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Lipid rafts regulate cellular CD40 receptor localization in vascular endothelial cells
Biochemical and Biophysical Research Communications 361 (2007) 768–774 www.elsevier.com/locate/ybbrc
Lipid rafts regulate cellular CD40 receptor localization in vascular endothelial cells Min Xia 1, Qing Wang 1, Huilian Zhu, Jing Ma, Mengjun Hou, Zhihong Tang, Juanjuan Li, Wenhua Ling * Department of Nutrition, School of Public Health, Sun Yat-Sen University (Northern Campus), 74 Zhongshan Road 2, Guangzhou, Guangdong Province 510080, PR China Received 16 July 2007 Available online 30 July 2007
Abstract Cholesterol enriched lipid rafts are considered to function as platforms involved in the regulation of membrane receptor signaling complex through the clustering of signaling molecules. In this study, we tested whether these specialized membrane microdomains affect CD40 localization in vitro and in vivo. Here, we provide evidence that upon CD40 ligand stimulation, endogenous and exogenous CD40 receptor is rapidly mobilized into lipid rafts compared with unstimulated HAECs. Efficient binding between CD40L and CD40 receptor also increases amounts of CD40 protein levels in lipid rafts. Deficiency of intracellular conserved C terminus of the CD40 cytoplasmic tail impairs CD40 partitioning in raft. Raft disorganization after methyl-b-cyclodextrin treatment diminishes CD40 localization into rafts. In vivo studies show that elevation of circulating cholesterol in high-cholesterol fed rabbits increases the cholesterol content and CD40 receptor localization in lipid rafts. These findings identify a physiological role for membrane lipid rafts as a critical regulator of CD40-mediated signal transduction and raise the possibility that certain pathologic conditions may be treated by altering CD40 signaling with drugs affecting its raft localization. 2007 Elsevier Inc. All rights reserved. Keywords: CD40; Lipid rafts; Endothelial cells; Cholesterol
CD40 is 49 kDa integral membrane protein expressed on a variety of cells, including B-lymphocytes, monocytes, fibroblasts, epithelial, and endothelial cells, which shares significant sequence homology with the receptors for tumor necrosis factor receptor (TNFR) [1–3] CD40L, also referred as gp39 and recently renamed CD154, was first identified as a cell surface glycoprotein of 30–33 kDa and thought restricted to activated CD4 þ T-lymphocytes [1–3] Recent reports have implicated both receptor and ligand closely correlated with several inflammatory diseases such as atherosclerosis [1–3]. Atherosclerosis morbidity has been linked to a Western diet, which includes high levels of red meat and saturated *
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Corresponding author. Fax: +86 20 87330446. E-mail address: [email protected] (W. Ling). These authors contributed equally to this work.
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.07.102
fat with higher cholesterol. Cholesterol, a neutral lipid that is a prominent component of the Western diet, contributes to the unique biophysical properties of the lipid raft microdomain. However, little is known on the relationship between lipid rafts and atherogenesis. Membrane rafts are low density, detergent-insoluble membrane microdomains, enriched in sphingolipids, cholesterol, and glycosylphosphatidylinositol-linked proteins and found in all mammalian cell types [4]. Sphingolipids and cholesterol not only accumulate in detergent-resistant liquid-ordered lipid membrane microdomains, or membrane rafts, they are essential for raft formation [5,6]. These microdomains mechanistically contribute to signal transduction by raft proteins and play fundamental roles in diverse cellular functions, particularly in signal transduction [7,8]. Recently, several reports have demonstrated that CD40 recruitment to lipid rafts are thought to be intrinsic
M. Xia et al. / Biochemical and Biophysical Research Communications 361 (2007) 768–774
to some of the signaling functions of CD40 [9]. Several works, including ours, have highlighted the role of membrane rafts in the initiation of CD40 signal transduction and altered cholesterol distribution may influence CD40induced inflammation [10,11]. Substantial evidence exists that phenotypic modulation of the endothelium to an activated state contributes to the pathogenesis of atherosclerosis. In this study, we aimed to assess whether lipid raft organization influenced the CD40 recruitment in vitro and in vivo. Materials and methods Cell culture. HAECs were obtained from Genentech, and maintained in endothelial basal medium supplemented with various growth factors and 2% fetal bovine serum (FBS). Plasmid. Full-length wild-type human CD40 coding sequence was obtained by PCR and confirmed by sequencing. The primers used were: CGGGGTACCGCCACCATGG‘ITCGTCTGCC TCTGCAG for the upstream primer and WI 0 GTCGACTCACTGT CTCTCCTGCAC for the downstream primer. The upstream primer had a built-in BamHI site and the downstream primer a SalI site (underlined) to facilitate cloning into the expression vector pCMV vector. Mutant CD40 receptor construct that lacked either highly conserved C terminus of CD40 (residues 261–289, Delta 260) and the extensive deletion of the cytoplasmic domain (STOP222) were made as previously described [12]. The expression constructs encoding CD40-GFP was constructed by Dr. Tao Yue (UT Southwestern Medical Center) and donated as gift. The cells (1 · 106) were plated per well in a six-well plate with 2 mL of medium. Cells were transfected once they had reached 80–90% confluence. The transfection efficiency was estimated by using GFP-plasmids. Biochemical lipid raft separation. Rafts were isolated as described previously [13]. Briefly, postnuclear supernatant (PNS) from ECs (2 · 106) was solubilized in 1 mL buffer A [25 mmol/L Hepes (N-2-hydroxyethylpiperazine-N 0 -2-ethanesulfonic acid), 150 mmol/L NaCl, 1 mmol/L EGTA (ethyleneglycotetraacetic acid), protease inhibitors cocktail] containing 1% Brij 98 (Sigma–Aldrich) detergent for 5 min at 37 C and was chilled on ice before it was placed at the bottom of a step sucrose gradient (1.33–0.9–0.867–0.833–0.8–0.767–0.733–0.7–0.6 mol/L sucrose). Gradients were centrifuged at 200,000g for 16 h in a Ti90 Beckman rotor (Beckman Instruments) at 4 C. One-milliliter fractions were harvested from the top, except for the last fraction (no. 9), which contained 3 mL. The fraction contained the pooled fractions 1–4, and the heavy fraction (HF) consisted of