|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 1 January 2005, Vol. 105, No. 1, pp. 153-160. Prepublished online as a Blood First Edition Paper on August 24, 2004; DOI 10.1182/blood-2004-03-0990.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Acute cholesterol depletion impairs functional expression of tissue factor in fibroblasts: modulation of tissue factor activity by membrane cholesterolFrom the Biomedical Research Division, The University of Texas Health Center at Tyler, Tyler, TX.
Cholesterol, in addition to providing rigidity to the fluid membrane, plays a critical role in receptor function, endocytosis, recycling, and signal transduction. In the present study, we examined the effect of membrane cholesterol on functional expression of tissue factor (TF), a cellular receptor for clotting factor VIIa. Depletion of cholesterol in human fibroblasts (WI-38) with methyl-  -cyclodextrin–reduced TF activity at the cell surface. Binding studies with radiolabeled VIIa and TF monoclonal antibody (mAB) revealed that reduced TF activity in cholesterol-depleted cells stems from the impairment of VIIa interaction with TF rather than the loss of TF receptors at the cell surface. Repletion of cholesterol-depleted cells with cholesterol restored TF function. Loss of caveolar structure on cholesterol removal is not responsible for reduced TF activity. Solubilization of cellular TF in different detergents indicated that a substantial portion of TF in fibroblasts is associated with noncaveolar lipid rafts. Cholesterol depletion studies showed that the TF association with these rafts is cholesterol dependent. Overall, the data presented herein suggest that membrane cholesterol functions as a positive regulator of TF function by maintaining TF receptors, probably in noncaveolar lipid rafts, in a high-affinity state for VIIa binding.
Cholesterol is a lipid precursor for steroid hormones and bile salts and is present in cell membranes and circulation. Cholesterol in the membrane regulates flexibility and mechanical stability of the membrane.1 Further, cholesterol plays a critical role in differentiating and maintaining cell surface microdomains of differing lipid composition, particularly sphingolipid rafts. Lipid rafts are shown to contribute to the regulation of various cellular functions, including receptor function, endocytosis, intracellular trafficking of receptors, and signaling pathways.2-5 Tissue factor (TF) is the cellular receptor for clotting factor VIIa, and the formation of TF-VIIa complexes on cell surfaces triggers the coagulation cascade.6 Studies suggest that exposure of TF to circulating blood on rupture of atherosclerotic plaque plays an important role in the pathogenesis of thrombus formation at sites of plaque rupture, resulting in acute coronary events and myocardial infarction.7-10 Since cholesterol/oxidatively modified low-density lipoprotein (LDL) present in atherosclerotic plaques is thought to play an important role in the atherogenesis through its biologic effects, including TF expression, many earlier studies were focused on investigating the effect of cholesterol on TF expression. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to suppress plasma LDL cholesterol levels in patients with primary hypercholesterinemia, were shown to inhibit TF expression in both in vitro and in vivo.11,12 Consistent with this, dietary lipid lowering was found to reduce TF expression in rabbit atheroma.13 However, in vitro studies on effects of cholesterol on TF expression gave conflicting results. Cholesterol loading, by exposing monocytes/macrophages or endothelial cells to modified LDL or cholesterol, was shown to induce TF expression in some studies,14-19 whereas no effect was found in other studies.20-23 Most of these previous studies were focused primarily on investigating the role of LDL or cholesterol in modulating transcriptional or translational regulation of TF. At present, there is little information on how cholesterol regulates TF functional expression, independent of transcription/translational control. Studies show that cholesterol, either through a direct molecular interaction or other mechanisms, can have a strong influence on the affinity state, binding capacity, and signal transduction property of membrane receptors.2,24-34 Cholesterol- and sphingolipid-rich rafts in association with a structural protein, caveolin, form caveolae, flask-shaped invaginations of 50- to 100-nm diameter in the plasma membrane.5 These structures are present in many cell types, including endothelial cells35,36 and smooth muscle cells.37 The structure of caveolae is dependent on cholesterol,4,5 as the removal of cholesterol disrupts caveolae.31,32 Studies suggest that TF in smooth muscle cells was associated with caveolae and speculated that caveolae-associated TF may function as a latent pool, which can become active when the vessel wall integrity is lost.37 Studies of Ruf and colleagues (Sevinsky et al35) demonstrated that TF redistributes into caveolae following a series of events, which include binding of VIIa to TF, generation of factor Xa, and subsequent formation of a transient ternary complex with tissue factor pathway inhibitor (TFPI) localized in glycosphingolipid-rich microdomains.
