations (as indicated) of cholesterol at 37°C for1 hour and then further incubated with 0.0625 or 0.125 pM TGF-�1 at 37°C for 18 hours. DNA synthesis was then determinedby measurement of [methyl-3H]thymidine incorporation intocellular DNA (Fig. 3B). It is important to note that the optimalconcentrations of TGF-�1 for growth inhibition are much lowerthan those for transcription activation. The former are in therange of 0.1 to 2 pM whereas the latter are in the range of 10 to100 pM. TGF-�1 at 0.0625 and 0.125 pM inhibited DNA

synthesis in Mv1Lu cells by ~30% and ~40%, respectively.Treatment with increasing concentrations of cholesterolcorrespondingly reversed DNA synthesis inhibition induced byTGF-�1 (Fig. 3B). Cholesterol (8 �g/ml) effectively reversedthe inhibition of DNA synthesis induced by 0.0625 and 0.125pM TGF-�1. Together with the results shown above, this resultsuggests that cholesterol is an effective TGF-�1 antagonist.

Cholesterol increases accumulation of TGF-� receptorsin lipid rafts and caveolae, resulting in enhanced TGF-�-induced degradationWe previously showed that TGF-� responsiveness is determinedby the localization of T�R-I and T�R-II in lipid raft andcaveolae and non-lipid raft microdomains of the plasma

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Fig. 3. Effects of cholesterol and lovastatin on the TGF-�1-stimulated luciferase activity (A) and TGF-�1-induced growth inhibition (B) inMv1Lu cells. (A) Cells stably expressing a luciferase reporter gene were treated with increasing concentrations (as indicated) of cholesterol at37°C for 1 hour (a) or with 1 �M lovastatin at 37°C for 16 hours ± cholesterol (20 �g/ml) at 37°C for 1 hour (b) and then further incubatedwith 50 pM TGF-�1 for 6 hours. The luciferase activity of the cell lysates (20 �g 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 independentanalyses. *Significantly lower or higher than that in cells treated with TGF-�1 only: P<0.001. (B) Cells were incubated with 0.0625 and 0.125pM 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 as100%. TGF-�1 at 0.0625 and 0.125 pM inhibited DNA synthesis by ~30% and ~40%, respectively. The degree (%) of cholesterol-mediatedreversal of TGF-�1 growth inhibition was estimated by the equation: % reversal=[1–(T1–T2/T3–T4)]�100, where T1 is the thymidineincorporation in cells treated with cholesterol alone; T2, the thymidine incorporation in cells treated with cholesterol plus TGF-�1; T3, thethymidine incorporation in cells treated with vehicle only and T4, the thymidine incorporation in cells treated with TGF-�1 alone. Theexperiments were carried out in triplicate.

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membranes (Huang and Huang, 2005; Chen et al., 2006). To testthe effect of cholesterol on the plasma microdomain localization

of the TGF-� receptors, we analyzed the lipid raft, caveolae andnon-lipid raft localization of T�R-I and T�R-II in the plasmamembrane of untreated cells or cells treated with cholesterol (50�g/ml), using sucrose density gradient ultracentrifugationanalysis (Ito et al., 2004; Chen et al., 2006) andimmunofluorescence microscopy. As shown in Fig. 4, T�R-Iwas mainly present in non-lipid raft fractions (fractions 7 and 8)whereas T�R-II was present in both the non-lipid raft and lipidraft-caveolae fractions (4 and 5), which contained transferrinreceptor 1 (TfR-1) and caveolin-1, respectively. TfR-1 andcaveolin-1 localization did not change with any of the treatmentprotocols. After treatment with cholesterol, T�R-I and T�R-IIwere found to be enriched in the lipid raft-caveolae fractions (4and 5) of the plasma membrane in Mv1Lu cells compared to thesame fractions before cholesterol treatment (Fig. 4, cholesterolversus control). Addition of TGF-�1 to the medium induceddegradation of T�R-II associated with lipid rafts/caveolae(fractions 4 and 5), which contained ~50% of the total T�R-IIprotein of plasma membranes in Mv1Lu cells (Fig. 4, TGF-�1verses control). Pretreatment with cholesterol further enhancedTGF-�1-induced degradation of T�R-II in lipid rafts andcaveolae (Fig. 4; fractions 4 and 5, cholesterol + TGF-�1 versesTGF-�1). After treatment with cholesterol, T�R-I was found tocolocalize with caveolin-1 at the cell surface as determine byimmunofluorescence confocal microscopy (Fig. 5j). Treatmentwith both cholesterol and TGF-�1 resulted in the colocalizationof T�R-I and caveolin-1 in endocytic vesicles (Fig. 5l). In NRKcells, cholesterol treatment also increased lipid raft/caveolaeaccumulation of both T�R-I and T�R-II and facilitated TGF-�1-induced degradation of these receptors as determined bywestern blot analysis of the sucrose density gradient fractionsusing antibodies to T�R-I (ALK-5) and T�R-II and (data notshown). These results suggest that cholesterol treatmentenhances TGF-�-induced and lipid raft/caveolae-mediatedinternalization and degradation of TGF-� receptors.