pooled fractions 8 and 9. For determination ganglioside GM1 content of density gradient fractions, 2.5 · 107 HAECs were incubated for 10 min at room temperature in 1 mL of culture medium containing 3.5 lg of HRP-labeled cholera toxin B subunit (or, as controls, untreated cells and cells incubated with an equivalent amount of unconjugated HRP). Cells were then subjected to lysis and density gradient centrifugation as described above. Gradient fractions were assayed for peroxidase activity by mixing 10 lL gradient fractions with 100 lL of 50 mM sodium phosphate, 25 mM citric acid (pH 5.0), 1 mg/mL o-phenylenediamine dihydrochloride, and 0.012% H2O2. Samples were incubated for 5 min at room temperature, and the reaction was stopped by adding 150 lL of 0.67 M sulfuric acid. Optical density of the samples was read at 405 nm in an enzyme-linked immunosorbent assay (ELISA) plate reader [14]. Immunoblotting. A total of 2 · 107 ECs in 1 mL medium were stimulated with 5 lg/mL of CD40 L for the indicated times at 37 C, washed with cold PBS, lysed in 4 mL of MNX buffer [1% Triton X-100 in 25 mM MES, 150 mM NaCl (pH 6.5)] and subjected to lipid raft isolation. Soluble and lipid raft fractions were detected using immunoblotting with the specific antibody. Confocal microscopy. Cellular surface CD40 receptor was detected using a recently described method [15], and lipid rafts were identified by
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the expression of the glycosphingolipid, GM1, which binds the B subunit of cholera toxin. Briefly, cells grown on glass coverslips were transfected with CD40-GFP construct, then incubated with CTxB-Alexa 488 (10 lg/ mL) alone or in the presence of CD40L (5 lg/mL) for 1 h at 4 C. Cells were fixed with 4% paraformaldehyde/PBS for 20 min and analyzed using a laser scanning confocal microscope (ZEISS). Cholesterol depletion. For cholesterol depletion, 2.5 · 105 cells/mL was pretreated with 20 mM methyl-b-cyclodextrin (MbCD) for 30 min at 37 C in serum-free medium. Cells were then washed three times with PBS and resuspended in complete culture medium [16]. Filipin staining. Cells were fixed in 3.2% PFA in PBS and then resuspended in PBS containing 30 lg/mL filipin, held at room temperature for 2 h, and directly analyzed on a flow cytometer [17]. Perfusion organ culture. Animal experiments were performed according to the guidelines of Sun Yat-Sen University for the care and use of laboratory animals which were approved by the Sun Yat-Sen University Animal Care Committee. Male New Zealand White rabbits (2–3 kg) were fed normal chow diet (control) or high-cholesterol diet (chow diet with 0.5% cholesterol) for 8 weeks. After treatment, the rabbits were anesthetized with ketamine (50 mg/kg IV) and xylazine (2 mg/kg IV) and arterial segments were isolated as previously described [18]. Organ culture of the aortic segments was carried out under sterile conditions in an incubator containing 5% CO2 at 37 C for 24–26 h. Statistical analysis. Data are expressed as means ± SD. To compare the data between groups, ANOVA followed by post hoc statistical tests was used. A value of P < 0.05 was considered statistically significant.
Results CD40 receptor associates with cholesterol-rich lipid rafts after membrane treatment with its ligand-CD40L We initially assessed whether CD40 receptor localization in cholesterol-rich membrane lipid rafts during CD40 signaling by examining membrane localization of CD40 on HAECs using a biochemical approach [19]. As shown in Fig. 1A, none of CD40 receptor was found within lipid rafts, whereas CD40L-stimulation promoted the large majority of CD40 recruitment in this microdomain. As a control for proper separation, the membrane was probed with an antibody recognizing the raft marker protein Fyn, which is localized in rafts. As expected, Fyn appeared mainly in the light raft fractions, whereas the non-raft marker Rab5 was isolated in the heavy fractions (Fig. 1A). A similar distribution pattern of CD40 receptor in the rafts after CD40L-stimulation was observed versus nonraft fraction (Fig. 1B). As a control, the raft marker flotilin was predominantly found in the raft fractions, whereas the cytoplasmic protein tubulin was exclusively detected in the nonraft fraction. These results indicated that CD40 receptor was associated with lipid rafts during CD40 signaling challenged with CD40L. Next, the exogenous CD40 construct distribution in rafts was analyzed. We lysed HAECs transfected with CD40-GFP construct and fractionated the lysates by sucrose gradient centrifugation after CD40L-stimulation. Detection of GFP in lipid rafts indicated interaction of CD40 with cholesterol-rich membranes lipid rafts (Supplementary Fig. 1A). Lipid raft fractions were identified by the presence of the marker protein flotilin in light density factions 1, 2, 3, and 4 (Supplementary Fig. 1B). Confocal
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M. Xia et al. / Biochemical and Biophysical Research Communications 361 (2007) 768–774 Raft
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Fig. 1. CD40 receptor mobilizes to lipid rafts after membrane treatment with CD40L. (A) HAECs were left alone or stimulated with 5 lg/mL CD40L for 30 min, then solubilized in Brij 98 and subjected to sucrose gradient separation, as described in ‘‘Materials and methods’’. Sucrose fractions were analyzed by Western blot with anti-CD40, anti-Fyn, and anti-Rab5 antibodies. (B) Lipid rafts were also isolated using the short method. CD40, flotilin, and tubulin were detected by Western blot. NR indicates nonraft fraction; R, raft fraction. The typical experiment of blots was shown. (C) CD40 co-localizes with lipid rafts in HAECs. Lipid rafts were detected by confocal microscopy after labeling HAECs with CTxB-Alexa 488, which binds to the raft- associated glycosphingolipid GM1. GFP-CD40 transfected HAECs were also incubated alone (a–c) or with CD40L (d–f) for 30 min and observed by confocal microscopy. (a and c, CD40-GFP; b and d, lipid rafts; c and f, the merged image which revealed considerable co-localization of CD40 and lipid rafts). Scale bar = 10 lm.
image clearly showed that CD40 receptor co-localized with membrane rafts revealed by incubating HAECs with CTXB-Alexa 488 [17], which binds to GM1, a glycolipid preferentially concentrated within membrane lipid rafts (Fig. 1C). Taken together, these biochemical data demonstrated that endogenous and exogenously expressed CD40 receptor are partitioned to lipid membrane rafts during CD40 signaling.