In the present study, we investigated the role of membrane cholesterol on the regulation of TF receptor function by depleting the membrane cholesterol of fibroblasts with methyl-
Cell culture A human fibroblast cell line (WI-38), derived from normal embryonic lung tissue, was obtained from ATCC (Rockville, MD) and was cultured as described earlier.38 Radiolabeling of proteins VIIa and other proteins were labeled by using Iodo-Gen–coated tubes and Na125I according to the manufacturer's (Pierce Biotechnology, Rockford, IL) technical bulletin and as described previously.39 Our earlier studies40,41 established that the radiolabeled proteins were intact with no apparent degradation, and 125I-labeled VIIa retained 80% or more of the functional activity of the unlabeled material. Cholesterol depletion and loading of cholesterol
To deplete cholesterol, unless specified otherwise, monolayers of fibroblasts were treated with m Cholesterol determination Cells were removed from culture dishes by scraping them in buffer A, and the cell suspension was centrifuged for 5 minutes at 3000 rpm in an Eppendorf 5415 C microcentrifuge (Eppendorf AG, Hamburg, Germany). The cell pellets were suspended in TBS (Tris-buffered saline; 50 mM tris(hydroxymethyl)aminomethane [Tris]–HCl, 0.15 M NaCl, pH 7.5) containing 0.1% Tween-20. Cholesterol was determined spectrophotometrically using Cholesterol CII kit (Wako Chemicals, Richmond, VA), following the manufacturer's instructions. We also determined cholesterol levels in cell membrane fractions by first isolating the cell membranes by ultracentrifugation as described earlier 42 and suspending the membrane pellet in TBS containing 0.1% Tween-20. Binding studies Cell surface binding of 125I-VIIa (TF-specific) or 125I-TF mAb (TF9H10) was performed essentially as described previously.38 Determination of cell-surface TF-VIIa activity Monolayers of control cells, cholesterol-depleted or cholesterol-loaded cells were incubated with VIIa (10 nM) in buffer B (buffer A containing 5 mM CaCl2 and 1 mg/mL bovine serum albumin [BSA]) for 5 minutes at 37°C, followed by the addition of substrate factor X (175 nM). Unless otherwise specified, an aliquot was removed at a specific time point (usually at 5 minutes) into stopping buffer (TBS containing 1 mg/mL BSA and 10 mM ethylenediaminetetraacetic acid [EDTA]), and factor Xa in the sample was measured in a chromogenic assay as described earlier.43 Electron microscopy Following control and experimental treatments, cells were fixed for 1 hour at 4°C in 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. Following the fixation, cells were washed thrice with the cacodylate buffer and rinsed once with Milli-Q water. Cells were first stained with anti-TF mAB (a mixture TF9H10, TF9-5B7, and TF9-11D12, 30 µg/mL) for 90 minutes at 4°C in phosphate-buffered saline (PBS) containing 0.2% BSA, followed by secondary antibody, gold (10 nm particle size)–conjugated goat anti–mouse immunoglobulin G (IgG; 25-fold dilution) for 90 minutes at 4°C in PBS containing 0.2% BSA. After quick washes in PBS, the cells were refixed in paraformaldehyde as described earlier in this section and exposed to 1% OsO4 for 1 hour at room temperature in the cacodylate buffer. The fixed cells were stained in 1% aqueous uranyl acetate for 30 minutes in the dark at 4°C, washed in deionized water, subsequently dehydrated in graded ethanol, and embedded in epoxy resin. Thin sections (0.5 µm) were cut perpendicular to the dish. The sections were mounted on copper grids (300 mesh size) and stained in 0.5% aqueous uranyl acetate for 10 minutes, followed by 2% lead citrate for 5 to 10 minutes. Grids were washed thoroughly in deionized water and dried. Sections were viewed and photographed with a JOEL 12 EX electron microscope fitted with a BIOTEM SCAN camera (JOEL USA, Peabody, MA) at 30 000x magnification under 60 kV acceleration. Micrographs shown in Figures 1 and 5 were reproduced from original photographs without any manipulation.