Cholesterol and LDL appeared to enhance TGF-�1-induceddegradation of T�R-II in aconcentration-dependent manner(Fig. 6Aa and Fig. 6Ba,respectively). Cholesterol (10

Journal of Cell Science 120 (20)

Fig. 4. Sucrose density gradient analysis of T�R-II in the plasmamembrane of Mv1Lu cells treated with or without cholesterol andstimulated with and without TGF-�1. Cells were treated with orwithout 50 �g/ml cholesterol at 37°C for 1 hour and furtherincubated with and without 50 pM TGF-�1 for 2 hours. The celllysates from these treated cells were subjected to sucrose densitygradient ultracentrifugation. The sucrose gradient fractions were thenanalyzed by western blot analysis using anti-T�R-I, anti-T�R-II,anti-TfR-1 and anti-caveolin-1 antibodies. The arrow indicates thelocations of T�R-I, T�R-II, caveolin-1 and TfR-1. Fractions 4 and 5contained lipid rafts/caveolae whereas fractions 7 and 8 are non-lipidraft fractions. Treatment with cholesterol alone did not affect thetotal amounts of TGF-� receptor proteins and cell proteins. Openarrowheads indicate the increased amount of T�R-I or T�R-II in thefraction as compared with that of untreated control. *The decreasedamount of T�R-II in the fraction as compared with that of untreatedcontrol. #The decreased amount of T�R-II in the fraction ascompared with that of treatment with cholesterol or TGF-�1 alone.

Fig. 5. Immunofluorescent localizationof T�R-I and caveolin-1 in Mv1Lucells treated with and withoutcholesterol and TGF-�1. Cells weretreated with or without 50 �g/mlcholesterol at 37°C for 1 hour andincubated with and without 100 pMTGF-�1 at 37°C for 30 minutes. Thecells were then fixed with coldmethanol and incubated with a goatantibody to T�R-I (e-h) and rabbitantibody to caveolin-1 (a-d) followedby incubation with Rhodamine-conjugated donkey anti-goat antibodyor FITC-conjugated mouse anti-rabbitantibody. The fluorescence in cells wasexamined using a fluorescent confocalmicroscope. Bar, 20 �m. The arrowsindicate colocalization of T�R-I andcaveolin-1 at the cell surface (j).

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3515Cholesterol and TGF-� activity

�g/ml) and LDL (15 �g protein/ml) enhanced TGF-�1-induced degradation of T�R-II by ~70% (Fig. 6Ab and Fig.6Bb, respectively). Treatment with cholesterol or LDL alonedid not induce T�R-II degradation (Fig. 6Ab,Bb). Thecholesterol-enhanced TGF-�1-induced degradation of T�R-IIwas abolished by incubation of cells with 1% �-CD prior toTGF-�1 stimulation (Fig. 6Ab). Cholesterol-depleting agents(e.g. nystatin) have been shown to inhibit lipid-raft-mediateddegradation of receptor-bound TGF-� (Le Roy and Wrana,2005; Chen et al., 2006). These results suggest that cholesterolor LDL treatment increases lipid raft/caveolae accumulationand TGF-�1-induced degradation of T�R-II and that thecholesterol effect is reversible.

Statins increase accumulation of TGF-� receptors innon-lipid raft microdomains, resulting in attenuated TGF-�-induced degradationTo define the role of endogenous cholesterol in determining thelocalization of T�R-II in plasma membrane microdomains,Mv1Lu cells were treated with lovastatin (1 �M), fluvastatin(1 �M) or nystatin (25 �g/ml) and cholesterol (50 �g/ml) at37°C for 16 hours or 1 hour, respectively. The plasmamembrane microdomain localization of T�R-II wasdetermined by sucrose density gradient ultracentrifugationfollowed by western blot analysis. As shown in Fig. 7A,treatment with lovastatin, fluvastatin or nystatin increasedaccumulation of T�R-II in non-lipid raft microdomains(fraction 7 or 8). The fluvastatin-induced increasedaccumulation of T�R-II in non-lipid raft microdomainsappeared to attenuate degradation of T�R-II induced by TGF-�1 (Fig. 6B, bottom). It is important to note that treatment ofMv1Lu cells with fluvastatin at 37°C for 16 hours increasesthe total amount of T�R-II protein by approx. twofold whencompared with those of cholesterol-treated and control cells(Fig. 7A,B, top). This could be due to decreased degradationand/or increased biosynthesis of T�R-II in cells treated withfluvastatin. Taken together with the results shown in Fig. 2F,G,

these results suggest that treatment of cells with the statinsincreases accumulation of T�R-II in non-lipid raftmicrodomains and presumably increases endosomal signaling(Di Guglielmo et al., 2003; Chen et al., 2006), resulting inenhanced TGF-� responsiveness.