CD40 protein levels increase in lipid rafts after the interaction of ligand with CD40 receptor To test whether binding of the ligand to its receptor changes the extent of CD40 raft partitioning, we incubated HAECs with wide type CD40 (wtCD40) or CD40 mutant (mCD40, Delta 260) construct and then stimulated with CD40L. Interaction with the wtCD40 by CD40L led to almost all of the CD40 translocation from
M. Xia et al. / Biochemical and Biophysical Research Communications 361 (2007) 768–774
the disordered membrane environment into the ordered membrane raft domains. However, mCD40 transfection markedly abrogated CD40 translocation and only a little CD40 could be detected in lipid rafts (Fig. 2A and B). We then analyzed for the efficient interaction between CD40 receptor and CD40L by flow cytometry. Transfection of HAECs with mCD40 resulted in slightly detectable CD40L binding, which was significantly augmented by transfection with wtCD40 receptor (Supplementary Fig. 2A). Functional binding of CD40L to CD40 receptor was also substantiated by immunoprecipitation. Compared with mCD40, wtCD40 significantly elevated CD40 and CD40L interaction in HAECs (Supplementary Fig. 2B). Intracellular conserved C terminus of the CD40 cytoplasmic tail is essential for CD40 partitioning in rafts The fact that C-terminal intracellular CD40 receptor is highly conserved between species indicates an important function of this domain. Here, we tested a potential involvement of the intracellular domain of C-terminal tail in CD40 receptor partitioning into rafts. For this purpose,
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HAECs were transfected with deletion mutants including Delta 260 and STOP222. To compare the relative proportion of raft localized CD40 receptor, HAECs were subjected to biochemical raft separation and CD40 localization was determined by Western blot. Compared with wtCD40 (Supplementary Fig. 3A), both deletion mutants of CD40 severely impaired the raft localization of CD40 receptor by CD40L stimulation (Supplementary Fig. 3B and C). Interestingly, although removal of the part of C-terminal amino acids of CD40 allowed residual CD40 raft localization (Supplementary Fig. 3B), deletion of the complete cytoplasmic domain completely abolished all the CD40 localization to the light raft fractions (Supplementary Fig. 3C). Therefore, we concluded that the intracellular domain of CD40 receptor was crucial for its targeting to or maintenance within the membrane rafts.
We then attempted to identify the role of lipid rafts in the regulation of CD40 expression and activity. To achieve this question, we employed a small cyclic oligosaccharide MbCD, which binds cholesterol reversibly and allows cholesterol to be depleted from cell membranes [20], to destroyed the lipid rafts in CD40-transfected HAECs, To substantiate the lowering of plasma membrane cholesterol by MbCD, HAECs were stained for FACS with the antibiotic filipin, which binds to cholesterol and has fluorescent properties [21]. Filipin staining decreased about 50% upon MbCD treatment whereas cholesterol replenishment reversed the effect of MbCD on filipin staining (Fig. 3A), showing the nonspecific effects of MbCD. Beside this, MbCD treatment dramatically reduced the amount of cell surface GM1 glycosphingolipids (Fig. 3B) and resulted in a significant shift of the lipid raft marker flotilin from the raft fraction to the nonraft fraction (Fig. 3C), indicating the efficacy of the lipid raft disruption. Accordingly, we also found that MbCD almost completely abolished the translocation of CD40 from nonraft fraction to rafts, demonstrating that lipid rafts may regulate the redistribution of CD40 within the plasma membrane (Fig. 3C).
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Fig. 2. Inducible translocation of CD40 protein in lipid membrane rafts by functional CD40/CD40L interaction. HAECs were transfected with human CD40 (wtCD40) or mutant CD40 (mCD40) construct, then stimulated with 5 lg/mL CD40L for 30 min before solubilization in Brij 98 and were subjected to sucrose gradient separation. Single fractions were analyzed by Western blot with anti-CD40, anti-Fyn, and anti-Rab5 antibodies (A). Immunoblots were performed on pooled heavy (8–9; HF) and light (1–4; raft) fractions to measure CD40 protein expression (B).
We next sought to determine whether lipid raft membranes isolated from aortas exhibited alterations attributable to the high-cholesterol regimen in hypercholesterolemic rabbits. Compared control group, serum cholesterol levels in cholesterol-fed rabbits increased about 4-fold after stable elevation using dietary modification for 8 weeks (Fig. 4A). Cholesterol content of lipid rafts was also significantly elevated in the high-cholesterol group in comparison with the normal group
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Fig. 3. Effects of depleting cholesterol by MbCD on organization of membrane lipid rafts of HAECs and CD40 localization. (A) Membrane cholesterol levels were measured by staining with filipin and flow cytometric measurement of filipin fluorescence on untreated cells (control), MbCD-treated cells (MbCD), or cells treated with MbCD that had been preloaded with cholesterol (MbCD-Chol). Values of the mean fluorescence of the filipin staining were determined by fluorospectrophotometer. **P < 0.01 compared to control. (B) Surface levels of raft-associated GM1 sphingolipids content of density gradient fractions was measured by bound HRP-labeled cholera toxin B subunit (CT-HRP) activity. (C) CD40-transfected HAECs were incubated for 30 min in the culture medium (control) or with MbCD and rested for 30 min in medium, then lipid rafts were isolated. CD40 and flotilin were detected by Western blot. NR indicates nonraft fraction; R, raft fraction.
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Fig. 4. Lipid rafts regulates CD40 receptor localization and endothelial activation in vivo. After treatment as described in ‘‘Materials and methods’’. The aortic endothelial cells were isolated, cultured and then were solubilized in Brij 98 before they were subjected to sucrose gradient separation. (A) Blood samples were obtained from rabbits under anesthesia, and serum was prepared by centrifugation. Serum total cholesterol levels was measured using an enzymatic assay kit. (B) Cholesterol content of lipid rafts were analyzed by enzymatic method. Results, expressed as fold of control, are representative of three independent experiments. **P < 0.01 compared to high cholesterol. (C) Sucrose fractions were analyzed by Western blot with anti-CD40, anti-Fyn, and anti-Rab5 antibodies.