Separation of Triton X-100–insoluble complexes by sucrose gradient ultracentrifugation Triton X-100–insoluble complexes were prepared by sucrose gradient ultracentrifugation fractionation essentially as described earlier.35,44 From each fraction, 30 µg protein was precipitated using 10% vol/vol trichloroacetic acid (TCA), and the pellets were suspended in 50 µL sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Aliquots (20 µL) were subjected to SDS-PAGE, followed by Western blot analysis. Detergent lysis and fractionation Cells were solubilized in various detergents and fractionated as described earlier.34 Briefly, control and cholesterol-depleted cells (2 T-75 flasks each) were harvested in ice-cold buffer A by detaching the cells from the bottom of the dish with a cell scraper. The cells were sedimented by centrifugation, resuspended in buffer A, and split into 3 equal aliquots. The cells in each aliquot were lysed in an equal volume of 1% ice-cold detergent, either Triton X-100, Brij 56 or Brij 58, by gentle mixing at 4°C for 30 minutes. The cell lysates were centrifuged at 800g for 10 minutes at 4°C to remove nuclei and cell debris. The postnuclear supernatants were centrifuged at 16 000g for 30 minutes at 4°C. Pellets, which contain insoluble membrane domains, were resuspended in buffer A containing 0.5% appropriate detergent. Both pellets and supernatants were subjected to SDS-PAGE on 12% polyacrylamide gels and processed for immunoblot analysis using standard approaches.
Ultrastructural localization of TF To determine the role of membrane cholesterol on cell surface TF expression, we first investigated the cellular distribution of TF in fibroblasts by immunogold electron microscopy. To avoid the possibility of nonspecific clustering due to secondary antibody cross-linking, we first fixed the cells before they were immunostained. Tissue factor was predominantly localized on the cell membrane and on cellular processes (Figure 1A-B). In general, cellular processes were stained heavily with anti-TF antibodies. It is interesting to note that when a cellular process from 1 cell comes in contact with another cell, the tip of the cellular process is decorated with TF (Figure 1D). Similar observations were also made with confocal microscopy using fibroblasts transfected with TF–green fluorescent protein (GFP; data not shown). In addition to localizing on the cell membrane and cellular processes, TF was also found in noncoated membrane invaginations, caveolae, mostly at the neck of caveolae (Figure 1C). Quantitation of the cellular distribution of TF from a total of 41 sections revealed that about 15% of gold particles were associated with caveolae. Depletion of cholesterol and loss of caveolar structure
We have used m
Removal of cholesterol from the plasma membrane by m Cholesterol depletion inhibits functional expression of TF at the cell surface
To determine the role of membrane cholesterol on TF functional expression, WI-38 cells were treated with varying concentrations of m
Our earlier studies39,45 suggest that negatively charged phospholipids in the outer leaflet of the cell membrane modulate cell surface TF interaction with VIIa and subsequently TF-VIIa activation of factor X. To address whether cholesterol depletion reduced the availability of negatively charged phospholipids at the cell surface, we evaluated the binding of annexin V, which was shown to bind specifically to negatively charged phospholipids,46,47 to untreated cells, and cells treated with m Evaluation of the modulatory effect of cholesterol on TF interaction with VIIa
To determine whether the reduced VIIa binding to TF in cholesterol-depleted cells represents the loss of TF receptors on the cell surface, we determined whether cholesterol depletion reduces the total number of TF receptors available on the cell surface. Monolayers of fibroblasts were treated with a control vehicle or 10 mM m
If the depletion of cholesterol has no effect on the total number of antibody-reactive TF sites on fibroblast cell membranes, but decreases VIIa binding and thus reduces functional activity, then at least 2 possibilities exist: cholesterol depletion either reduces the number of TF receptors that could support VIIa binding or alters the receptor from high- to low-affinity binding sites for VIIa without changing the number of binding sites. We examined these possibilities by performing dose-dependent VIIa binding (TF-specific) studies with control and cholesterol-depleted cells (Figure 4A) to determine Kd and Bmax for VIIa. Analysis of VIIa binding curves with curve-fitting program (Prism; GraphPad, San Diego, CA) revealed that cholesterol depletion reduced the TF affinity to VIIa (Kd: control, 6.0 ± 0.4 nM; m Disruption of caveolae is not responsible for impairment of TF activity in cholesterol-depleted cells As discussed (Figure 1F), cholesterol depletion disrupts caveolar structure. To determine whether the loss of caveolae or the cholesterol depletion per se is responsible for reduced TF functional activity, we treated fibroblasts with filipin, which does not remove cholesterol from the membrane but forms filipin-cholesterol complexes in the membrane and thereby alters the physical distribution of the cholesterol and disrupts caveolae.48 Ultrastructural analysis of control and filipin-treated cells by electron microscopy showed, as expected, filipin treatment reduced the number of caveolae on fibroblasts by about 60% (Figure 5). Quantitative analysis of 19 to 26 sections showed the following: number of caveolae/section for control, 15.1 ± 5.9, and for filipin-treated cells, 6.0 ± 2.9. Immunogold analysis of TF antigen showed no significant differences in the number of gold particles associated with cells in control and filipin-treated cells (gold-particles/section: control, 12.7 ± 1.1; filipin-treated, 11.0 ± 0.97). Next, we investigated the effect of filipin treatment on VIIa binding to cell surface TF and TF-VIIa activity. As shown in Figure 5C, filipin treatment slightly enhanced VIIa binding to fibroblasts but increased TF-VIIa activation of factor X markedly. These data serve as indirect evidence that the disruption of caveolae in cholesterol-depleted cells is not the cause for impaired TF functional expression observed in these cells. The increased TF-VIIa functional activity observed in filipin-treated cells could have been the result of increased concentration of cholesterol in membranes patches since filipin treatment is shown to result in cholesterol aggregation in the membrane48 or movement of TF from inactive glycosphingolipid-rich microdomains to active anionic phospholipid region of the membrane. Tissue factor is localized in Brij 58 detergent-resistant membrane domains (DRMs) To investigate whether TF is localized in cholesterol-sphingolipid rafts, fibroblasts were lysed in Triton X-100 and fractionated on a 5% to 30% sucrose gradient by ultracentrifugation. Fractions were subjected to SDS-PAGE and Western blot analysis using anti-human TF IgG and anti-caveolin IgG. The data revealed that less than 5% of TF was fractionated into low-density Triton X-100–insoluble complexes (as indicated by the presence of caveolin in these fractions). Solubility of a protein in Triton X-100 and/or inability to float after detergent extraction does not exclude a possibility that the protein is actually associated with cholesterol-sphingolipid rafts. Weak interaction of a protein with rafts may lead to its solubilization by the detergent. Further, cell type, detergent type, detergent/lipid ratio, and potential adhesion to the cytoskeleton may influence the raft protein association with DRMs and its migration to low density during sucrose gradient centrifugation.49,50 For example, T-cell antigen receptor51 and epidermal growth factor (EGF) receptor34 were shown to be associated with lipid rafts by fluorescence microscopy, but this interaction is not preserved during Triton X-100 extraction. Studies indicate other nonionic detergents, such as Brij 58 and Lubrol WX, are more suitable in preserving the interaction of receptors with cholesterol-sphingolipid rafts.34,52 Therefore, we next investigated the solubility of TF in the nonionic detergents Brij 56 and Brij 58. (Brij 58 has a higher hydrophilic-lipophilic balance than Triton X-100, whereas Brij 56 is similar to Triton X-100.52) Extraction of fibroblasts with Brij 58 resulted in a substantial amount of TF in the pellet, whereas minimal or no TF was found in the pellet when fibroblasts were extracted with Brij 56 or Triton X-100 (Figure 6A, top). Caveolin-1 was found exclusively in the pellet after lysis with both Brij 58 and Brij 56. Insolubility of TF in Brij 58 indicates that TF is localized in lipid rafts; however, the interaction between TF with lipid rafts may be weak.
Next, to investigate whether the association of TF with Brij 58-DRMs is cholesterol dependent, fibroblasts were cholesterol-depleted with m
In the present study, we show that the cholesterol content in the plasma membrane regulates TF functional expression by regulating TF interaction with ligand VIIa without altering TF levels at the cell surface. Data presented herein also show that in fibroblasts only a minor fraction of TF receptors is localized in caveolae, whereas a substantial portion of TF is localized in noncaveolar lipid rafts (DRMs) that are sensitive to extraction with Triton X-100 but not to extraction with Brij 58. The association of TF with these DRMs appears to be cholesterol dependent. Overall these data suggest that membrane cholesterol positively regulates TF coagulant function at the cell surface, probably by maintaining TF in a high-affinity state for VIIa binding.