A low ratio of TGF-� binding to T�R-II and T�R-I and alow level of P-Smad2 occur in aortic endothelium ofApoE-null mice fed a high-cholesterol diet and in BAECsWe previously demonstrated that the ratio of TGF-�1 bindingto T�R-II and T�R-I (as determined by 125I-TGF-�1 affinitylabeling) can be used as an indicator of TGF-� responsiveness(Huang and Huang, 2005; Chen et al., 2006). The magnitudeof the cellular responsiveness induced by TGF-� positivelycorrelates with the ratio of TGF-� binding to T�R-II and T�R-I in the same cell type (Chen et al., 2006). To define thephysiological relevance of the in vitro effect of cholesterol, weperformed 125I-TGF-�1 affinity labeling and determined TGF-� responsiveness in the aortic endothelium of wild-type miceand ApoE-null mice fed a high-cholesterol (2%) or normaldiet. ApoE-null mice fed a high-cholesterol diet exhibitedtypical atherosclerotic lesions (such as fatty streaks andplaques) in the aorta. For 125I-TGF-�1 affinity labeling, theaortas were cut lengthwise to expose the intimal endotheliumto 125I-TGF-�1 in binding buffer. After 2.5 hours at 0°C, aortaswere washed with phosphate-buffered saline (PBS) severaltimes and cross-linked with disuccinimidyl suberate (DSS); the125I-TGF-�1 affinity-labeled aortic endothelium was scrappedoff from the luminal surface of the aorta and extracted with 1%Triton X-100. The extracts, which contained factor VIII (anendothelial cell marker), were then analyzed by 7.5% SDS-PAGE and autoradiography. As shown in Fig. 8A, the aorticendothelium from wild-type mice exhibited a higher ratio (>1)of 125I-TGF-�1 binding to T�R-II and T�R-I (Fig. 8Aa, top,lane 1 and Fig. 8Aa, bottom). However, the aortic endotheliumfrom atherosclerotic mice (ApoE-null mice fed a high-cholesterol diet) exhibited a low ratio (<1) of 125I-TGF-�1

Fig. 6. Concentration dependence ofcholesterol (A) or LDL (B) inenhancing TGF-�1-induceddegradation of T�R-II in Mv1Lu cells.Cells were treated with severalconcentrations of cholesterol (A) orLDL (B), as indicated, at 37°C for 1hour, then incubated with and without1% �-CD at 37°C for 1 hour andfurther incubated with 50 pM TGF-�for 2 hours. The cell lysates were thensubjected to western blot analysisusing anti-T�R-II and anti-�-actinantibodies (a) and quantification bydensitometry (b). The ratio of therelative amounts of T�R-II and �-actinin cells treated without TGF-�1 wastaken as the 100% level of T�R-II. Thedata are representative of a total ofthree independent analyses; values aremean ± s.d. *Significantly lower thancontrol cells: P<0.001.

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binding to T�R-II and T�R-I (Fig. 8Aa, top, lane 2 and Fig.8Aa, bottom). A low ratio of 125I-TGF-�1 binding to T�R-IIand T�R-I was also found in cultured BAECs treated withcholesterol (50 �g/ml) as compared with that found inuntreated cells (Fig. 8Ab, top, lane 2 versus lane 1 and bottom).It is important to note that cholesterol treatment resulted inincreased 125I-TGF-�1 binding to T�R-I and decreased 125I-TGF-�1 binding to T�R-II in BAECs (Fig. 8Ab).

To examine TGF-� responsiveness, the levels of P-Smad2and VCAM-1 in the Triton X-100 extracts of the aorticendothelium from wild-type and atherosclerotic mice wereexamined by western blot analysis following 7.5% SDS-PAGE. The level of P-Smad2 has been used as an indicatorfor TGF-� responsiveness (Smad2/3 dependent) in aortas andother tissues (Phipps et al., 2004; Liao and Laufs, 2005). Theupregulation of VCAM-1 has been employed as a marker forearly atherosclerotic lesions (Nakashima et al., 1998). Asshown in Fig. 8B, ApoE-null mice fed a high cholesterol dietexhibited higher expression (~1.6 fold) of VCAM-1 than thatfound in wild-type mice (top, lanes 3 and 4 versus lanes 1 and2, and bottom). The levels of P-Smad2 were lower inatherosclerotic mice than those in wild-type mice fed a high-cholesterol diet (top, lanes 3 and 4 versus lanes 1 and 2). Thelevels of P-Smad2 in atherosclerotic mice were estimated tobe ~40% of those found in wild-type mice (Fig. 8B, bottom).The endothelium of the coronary artery from wild-type miceappeared to be plaque free (Fig. 9A), whereas an advancedplaque was observed in ApoE-null mice fed a high cholesteroldiet (Fig. 9B). P-Smad2 was not detected byimmunofluorescence microscopy in the endothelium of thecoronary artery from Apo-E-null mice fed a high cholesteroldiet (Fig. 9D). By contrast, P-Smad2 was found to be presentin the endothelium of the coronary artery from wild-type mice

(Fig. 9C). These results suggest that the aortic endothelium ofatherosclerotic mice has the characteristics of a low ratio ofTGF-�1 binding to T�R-II and T�R-I and suppressed TGF-� responsiveness, like those observed in cultured BAECstreated with cholesterol. Interestingly, vascular smooth musclecells derived from atherosclerotic plaques have also beenshown to exhibit a low ratio (<1) of TGF-�1 binding to T�R-II and T�R-I and suppressed TGF-� responsiveness(McCaffrey et al., 1997). The ratio of TGF-�1 binding toT�R-II and T�R-I in vascular smooth muscle cells fromnormal humans is >1 (McCaffrey et al., 1997). Vascular cellsin atherosclerotic mice and humans appear to have a low ratioof TGF-�1 binding to T�R-II and T�R-I and suppressed TGF-� responsiveness. This suggests that hypercholesterolemiamay contribute to atherosclerosis via suppression of TGF-�responsiveness.