Discussion (Fig. 4B). This demonstrates that elevated circulating cholesterol increases the cholesterol content of lipid raft membranes of endothelial cells. We next analyzed the CD40 distribution in rabbit aortic segments. Under normal state, rabbit aortic endothelial cells had nearly undetectable CD40 receptor in lipid rafts (Fig. 4C) which was markedly enhanced in aortas of high-cholesterol fed rabbits.
Lipid rafts are considered microdomains of ordered lipids that selectively partition functional groups of proteins within the membrane. The crucial role of lipid rafts in signal transduction of several receptors, such as TCR, BCR, and FccRs, has been well documented [22,23]. However, signals induced by CD40 in vascular endothelial cells, which is strongly relevant to atherogenesis, on these spe-
M. Xia et al. / Biochemical and Biophysical Research Communications 361 (2007) 768–774
cialized membrane microdomains has not been analyzed in detail. Here, we reported that endogenous and exogenously expressed CD40 receptor constitutively partitioned from the detergent-soluble fraction into membrane lipid rafts by CD40L-stimulation. The intracellular domain of CD40 receptor was crucial for its maintenance within the membrane rafts as partly or completely deleted of the cytoplasmic domain abolished CD40 receptor localization to the rafts. Disassembly of lipid rafts by MbCD interrupted the translocation of CD40 into lipid rafts. In vivo studies showed that both cholesterol content and CD40 localization in lipid rafts of endothelium were increased under the high-cholesterol regimen. These data supported the evolving concept that membrane lipid raft was a key component of biomechanical signal transduction and established it as a potentially novel regulator of CD40 signaling in the endothelium. Lipid rafts are a distinct biophysical plasma membrane compartment and they are highly dynamic, submicroscopic assemblies that float freely within the liquid bilayer in cell membranes [24]. Our current biochemical separation studies showed that endogenously expressed and exogenously transfected CD40 receptor almost exclusively detected in the detergent-insoluble fraction of HAECs by CD40L stimulation, indicating a constitutive association of CD40 with lipid rafts during CD40 signaling. Further functional investigations demonstrated that with the efficient binding of ligand to CD40 receptor, the amount of raft-associated CD40 protein increased, implying the translocation of additional CD40 protein into rafts. These findings indicated that the partition of CD40 receptor into lipid rafts was a dynamic process, not a static process. However, the molecular mechanism controlling this translocation was still unknown. Our current data also suggested an important role for the intracellular cytoplasmic portion of CD40 receptor in raft localization. This domain fulfilled an important role in CD40 sorting into intracellular secretory lysosomes in HAECs. It is conceivable that CD40 raft partitioning was regulated by other proteins binding to the intracellular CD40 domain, possibly in an inducible manner. Many studies have shown that lipid raft membrane microdomains are functionally modulated by increases/ decreases in cholesterol content [25,26]. Hypercholesterolemia, which is characterized by high levels of lipoprotein-containing cholesterol in plasma, is associated with endothelial activation and generally accepted as a major risk factor for the development of atherosclerosis. These findings raise the question whether hypercholesterolemia may influence CD40 partition in rafts which then alters endothelial function or not. More strikingly, the present study provided direct in vivo evidence that elevation in circulating cholesterol levels was capable of increasing the cholesterol content of lipid rafts and CD40 receptor localization. Our observations support that lipid rafts may serve as an important signaling platform in the endothelium and
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prominently contribute to CD40 signaling relevant to atherogenesis. Thus, modulating the extent of CD40 partitioning seemed to be another level at which the cells could regulated CD40 biological activity, suggesting that lipid rafts microdomain is a promising subcellular location for the identification of novel molecular targets. This concept may be adapted to clinical applications with applicable drugs (or natural products) that would influence raft composition and thereby affected CD40-activated signaling. This discovery may represent a therapeutic option to inhibit CD40-related cardiovascular diseases (especially atherosclerosis). Acknowledgments This work was supported by the research grants from National Natural Science Foundation of China Research Grant 30571568 and China Medical Board of New York Inc. (Grant CMB 98-677). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2007. 07.102. References [1] R.P. Phipps, Atherosclerosis: the emerging role of inflammation and the CD40–CD40 ligand system, Proc. Natl. Acad. Sci. USA 97 (2000) 6930–6932. [2] U. Schonbeck, P. Libby, CD40 signaling and plaque instability, Circ. Res. 89 (2001) 1092–1103. [3] S.R. Bennett, F.R. Rrbone, F. Karamalis, R.A. Flavell, J.F. Miller, W.R. Heath, Help for cytotoxic-T-cell responses is mediated by CD40 signaling, Nature 393 (1998) 478–480. [4] C.R. Longo, M.B. Arvelo, V.I. Patel, S. Daniel, J. Mahiou, S.T. Grey, C. Ferran, A20 protects from CD40–CD40 ligand-mediated endothelial cell activation and apoptosis, Circulation 108 (2003) 1113–1118. [5] J.F. Hancock, Lipid rafts: contentious only from simplistic standpoints, Nature 7 (2006) 456–462. [6] K. Simons, E. Ikonen, Functional rafts in cell membranes, Nature 387 (1997) 569–572. [7] M. Rothe, V. Sarma, V.M. Dixit, D.V. Goeddel, TRAF2-mediated activation of NF-kappa B by TNF receptor 2 and CD40, Science 269 (1995) 1424–1427. [8] H.Y. Song, C.H. Regnier, C.J. Kirschning, d.V. Goeddel, M. Rothe, Tumor necrosis factor (TNF)-mediated kinase cascades: bifurcation of nuclear factor-kappaB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2, Proc. Natl. Acad. Sci. USA 94 (1997) 9792–9796. [9] J.R. Arron, Y. Pewzner-Jung, M.C. Walsh, T. Kobayashi, Y. Choi, Regulation of the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF)2 by TRAF1 reveals mechanisms of TRAF2 signaling, J. Exp. Med. 196 (2002) 923–934. [10] P. Xie, B.S. Hostager, M.E. Munroe, C.R. Moore, G.A. Bishop, Cooperation between TNF receptor-associated factors 1 and 2 in CD40 signaling, J. Immunol. 176 (2006) 5388–5400. [11] M. Xia, W. Ling, H. Zhu, Q. Wang, J. Ma, M. Hou, Z. Tang, L. Li, Q. Ye, Anthocyanin prevents CD40-activated proinflammatory