Cholesterol, which plays an important role in the structure of biologic membranes, is known to modulate the activity of various membrane-embedded receptor proteins, including the transferrin receptor,53 the nicotinic acetylcholine receptor,54 insulin receptor,31 EGF receptor,34 and several G-coupled protein receptors24 (reviewed in Burger et al2). There are at least 2 defined mechanisms by which cholesterol is shown to modulate receptor function: (1) changes in membrane fluidity or (2) specific interaction between cholesterol and the receptor. Since cholesterol is essential in maintaining the rigidity of cell membranes, removal of cholesterol from the plasma membrane by m
Since changes in lipids regulate membrane fluidity, fluidization of membrane by cholesterol depletion may alter phospholipid distribution of the cell membrane. Earlier studies from others58,59 and us45,60 showed that the increased exposure of phosphatidylserine (PS) at the outer cell membrane enhances TF functional expression. If m
Comparison of VIIa binding in control and m
While the present manuscript was being prepared, a manuscript describing data that contrast the present data has been published online.64 These data show that treatment of HEK293 cells and dermal fibroblasts with m
Ultrastructural localization of TF in smooth muscle cells (SMCs) showed that about 20% of TF in these cells was associated with caveolae.37 On the basis of increased TF activity and enlargement of caveolar structures in SMCs following their detachment, Mulder et al37 speculated that caveolae-associated TF might function as a latent pool of procoagulant activity, which can rapidly be activated at sites in which vessel wall integrity is lost.37 In recent years, cholesterol depletion by m Advances suggest that cholesterol exerts many of its actions mainly by maintaining sphingolipid rafts, which function to segregate and concentrate specific membrane proteins.67 Studies showed that raft-associated proteins, based on the raft structures, their interaction with raft lipids, or other proteins within the same raft, might exhibit differential sensitivity to extraction with different detergents.34,51,52 Consistent with this hypothesis, we found that TF in fibroblasts was soluble in Triton X-100 and Brij 56 (a detergent that is similar to Triton X-100) but partly resistant to extraction with Brij 58, a detergent with a higher hydrophilic-lipophilic balance than Triton X-100. In contrast to TF, caveolin-1 is associated completely with insoluble membrane domains on extraction with all 3 detergents. At present, it is not entirely clear whether differential behavior of caveolin-1 and TF during Triton X-100 extraction is caused by their localization on different membrane domains or dissociation of TF from caveolar membrane domains. Since ultrastructural localization of TF clearly indicated that only a minor fraction of TF present at the cell surface is associated with caveolae, it is reasonable to conclude that differential behavior of caveolin-1 and TF in Triton X-100 reflects TF association with noncaveolar cholesterol-rich membrane domains. The observation that depletion of cholesterol increased the solubility of TF in Brij 58 supports the notion that cholesterol is an integral part of these membrane domains. In conclusion, the data presented in the manuscript demonstrate for the first time that membrane cholesterol modulates interaction of TF receptor with VIIa and subsequently TF-VIIa activation of factor X. These data may provide an additional explanation on how therapeutic intervention to lower cholesterol reduces the incidence of acute coronary events associated with atherosclerosis. Since studies show that TF-VIIa, in addition to triggering blood coagulation, plays a role in many pathophysiological processes, it is interesting to examine how cholesterol modulates other functions of TF-VIIa. These and similar studies in the future may provide clues in understanding the unexplained benefits of cholesterol-lowering drugs and may stimulate new studies in evaluating potential benefits, in addition to reducing atherosclerosis, associated with therapeutic intervention of lowering cholesterol.
We acknowledge the excellent technical assistance provided by Mylinh Ngyuen. We are thankful for Dr Ronald Dodson's laboratory at the Health Center for helping in electron microscopy.
Submitted March 16, 2004; accepted August 12, 2004.
Prepublished online as Blood First Edition Paper, August 24, 2004; DOI 10.1182/blood-2004-03-0990.
Supported by grants from National Institute of Health (HL58869) and American Heart Association, Texas Affiliate (0355096Y).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: L. Vijaya Mohan Rao, Biomedical Research, University of Texas Health Center at Tyler, 11937 US Hwy 271, Tyler, TX 75708; e-mail: vijay.rao{at}uthct.edu.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
indicates control; 
, cholesterol-deplete; 
, cholesterol-laden. In D and E, 