DiscussionSeveral lines of evidence presented herein indicate thatcholesterol is an effective TGF-�1 antagonist capable ofsuppressing TGF-� responsiveness (Smad2/3 dependent) invarious cell types: (1) cholesterol treatment suppresses TGF-�1-induced signaling, such as Smad2 phosphorylation andnuclear translocation; (2) cholesterol treatment antagonizesTGF-�1-induced PAI-1 expression in Mv1Lu, BAE and NRKcells; (3) cholesterol treatment suppresses the luciferaseactivity in TGF-�1-stimulated Mv1Lu cells expressing aluciferase reporter gene driven by the PAI-1 promoter; (4)cholesterol treatment reverses TGF-�-induced growthinhibition in Mv1Lu cells; and (5) the effect of cholesterol onTGF-� responsiveness is rapid, reversible and specific. Theeffect reaches maximum after incubation of cells withcholesterol at 37°C for 30 minutes (unpublished results). The

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Fig. 7. Effects of the treatments with lovastatin, fluvastatin and nystatin on the plasma-membrane microdomain localization (A) and TGF-�1-induced degradation of T�R-II (B) in Mv1Lu cells. Cells were treated with or without lovastatin (1 �M), fluvastatin (1 �M) or nystatin (25�g/ml) at 37°C for 16 hours or 1 hour, respectively. The treated cells were directly analyzed by sucrose density gradient ultracentrifugationanalysis (A) or further incubated with 50 pM TGF-� at 37°C for several time periods as indicated (B). Western blot analyses of the sucrosedensity gradient fractions (A) and of TGF-�1-treated cell lysates (B) were performed using anti-T�R-II, anti-caveolin-1, anti-TfR-1 and anti-�-actin antibodies. The open arrowheads indicate the increased amount of T�R-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 treatedwithout fluvastatin: P<0.05.

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cholesterol effect can be reversed by incubation of cells with1% �-CD prior to TGF-�1 stimulation. The cholesterol effectis also specific. Cholesterol effectively suppresses TGF-�responsiveness whereas related oxysterol derivatives(including 25-hydroxycholesterol, 7-dehydrocholesterol,cholest-5-one-7-one, 7-ketocholesterol, 7�,8�-epoxy -cholesterol and 7�-hydroxycholesterol) do not show suchactivity (unpublished results). However, cholesterol is differentfrom conventional TGF-� antagonists. Cholesterol does notbind to TGF-� or compete with TGF-� for binding to TGF-�

receptors under the experimental conditions (unpublishedresults).

Di Guglielmo et al. (Di Guglielmo et al., 2003) and Chen etal. (Chen et al., 2006) demonstrated that TGF-� responsivenessis determined by TGF-� partitioning between lipid raft/caveolae-mediated and clathrin-mediated endocytosis. Lipid-raft/caveolae-mediated endocytosis facilitates TGF-�degradation and thus suppresses TGF-� responsiveness.Clathrin-mediated endocytosis results in Smad2/3-dependentendosomal signaling, promoting TGF-� responsiveness. Based

on the dominance model for the signal that controls TGF-� partitioning between the two distinct endocytosispathways (Huang et al., 2005; Chen et al., 2006), wehypothesize that two major T�R-I-T�R-II complexes,Complex I and Complex II, are present in the non-lipid raftand lipid raft/caveolae microdomains of the plasmamembrane, respectively (Fig. 10). T�R-I and T�R-II haveaffinity for each other and form complexes in the absence

Fig. 8. A lower ratio of 125I-TGF-�1binding to T�R-II and T�R-I (A) andsuppressed TGF-� responsiveness (B) inthe aortic endothelium of ApoE-nullmice fed a high-cholesterol diet and incultured BAECs treated withcholesterol. (A) 125I-TGF-� affinitylabeling. (a) The aortic endotheliumfrom wild-type and ApoE-null(ApoE–/–) mice fed a high-cholesteroldiet (lanes 1 and 2, respectively) andBAECs treated with and without 50�g/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 andautoradiography (top), and quantifiedusing a PhosphoImager (bottom). Arepresentative of a total of five animalseach analyzed or of three independentBAEC analyses is shown. The numberon the top of the bar charts is theestimated ratio of 125I-TGF-�1 bindingto T�R-II and T�R-I. (B) Western blotanalysis. 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 �g) of the Triton X-100 extracts were then subjected towestern blot analysis using antibodies to Smad2, P-Smad2, VCAM-1 and �-actin (top). Two representatives (lanes 1 and 2, and 3 and 4) of atotal of five animals each analyzed are shown (top). The relative levels of P-Smad2 (P-Smad2/Smad2) and VCAM-1 (VCAM-1/�-actin) wereestimated (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.