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Lipid rafts regulate cellular CD40 receptor localization in vascular endothelial cells Min Xia 1, Qing Wang 1, Huilian Zhu, Jing Ma, Mengjun Hou, Zhihong Tang, Juanjuan Li, Wenhua Ling * Department of Nutrition, School of Public Health, Sun Yat-Sen University (Northern Campus), 74 Zhongshan Road 2, Guangzhou, Guangdong Province 510080, PR China Received 16 July 2007 Available online 30 July 2007
Abstract Cholesterol enriched lipid rafts are considered to function as platforms involved in the regulation of membrane receptor signaling complex through the clustering of signaling molecules. In this study, we tested whether these specialized membrane microdomains affect CD40 localization in vitro and in vivo. Here, we provide evidence that upon CD40 ligand stimulation, endogenous and exogenous CD40 receptor is rapidly mobilized into lipid rafts compared with unstimulated HAECs. Efficient binding between CD40L and CD40 receptor also increases amounts of CD40 protein levels in lipid rafts. Deficiency of intracellular conserved C terminus of the CD40 cytoplasmic tail impairs CD40 partitioning in raft. Raft disorganization after methyl-b-cyclodextrin treatment diminishes CD40 localization into rafts. In vivo studies show that elevation of circulating cholesterol in high-cholesterol fed rabbits increases the cholesterol content and CD40 receptor localization in lipid rafts. These findings identify a physiological role for membrane lipid rafts as a critical regulator of CD40-mediated signal transduction and raise the possibility that certain pathologic conditions may be treated by altering CD40 signaling with drugs affecting its raft localization. 2007 Elsevier Inc. All rights reserved. Keywords: CD40; Lipid rafts; Endothelial cells; Cholesterol
CD40 is 49 kDa integral membrane protein expressed on a variety of cells, including B-lymphocytes, monocytes, fibroblasts, epithelial, and endothelial cells, which shares significant sequence homology with the receptors for tumor necrosis factor receptor (TNFR) [1–3] CD40L, also referred as gp39 and recently renamed CD154, was first identified as a cell surface glycoprotein of 30–33 kDa and thought restricted to activated CD4 þ T-lymphocytes [1–3] Recent reports have implicated both receptor and ligand closely correlated with several inflammatory diseases such as atherosclerosis [1–3]. Atherosclerosis morbidity has been linked to a Western diet, which includes high levels of red meat and saturated *
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Corresponding author. Fax: +86 20 87330446. E-mail address: [email protected] (W. Ling). These authors contributed equally to this work.
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.07.102
fat with higher cholesterol. Cholesterol, a neutral lipid that is a prominent component of the Western diet, contributes to the unique biophysical properties of the lipid raft microdomain. However, little is known on the relationship between lipid rafts and atherogenesis. Membrane rafts are low density, detergent-insoluble membrane microdomains, enriched in sphingolipids, cholesterol, and glycosylphosphatidylinositol-linked proteins and found in all mammalian cell types [4]. Sphingolipids and cholesterol not only accumulate in detergent-resistant liquid-ordered lipid membrane microdomains, or membrane rafts, they are essential for raft formation [5,6]. These microdomains mechanistically contribute to signal transduction by raft proteins and play fundamental roles in diverse cellular functions, particularly in signal transduction [7,8]. Recently, several reports have demonstrated that CD40 recruitment to lipid rafts are thought to be intrinsic
M. Xia et al. / Biochemical and Biophysical Research Communications 361 (2007) 768–774
to some of the signaling functions of CD40 [9]. Several works, including ours, have highlighted the role of membrane rafts in the initiation of CD40 signal transduction and altered cholesterol distribution may influence CD40induced inflammation [10,11]. Substantial evidence exists that phenotypic modulation of the endothelium to an activated state contributes to the pathogenesis of atherosclerosis. In this study, we aimed to assess whether lipid raft organization influenced the CD40 recruitment in vitro and in vivo. Materials and methods Cell culture. HAECs were obtained from Genentech, and maintained in endothelial basal medium supplemented with various growth factors and 2% fetal bovine serum (FBS). Plasmid. Full-length wild-type human CD40 coding sequence was obtained by PCR and confirmed by sequencing. The primers used were: CGGGGTACCGCCACCATGG‘ITCGTCTGCC TCTGCAG for the upstream primer and WI 0 GTCGACTCACTGT CTCTCCTGCAC for the downstream primer. The upstream primer had a built-in BamHI site and the downstream primer a SalI site (underlined) to facilitate cloning into the expression vector pCMV vector. Mutant CD40 receptor construct that lacked either highly conserved C terminus of CD40 (residues 261–289, Delta 260) and the extensive deletion of the cytoplasmic domain (STOP222) were made as previously described [12]. The expression constructs encoding CD40-GFP was constructed by Dr. Tao Yue (UT Southwestern Medical Center) and donated as gift. The cells (1 · 106) were plated per well in a six-well plate with 2 mL of medium. Cells were transfected once they had reached 80–90% confluence. The transfection efficiency was estimated by using GFP-plasmids. Biochemical lipid raft separation. Rafts were isolated as described previously [13]. Briefly, postnuclear supernatant (PNS) from ECs (2 · 106) was solubilized in 1 mL buffer A [25 mmol/L Hepes (N-2-hydroxyethylpiperazine-N 0 -2-ethanesulfonic acid), 150 mmol/L NaCl, 1 mmol/L EGTA (ethyleneglycotetraacetic acid), protease inhibitors cocktail] containing 1% Brij 98 (Sigma–Aldrich) detergent for 5 min at 37 C and was chilled on ice before it was placed at the bottom of a step sucrose gradient (1.33–0.9–0.867–0.833–0.8–0.767–0.733–0.7–0.6 mol/L sucrose). Gradients were centrifuged at 200,000g for 16 h in a Ti90 Beckman rotor (Beckman Instruments) at 4 C. One-milliliter fractions were harvested from the top, except for the last fraction (no. 9), which contained 3 mL. The fraction contained the pooled fractions 1–4, and the heavy fraction (HF) consisted of pooled fractions 8 and 9. For determination ganglioside GM1 content of density gradient fractions, 2.5 · 107 HAECs were incubated for 10 min at room temperature in 1 mL of culture medium containing 3.5 lg of HRP-labeled cholera toxin B subunit (or, as controls, untreated cells and cells incubated with an equivalent amount of unconjugated HRP). Cells were then subjected to lysis and density gradient centrifugation as described above. Gradient fractions were assayed for peroxidase activity by mixing 10 lL gradient fractions with 100 lL of 50 mM sodium phosphate, 25 mM citric acid (pH 5.0), 1 mg/mL o-phenylenediamine dihydrochloride, and 0.012% H2O2. Samples were incubated for 5 min at room temperature, and the reaction was stopped by adding 150 lL of 0.67 M sulfuric acid. Optical density of the samples was read at 405 nm in an enzyme-linked immunosorbent assay (ELISA) plate reader [14]. Immunoblotting. A total of 2 · 107 ECs in 1 mL medium were stimulated with 5 lg/mL of CD40 L for the indicated times at 37 C, washed with cold PBS, lysed in 4 mL of MNX buffer [1% Triton X-100 in 25 mM MES, 150 mM NaCl (pH 6.5)] and subjected to lipid raft isolation. Soluble and lipid raft fractions were detected using immunoblotting with the specific antibody. Confocal microscopy. Cellular surface CD40 receptor was detected using a recently described method [15], and lipid rafts were identified by
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the expression of the glycosphingolipid, GM1, which binds the B subunit of cholera toxin. Briefly, cells grown on glass coverslips were transfected with CD40-GFP construct, then incubated with CTxB-Alexa 488 (10 lg/ mL) alone or in the presence of CD40L (5 lg/mL) for 1 h at 4 C. Cells were fixed with 4% paraformaldehyde/PBS for 20 min and analyzed using a laser scanning confocal microscope (ZEISS). Cholesterol depletion. For cholesterol depletion, 2.5 · 105 cells/mL was pretreated with 20 mM methyl-b-cyclodextrin (MbCD) for 30 min at 37 C in serum-free medium. Cells were then washed three times with PBS and resuspended in complete culture medium [16]. Filipin staining. Cells were fixed in 3.2% PFA in PBS and then resuspended in PBS containing 30 lg/mL filipin, held at room temperature for 2 h, and directly analyzed on a flow cytometer [17]. Perfusion organ culture. Animal experiments were performed according to the guidelines of Sun Yat-Sen University for the care and use of laboratory animals which were approved by the Sun Yat-Sen University Animal Care Committee. Male New Zealand White rabbits (2–3 kg) were fed normal chow diet (control) or high-cholesterol diet (chow diet with 0.5% cholesterol) for 8 weeks. After treatment, the rabbits were anesthetized with ketamine (50 mg/kg IV) and xylazine (2 mg/kg IV) and arterial segments were isolated as previously described [18]. Organ culture of the aortic segments was carried out under sterile conditions in an incubator containing 5% CO2 at 37 C for 24–26 h. Statistical analysis. Data are expressed as means ± SD. To compare the data between groups, ANOVA followed by post hoc statistical tests was used. A value of P < 0.05 was considered statistically significant.
Results CD40 receptor associates with cholesterol-rich lipid rafts after membrane treatment with its ligand-CD40L We initially assessed whether CD40 receptor localization in cholesterol-rich membrane lipid rafts during CD40 signaling by examining membrane localization of CD40 on HAECs using a biochemical approach [19]. As shown in Fig. 1A, none of CD40 receptor was found within lipid rafts, whereas CD40L-stimulation promoted the large majority of CD40 recruitment in this microdomain. As a control for proper separation, the membrane was probed with an antibody recognizing the raft marker protein Fyn, which is localized in rafts. As expected, Fyn appeared mainly in the light raft fractions, whereas the non-raft marker Rab5 was isolated in the heavy fractions (Fig. 1A). A similar distribution pattern of CD40 receptor in the rafts after CD40L-stimulation was observed versus nonraft fraction (Fig. 1B). As a control, the raft marker flotilin was predominantly found in the raft fractions, whereas the cytoplasmic protein tubulin was exclusively detected in the nonraft fraction. These results indicated that CD40 receptor was associated with lipid rafts during CD40 signaling challenged with CD40L. Next, the exogenous CD40 construct distribution in rafts was analyzed. We lysed HAECs transfected with CD40-GFP construct and fractionated the lysates by sucrose gradient centrifugation after CD40L-stimulation. Detection of GFP in lipid rafts indicated interaction of CD40 with cholesterol-rich membranes lipid rafts (Supplementary Fig. 1A). Lipid raft fractions were identified by the presence of the marker protein flotilin in light density factions 1, 2, 3, and 4 (Supplementary Fig. 1B). Confocal
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M. Xia et al. / Biochemical and Biophysical Research Communications 361 (2007) 768–774 Raft
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Fig. 1. CD40 receptor mobilizes to lipid rafts after membrane treatment with CD40L. (A) HAECs were left alone or stimulated with 5 lg/mL CD40L for 30 min, then solubilized in Brij 98 and subjected to sucrose gradient separation, as described in ‘‘Materials and methods’’. Sucrose fractions were analyzed by Western blot with anti-CD40, anti-Fyn, and anti-Rab5 antibodies. (B) Lipid rafts were also isolated using the short method. CD40, flotilin, and tubulin were detected by Western blot. NR indicates nonraft fraction; R, raft fraction. The typical experiment of blots was shown. (C) CD40 co-localizes with lipid rafts in HAECs. Lipid rafts were detected by confocal microscopy after labeling HAECs with CTxB-Alexa 488, which binds to the raft- associated glycosphingolipid GM1. GFP-CD40 transfected HAECs were also incubated alone (a–c) or with CD40L (d–f) for 30 min and observed by confocal microscopy. (a and c, CD40-GFP; b and d, lipid rafts; c and f, the merged image which revealed considerable co-localization of CD40 and lipid rafts). Scale bar = 10 lm.
image clearly showed that CD40 receptor co-localized with membrane rafts revealed by incubating HAECs with CTXB-Alexa 488 [17], which binds to GM1, a glycolipid preferentially concentrated within membrane lipid rafts (Fig. 1C). Taken together, these biochemical data demonstrated that endogenous and exogenously expressed CD40 receptor are partitioned to lipid membrane rafts during CD40 signaling.