Fig. 9. Immunofluorescent localization of P-Smad2 in thecoronary artery from wild-type and ApoE-null mice fed a highcholesterol diet. (A,B) Representative photographs of thecoronary artery from wild-type (A) mice exhibited a plaque-freesection; that from ApoE-null mice fed a high cholesterol diet (B)showed an advanced plaque. (C,D) Immunofluorescent confocalmicroscopic analysis of the tissue cross sections revealed that P-Smad2 is present in wild-type mice (C) whereas no P-Smad2 wasdetected in the endothelium of the coronary artery from ApoE-null mice fed a high cholesterol diet (D). *The location of theartery lumen. The magnification is 200� (A and B); bar, 20 �m(C,D). The arrows in C indicate the localization of P-Smad2 inthe artery endothelium.

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of ligand (Chen et al., 1997). Complex I contains more T�R-II than T�R-I whereas Complex II contains more T�R-I thanT�R-II (Chen et al., 2006). The ratio of T�R-II and T�R-I inthe complexes can be determined by 125I-TGF-�1 affinitylabeling (Chen et al., 2006). In the presence of TGF-�,Complex I undergoes clathrin-mediated endocytosis, resultingin promotion of Smad2/3-dependent signaling and cellularresponsiveness. Complex II undergoes lipid raft/caveolae-mediated endocytosis, resulting in enhanced TGF-�-induceddegradation and less cellular responsiveness. Complex II mayalso be capable of mediating Smad2/3-independent signaling,which leads to different cellular responsiveness in fibroblasts(Pannu et al., 2007). In this study, we demonstrate thatcholesterol treatment increases accumulation of T�R-I andT�R-II (as Complex II) to lipid rafts/caveolae, resulting inenhanced TGF-� degradation and attenuated TGF-�responsiveness (Fig. 10). Depletion of cholesterol from theplasma membrane by cholesterol-lowering agents (statins) orcholesterol-depleting agents (�-CD and nystatin) leads todecreased formation of or destabilization of lipidrafts/caveolae, thereby increasing localization of T�R-I andT�R-II (as Complex I) in non-lipid raft microdomains and

promotion of TGF-�-induced endosomal signaling andresponsiveness (Fig. 10). The cholesterol effect on the lipidraft/caveolae localization of T�R-I and T�R-II appears to bespecific as compared with other TGF-� receptor types.Treatments with cholesterol and TGF-�1, alone or together, donot influence the plasma membrane microdomain localizationof T�R-III and T�R-V (unpublished results), which wasrecently identified as low density lipoprotein receptor-relatedprotein-1 (Huang et al., 2003). Both T�R-III and T�R-V aremainly localized in non-lipid raft microdomains of the plasmamembrane.

In this communication, we also demonstrate that, similar totreatment with free cholesterol, treatment with LDL and VLDLcauses suppression of TGF-� responsiveness (Smad2/3dependent). This effect of LDL or VLDL can be abolished inthe presence of cholesterol-binding compounds (e.g. �-CD andnystatin), suggesting that it is mediated specifically bycholesterol, rather than by some other lipoprotein constituents.However, treatment with lovastatin and fluvastatin increasesaccumulation of TGF-� receptors to non-lipid raftmicrodomains and attenuates TGF-�-induced degradation ofthe TGF-� receptors, resulting in enhanced TGF-�

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Degradation pathwaySignal transduction pathway

Early endosome

Caveolin-positive vesicle

IIII IIII

TGF-β

TGF-βTGF-β

IIII

Complex I Complex II

IIIIIIIIII

TGF-β

II

Lipid raft/Caveolae

Clathrin-coated pit

Chol (statins)

Chol (LDL)

IIIIIIIIIIII

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 T�R-I–T�R-II complexes (Complex I and Complex II) present on the cell face. Complex I and Complex II are mainlylocalized in the non-lipid raft and lipid raft/caveolae microdomains of the plasma membrane, respectively. The numbers of T�R-I and T�R-IImolecules (blue rectangles) in Complex I and Complex II shown in the model are arbitrary and intended to indicate that Complex I andComplex II contain T�R-II>T�R-I and T�R-I>T�R-II, respectively. The ratio of T�R-II to T�R-I can be determined by 125I-TGF-�1 affinitylabeling (Chen et al., 2006). Cholesterol increases the formation and/or stabilization of lipid rafts/caveolae by integration into the plasmamembrane, thereby increasing the localization of T�R-I and T�R-II in lipid rafts/caveolae (as Complex II), facilitating rapid degradation ofTGF-� and attenuating TGF-� responsiveness (Smad dependent). Complex II may also be capable of mediating Smad2/3-indepentent signalingwhich leads to different cellular responsiveness such as fibrogenesis in fibroblasts (Pannu et al., 2007). Depletion of cholesterol in the plasmamembrane, by treating cells with cholesterol-lowering agents (e.g. statins) or cholesterol-depleting agents (e.g. �-CD), facilitates thelocalization of T�R-I and T�R-II in non-lipid raft microdomains. In the presence of ligand, Complex I undergoes clathrin-mediatedendocytosis, 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-� receptorcomplexes contain more Complex I than Complex II in the aortic endothelium.