CD40 protein levels increase in lipid rafts after the interaction of ligand with CD40 receptor To test whether binding of the ligand to its receptor changes the extent of CD40 raft partitioning, we incubated HAECs with wide type CD40 (wtCD40) or CD40 mutant (mCD40, Delta 260) construct and then stimulated with CD40L. Interaction with the wtCD40 by CD40L led to almost all of the CD40 translocation from
M. Xia et al. / Biochemical and Biophysical Research Communications 361 (2007) 768–774
the disordered membrane environment into the ordered membrane raft domains. However, mCD40 transfection markedly abrogated CD40 translocation and only a little CD40 could be detected in lipid rafts (Fig. 2A and B). We then analyzed for the efficient interaction between CD40 receptor and CD40L by flow cytometry. Transfection of HAECs with mCD40 resulted in slightly detectable CD40L binding, which was significantly augmented by transfection with wtCD40 receptor (Supplementary Fig. 2A). Functional binding of CD40L to CD40 receptor was also substantiated by immunoprecipitation. Compared with mCD40, wtCD40 significantly elevated CD40 and CD40L interaction in HAECs (Supplementary Fig. 2B). Intracellular conserved C terminus of the CD40 cytoplasmic tail is essential for CD40 partitioning in rafts The fact that C-terminal intracellular CD40 receptor is highly conserved between species indicates an important function of this domain. Here, we tested a potential involvement of the intracellular domain of C-terminal tail in CD40 receptor partitioning into rafts. For this purpose,
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HAECs were transfected with deletion mutants including Delta 260 and STOP222. To compare the relative proportion of raft localized CD40 receptor, HAECs were subjected to biochemical raft separation and CD40 localization was determined by Western blot. Compared with wtCD40 (Supplementary Fig. 3A), both deletion mutants of CD40 severely impaired the raft localization of CD40 receptor by CD40L stimulation (Supplementary Fig. 3B and C). Interestingly, although removal of the part of C-terminal amino acids of CD40 allowed residual CD40 raft localization (Supplementary Fig. 3B), deletion of the complete cytoplasmic domain completely abolished all the CD40 localization to the light raft fractions (Supplementary Fig. 3C). Therefore, we concluded that the intracellular domain of CD40 receptor was crucial for its targeting to or maintenance within the membrane rafts.
We then attempted to identify the role of lipid rafts in the regulation of CD40 expression and activity. To achieve this question, we employed a small cyclic oligosaccharide MbCD, which binds cholesterol reversibly and allows cholesterol to be depleted from cell membranes [20], to destroyed the lipid rafts in CD40-transfected HAECs, To substantiate the lowering of plasma membrane cholesterol by MbCD, HAECs were stained for FACS with the antibiotic filipin, which binds to cholesterol and has fluorescent properties [21]. Filipin staining decreased about 50% upon MbCD treatment whereas cholesterol replenishment reversed the effect of MbCD on filipin staining (Fig. 3A), showing the nonspecific effects of MbCD. Beside this, MbCD treatment dramatically reduced the amount of cell surface GM1 glycosphingolipids (Fig. 3B) and resulted in a significant shift of the lipid raft marker flotilin from the raft fraction to the nonraft fraction (Fig. 3C), indicating the efficacy of the lipid raft disruption. Accordingly, we also found that MbCD almost completely abolished the translocation of CD40 from nonraft fraction to rafts, demonstrating that lipid rafts may regulate the redistribution of CD40 within the plasma membrane (Fig. 3C).
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Fig. 2. Inducible translocation of CD40 protein in lipid membrane rafts by functional CD40/CD40L interaction. HAECs were transfected with human CD40 (wtCD40) or mutant CD40 (mCD40) construct, then stimulated with 5 lg/mL CD40L for 30 min before solubilization in Brij 98 and were subjected to sucrose gradient separation. Single fractions were analyzed by Western blot with anti-CD40, anti-Fyn, and anti-Rab5 antibodies (A). Immunoblots were performed on pooled heavy (8–9; HF) and light (1–4; raft) fractions to measure CD40 protein expression (B).
We next sought to determine whether lipid raft membranes isolated from aortas exhibited alterations attributable to the high-cholesterol regimen in hypercholesterolemic rabbits. Compared control group, serum cholesterol levels in cholesterol-fed rabbits increased about 4-fold after stable elevation using dietary modification for 8 weeks (Fig. 4A). Cholesterol content of lipid rafts was also significantly elevated in the high-cholesterol group in comparison with the normal group
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Fig. 3. Effects of depleting cholesterol by MbCD on organization of membrane lipid rafts of HAECs and CD40 localization. (A) Membrane cholesterol levels were measured by staining with filipin and flow cytometric measurement of filipin fluorescence on untreated cells (control), MbCD-treated cells (MbCD), or cells treated with MbCD that had been preloaded with cholesterol (MbCD-Chol). Values of the mean fluorescence of the filipin staining were determined by fluorospectrophotometer. **P < 0.01 compared to control. (B) Surface levels of raft-associated GM1 sphingolipids content of density gradient fractions was measured by bound HRP-labeled cholera toxin B subunit (CT-HRP) activity. (C) CD40-transfected HAECs were incubated for 30 min in the culture medium (control) or with MbCD and rested for 30 min in medium, then lipid rafts were isolated. CD40 and flotilin were detected by Western blot. NR indicates nonraft fraction; R, raft fraction.
Rab5 Fraction:
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Fig. 4. Lipid rafts regulates CD40 receptor localization and endothelial activation in vivo. After treatment as described in ‘‘Materials and methods’’. The aortic endothelial cells were isolated, cultured and then were solubilized in Brij 98 before they were subjected to sucrose gradient separation. (A) Blood samples were obtained from rabbits under anesthesia, and serum was prepared by centrifugation. Serum total cholesterol levels was measured using an enzymatic assay kit. (B) Cholesterol content of lipid rafts were analyzed by enzymatic method. Results, expressed as fold of control, are representative of three independent experiments. **P < 0.01 compared to high cholesterol. (C) Sucrose fractions were analyzed by Western blot with anti-CD40, anti-Fyn, and anti-Rab5 antibodies.