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responsiveness. The abilities of LDL, VLDL and statins(lovastatin and fluvastatin) to suppress and enhance TGF-�responsiveness, respectively, in cultured cells may becorrelated with their capacities to cause and preventatherosclerosis in human patients, respectively.

The finding that cholesterol is a TGF-� antagonist for Smad-dependent cellular responsiveness not only provides onemolecular mechanism by which hypercholesterolemiacontributes to atherosclerosis, but also suggests explanationsfor many questions regarding diseases associated withhypercholesterolemia and pleiotropic effects of statins(Buchwald, 1992; Liao and Laufs, 2005; Jacobs et al., 2006).For example, why do patients with hypercholesterolemia tendto develop cancer? Since cholesterol is a TGF-� antagonist,hypercholesterolemia may suppress TGF-� growth inhibitoryactivity in targeted epithelial cells, contributing tocarcinogenesis. Why does statin therapy prevent or reduce theincidence of cancer occurrence? Cholesterol reductionafforded by statin therapy (Liao and Laufs, 2005) may enhancethe activity of TGF-�, a known tumor suppressor (Wang et al.,1995; Piek and Roberts, 2001; Derynck et al., 2001). Statinshave been reported to exhibit pleiotropic effects not thought tobe mediated by their cholesterol-lowering actions (Liao andLaufs, 2005). It is possible that some of these activities aremediated by their abilities to decrease cholesterol content inthe plasma membrane, affecting the structure and function oflipid rafts/caveolae, which are known to modulate signalingmediated by G protein-coupled receptors, receptor tyrosinekinases, TGF-� receptors (T�R-I and T�R-II) and possiblyothers (Galbiati et al., 2001; Simons and Toomre, 2000; DiGuglielmo et al., 2003; Gomez-Mouton et al., 2004; Le Royand Wrana, 2005; Huang and Huang, 2005; Chen et al., 2006).The finding that cholesterol contributes to atherosclerosis bysuppressing TGF-� responsiveness in vascular cells may leadto the development of novel therapies for treating or preventingatherosclerosis. We suspect that patients with normal levels ofcholesterol who receive high-dose statin therapy but stilldevelop atherosclerosis (Brown and Goldstein, 2006) may havesuppressed TGF-� responsiveness in vascular cells (due to anunidentified mechanism) and/or low-level TGF-� activity inblood, both of which contribute to the development ofatherosclerotic cardiovascular disease. Thus, therapeuticagents that enhance endogenous TGF-� activity in bloodand/or TGF-� responsiveness in vessel-wall cells couldprovide a novel strategy to treat or prevent atherosclerosis.

Materials and MethodsMaterialsNa125I (17 Ci/mg) and [methyl-3H]thymidine (67 Ci/mmol) were purchased fromICN Radiochemicals (Irvine, CA, USA). High molecular mass protein standards(myosin, 205 kDa; �-galactosidase, 116 kDa; phosphorylase, 97 kDa; bovine serumalbumin, 66 kDa), cholesterol (>99% pure), DAPI, chloramine-T, bovine serumalbumin (BSA), human low density lipoprotein (LDL; which was composed of ~20-25% protein and 75-80% lipid including 9% free cholesterol, 42% cholesterol ester,20-24% phospholipids and 5% triglycerides), human very low density lipoprotein(VLDL), human high density lipoprotein (HDL), fluvastatin, lovastatin, cholesterol,disuccinimidyl suberate (DSS), nystatin and �-cyclodextrin (�-CD) were obtainedfrom Sigma (St Louis, MO). Cholesterol did not undergo detectable oxidation underthe experimental conditions. Like cholesterol, LDL and VLDL (but not HDL)suppressed TGF-� responsiveness. However, cholesterol was mainly used in theexperiments in order to exclude the possibility that other lipid and proteincomponents in the lipoproteins might influence the assays employed. P-Smad2antibody was obtained from Cell Signaling Technology, Inc. (Danvers, MA). TGF-�1 was purchased from Austral Biologicals (San Ramon, CA). Rabbit polyclonalantibodies to caveolin-1, Smad2, �-actin, T�R-I (ALK-5) and T�R-II were obtained

from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonalantibodies to TfR-1 and VCAM-1 were obtained from Zymed Laboratories (SanFrancisco, CA) and Chemicon International, Inc. (Temecula, CA), respectively. Theluciferase assay system was obtained from Promega (Madison, WI).

Cell cultureMv1Lu cells, MLECs-clone 32 cells (which are Mv1Lu cells stably expressing theluciferase reporter gene driven by the PAI-1 promoter) (Abe et al., 1994), BAECsand NRK cells were maintained in DMEM or DMEM-F12 containing 10% fetalcalf serum with or without bFGF.

Western blot analysis of cultured cellsCells grown to near confluence on 12-well dishes were treated with cholesterol,vehicle (ethanol), or LDL in serum-free DMEM (0.5 ml/well) at 37°C for 1 hour.The final concentration of ethanol in the medium was 0.2%. Serum-free DMEMwas used to avoid the potential influence of serum components in the assay systems.The effect on TGF-� responsiveness reached maximum after treatment of cells withcholesterol or LDL at 37°C for 30 minutes but the treatment (at 37°C for 1 hour)of cells with cholesterol or lipoproteins was used throughout the experiments. Thetreated cells were further incubated with 50 or 100 pM TGF-�1 at 37°C for 30minutes (for determining Smad2 phosphorylation) or for 2 hours (for determiningT�R-II). Treated cells were lysed and cell lysates with equal amounts of proteinwere analyzed by 7.5% SDS-PAGE and western blotting using anti-Smad2, anti-P-Smad2, anti-�-actin, anti-caveolin-1 anti-T�R-I or anti-T�R-II antibodies, asdescribed previously (Huang et al., 2003). The antigens on the blots were visualizedby using horseradish peroxidase-conjugated anti-rabbit IgG antibody and the ECLsystem as described (Huang et al., 2003). The relative intensities of antigen bandson X-ray films were quantified by densitometry.

Northern blot analysisCells grown to confluence on 12-well dishes in DMEM containing 10% fetal calfserum were treated with several concentrations of cholesterol in ethanol in serum-free DMEM (0.5 ml/well) at 37°C for 1 hour. The final concentration of ethanol inthe medium was 0.2%. The cholesterol-treated cells were then incubated with 100pM TGF-�1 at 37°C for 2 hours. The transcripts of PAI-1 and G3PDH (as control)in the cell lysates were examined by northern blot analysis and quantified with aPhosphoImager which yields a linearity from 9,000 to 100,000 arbitrary units of thetranscript intensity.

[Methyl-3H]thymidine incorporationThe growth of cholesterol-treated cells was determined by measurement of [methyl-3H]thymidine incorporation into cellular DNA as described previously (Huang etal., 2003). Briefly, cells grown to near confluence on 48-well dishes were treatedwith several concentrations of cholesterol at 37°C for 1 hour in serum-free DMEM.The final concentration of ethanol (the solvent vehicle for cholesterol) in themedium was 0.2%. Treated cells were then incubated with 0.0625 pM or 0.125 pMof TGF-�1 in DMEM containing 0.1% fetal calf serum at 37°C for 18 hours. The[methyl-3H]thymidine incorporation into cellular DNA was then determined byincubation of cells with [methyl-3H]thymidine at 37°C for 2 hours in DMEMcontaining 0.1% fetal calf serum. The optimal concentrations of TGF-�1 to inhibitcell growth are in the range of 0.1 to 2 pM. Under the experimental conditions,cholesterol did not affect cell viability.

Luciferase activity assay and indirect immunofluorescentstainingMv1Lu cells stably expressing the luciferase reporter gene driven by the PAI-1promoter (MLECs – Clone 32) (Abe et al., 1994) grown to near confluence on 12-well dishes were treated with different concentrations of cholesterol, with andwithout 50 �g/ml cholesterol or with 1 �M lovastatin at 37°C for 1 hour or 16hours, respectively. Treated cells were further incubated with 50 pM TGF-�1 at37°C for 6 hours and lysed in 100 �l of lyses buffer (Promega). The cell lysates(~20 �g protein) were then assayed using the luciferase kit from Promega. Forindirect immunofluorescent staining, cells grown on cover glasses were treated withcholesterol (50 �g/ml) or with solvent vehicle at 37°C for 1 hour, stimulated with50 pM TGF-�1 at 37°C for 30 minutes, fixed in cold 100% methanol and thenincubated with antibody to P-Smad2 overnight. The antigen was visualized byincubation with Rhodamine-conjugated goat antibody to rabbit IgG followed byimmunofluorescence microscopy. Cell nuclei were stained by DAPI staining. Cellswith P-Smad2 nuclear localization were counted. The experiment was performed intriplicate.

Immunofluorescent confocal microscopyMv1Lu cells grown on coverslips overnight (50% confluency) were pretreated with50 �g/ml cholesterol at 37°C for 1 hour and than incubated with 100 pM TGF-�1for 30 minutes. After TGF-�1 stimulation, cells were fixed in methanol at –20°Cfor 15 minutes, washed with PBS and then blocked by 0.2% gelatin in PBS for 1hour. Cells were incubated overnight at 4°C in a humidified chamber with a goatantibody against T�R-I (G-16; Santa Cruz Biotechnology) and rabbit antibody

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against caveolin-1 (BD Transduction Laboratories) at 1:100 dilution. After extensivewashing, cells were incubated with Rhodamine-conjugated donkey anti-goatantibody and FITC-conjugated mouse anti-rabbit antibody at a 1:50 dilution for 1hour. Images were acquired using a Leica TCS SP confocal microscope (LeicaMicrosystems Ltd., Heidelberg, Germany). The measurements of colocalization ratewere analyzed using a Leica Application Suite.