Discussion (Fig. 4B). This demonstrates that elevated circulating cholesterol increases the cholesterol content of lipid raft membranes of endothelial cells. We next analyzed the CD40 distribution in rabbit aortic segments. Under normal state, rabbit aortic endothelial cells had nearly undetectable CD40 receptor in lipid rafts (Fig. 4C) which was markedly enhanced in aortas of high-cholesterol fed rabbits.
Lipid rafts are considered microdomains of ordered lipids that selectively partition functional groups of proteins within the membrane. The crucial role of lipid rafts in signal transduction of several receptors, such as TCR, BCR, and FccRs, has been well documented [22,23]. However, signals induced by CD40 in vascular endothelial cells, which is strongly relevant to atherogenesis, on these spe-
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cialized membrane microdomains has not been analyzed in detail. Here, we reported that endogenous and exogenously expressed CD40 receptor constitutively partitioned from the detergent-soluble fraction into membrane lipid rafts by CD40L-stimulation. The intracellular domain of CD40 receptor was crucial for its maintenance within the membrane rafts as partly or completely deleted of the cytoplasmic domain abolished CD40 receptor localization to the rafts. Disassembly of lipid rafts by MbCD interrupted the translocation of CD40 into lipid rafts. In vivo studies showed that both cholesterol content and CD40 localization in lipid rafts of endothelium were increased under the high-cholesterol regimen. These data supported the evolving concept that membrane lipid raft was a key component of biomechanical signal transduction and established it as a potentially novel regulator of CD40 signaling in the endothelium. Lipid rafts are a distinct biophysical plasma membrane compartment and they are highly dynamic, submicroscopic assemblies that float freely within the liquid bilayer in cell membranes [24]. Our current biochemical separation studies showed that endogenously expressed and exogenously transfected CD40 receptor almost exclusively detected in the detergent-insoluble fraction of HAECs by CD40L stimulation, indicating a constitutive association of CD40 with lipid rafts during CD40 signaling. Further functional investigations demonstrated that with the efficient binding of ligand to CD40 receptor, the amount of raft-associated CD40 protein increased, implying the translocation of additional CD40 protein into rafts. These findings indicated that the partition of CD40 receptor into lipid rafts was a dynamic process, not a static process. However, the molecular mechanism controlling this translocation was still unknown. Our current data also suggested an important role for the intracellular cytoplasmic portion of CD40 receptor in raft localization. This domain fulfilled an important role in CD40 sorting into intracellular secretory lysosomes in HAECs. It is conceivable that CD40 raft partitioning was regulated by other proteins binding to the intracellular CD40 domain, possibly in an inducible manner. Many studies have shown that lipid raft membrane microdomains are functionally modulated by increases/ decreases in cholesterol content [25,26]. Hypercholesterolemia, which is characterized by high levels of lipoprotein-containing cholesterol in plasma, is associated with endothelial activation and generally accepted as a major risk factor for the development of atherosclerosis. These findings raise the question whether hypercholesterolemia may influence CD40 partition in rafts which then alters endothelial function or not. More strikingly, the present study provided direct in vivo evidence that elevation in circulating cholesterol levels was capable of increasing the cholesterol content of lipid rafts and CD40 receptor localization. Our observations support that lipid rafts may serve as an important signaling platform in the endothelium and
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prominently contribute to CD40 signaling relevant to atherogenesis. Thus, modulating the extent of CD40 partitioning seemed to be another level at which the cells could regulated CD40 biological activity, suggesting that lipid rafts microdomain is a promising subcellular location for the identification of novel molecular targets. This concept may be adapted to clinical applications with applicable drugs (or natural products) that would influence raft composition and thereby affected CD40-activated signaling. This discovery may represent a therapeutic option to inhibit CD40-related cardiovascular diseases (especially atherosclerosis). Acknowledgments This work was supported by the research grants from National Natural Science Foundation of China Research Grant 30571568 and China Medical Board of New York Inc. (Grant CMB 98-677). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2007. 07.102. References [1] R.P. Phipps, Atherosclerosis: the emerging role of inflammation and the CD40–CD40 ligand system, Proc. Natl. Acad. Sci. USA 97 (2000) 6930–6932. [2] U. Schonbeck, P. Libby, CD40 signaling and plaque instability, Circ. Res. 89 (2001) 1092–1103. [3] S.R. Bennett, F.R. Rrbone, F. Karamalis, R.A. Flavell, J.F. Miller, W.R. Heath, Help for cytotoxic-T-cell responses is mediated by CD40 signaling, Nature 393 (1998) 478–480. [4] C.R. Longo, M.B. Arvelo, V.I. Patel, S. Daniel, J. Mahiou, S.T. Grey, C. Ferran, A20 protects from CD40–CD40 ligand-mediated endothelial cell activation and apoptosis, Circulation 108 (2003) 1113–1118. [5] J.F. Hancock, Lipid rafts: contentious only from simplistic standpoints, Nature 7 (2006) 456–462. [6] K. Simons, E. Ikonen, Functional rafts in cell membranes, Nature 387 (1997) 569–572. [7] M. Rothe, V. Sarma, V.M. Dixit, D.V. Goeddel, TRAF2-mediated activation of NF-kappa B by TNF receptor 2 and CD40, Science 269 (1995) 1424–1427. [8] H.Y. Song, C.H. Regnier, C.J. Kirschning, d.V. Goeddel, M. Rothe, Tumor necrosis factor (TNF)-mediated kinase cascades: bifurcation of nuclear factor-kappaB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2, Proc. Natl. Acad. Sci. USA 94 (1997) 9792–9796. [9] J.R. Arron, Y. Pewzner-Jung, M.C. Walsh, T. Kobayashi, Y. Choi, Regulation of the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF)2 by TRAF1 reveals mechanisms of TRAF2 signaling, J. Exp. Med. 196 (2002) 923–934. [10] P. Xie, B.S. Hostager, M.E. Munroe, C.R. Moore, G.A. Bishop, Cooperation between TNF receptor-associated factors 1 and 2 in CD40 signaling, J. Immunol. 176 (2006) 5388–5400. [11] M. Xia, W. Ling, H. Zhu, Q. Wang, J. Ma, M. Hou, Z. Tang, L. Li, Q. Ye, Anthocyanin prevents CD40-activated proinflammatory
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