Separation of lipid raft and non-lipid raft microdomains ofplasma membranes by sucrose density gradientultracentrifugationMv1Lu or NRK cells were grown to near confluence in 100 mm dishes (5-10�106

cells per dish). Cells were incubated with cholesterol (50 �g/ml) at 37°C for 1 hourand then incubated with TGF-�1 (100 pM) for 1 hour. After two washes with ice-cold phosphate-buffered saline, cells were scraped into 0.85 ml of 500 mM sodiumcarbonate, pH 11.0. Homogenization was carried out with 10 strokes of a tight-fitting Dounce homogenizer followed by three 20-second bursts of an ultrasonicdisintegrator (Soniprep 150; Fisher Scientific) to disrupt cell membranes, asdescribed previously (Ito et al., 2004; Chen et al., 2006). The homogenates wereadjusted to 45% sucrose by addition of 0.85 ml of 90% sucrose in 25 mM 2-(N-morpholino) ethanesulfonic acid, pH 6.5, 0.15 M NaCl (MBS), and placed at thebottom of an ultracentrifuge tube. A discontinuous sucrose gradient was generatedby overlaying 1.7 ml of 35% sucrose and 1.7 ml of 5% sucrose in MBS on the topof the 45% sucrose solution and it was then centrifuged at 200,000 g for 16–20hours in an SW55 TI rotor (Beckman Instruments, Palo Alto, CA, USA). A light-scattering band was observed at the 5 and 35% sucrose interface. Ten 0.5-mlfractions were collected from the top of the tube, and a portion of each fraction wasanalyzed by SDS-PAGE followed by western blot analysis using antibodies to T�R-I (ALK-5), T�R-II, TfR-1 and caveolin-1. The relative amounts of T�R-I, T�R-II,TfR-1 and caveolin-1 on the blot were quantified by densitometry. The proteinrecovery and caveolin-1 and TfR-1 localization (fractions 4 and 5, and 7 and 8,respectively) did not significantly change with any of the treatment protocols.

125I-TGF-�1 affinity labeling and the determination of P-Smad2and VCAM-1 levels in aortic endothelium from ApoE-null andwild-type miceFemale ApoE-null and wild-type mice (C57BL/6 background; 6- to 8-weeks old)were fed a high cholesterol (2%) or normal diet for 4-5 weeks. ApoE-null mice feda high-cholesterol diet exhibited typical atherosclerotic lesions (such as fatty streaksand plaques) in the aorta as described previously (Palinski et al., 1994). By contrast,ApoE-null mice fed a normal diet, and wild-type mice fed either a high-cholesteroldiet or a normal diet, did not have significant atherosclerotic lesions in the aortasin the experimental period. The aortas (~2 cm) removed from the animals were cutlengthwise to expose intimal endothelium to binding buffer (1 ml) containing 100pM 125I-TGF-�1 (Huang et al., 2003). After 2.5 hours on ice, 125I-TGF-�1 affinitylabeling was performed using DSS, as described previously (Huang et al., 2003;Chen et al., 2006). The aortas were then washed with binding buffer. The aorticendothelia were then scraped off using a razor and extracted with 1% Triton X-100in the binding buffer. The Triton X-100 extracts with equal amounts of protein wereanalyzed by 7.5% SDS-PAGE and autoradiography. The Triton X-100 extracts werefound to contain factor VIII, an endothelial cell marker (based on western blotanalysis). To determine the relative amounts of P-Smad2, Smad2, VCAM-1 and �-actin, the aortic endothelia from ApoE-null and wild-type mice were extracted with1% Triton X-100 in the binding buffer. The Triton X-100 extracts, with equalamounts of protein, were subjected to 7.5% SDS-PAGE followed by western blotanalysis using antibodies to P-Smad2, Smad2 and VCAM-1/�-actin. The relativeamounts of P-Smad2, Smad2, VCAM-1 and �-actin were quantified bydensitometry as described above.

Immunofluorescent localization of P-Smad2 in coronaryarteriesTissues cross sections (5 �m thick) were stained with Hematoxylin and Eosin(H&E). The tissue sections were subjected to immunostaining with rabbit anti-P-Smad2 antibody after deparaffinization and antigen retrieval by heating in amicrowave. The tissue slides were first blocked with 5% BSA and immunostainedwith anti-P-Smad2 antibody (1:100 dilution) overnight and detected with FITC-conjugated goat anti-rabbit antibody (1:300 dilution) at room temperature for 1 hour.The tissue slides were viewed using a fluorescent confocal microscopy andphotographed.

Statistical analysisThe values (except in Fig. 8B) are presented as mean ± s.d. Two-tailed unpairedStudent’s t-test was used to determine the significance of differences betweengroups. P<0.05 was considered significant. Comparisons between the two groupsin Fig. 7B was conducted with the Mann-Whitney test.

We thank Daniel B. Rafkin for providing Mv1Lu cells expressingthe PAI-1 promoter-driven luciferase, Tomasz Heyduk for

immunofluorescent confocal microscopy, and William S. Sly, AbdulWaheed and Frank E. Johnson for critical review of the manuscript,and John McAlpin for typing the manuscript. This work wassupported by U.S.P.H.S. (National Institutes of Health) grantsCA38808 (J.S.H.), AR052578 (S.S.H.) and EY07361 (S.J.F.), by anunrestricted departmental grant from Research to Prevent Blindness(S.J.F.), and by the Norman J. Stupp Charitable Trust (S.J.F.).

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