Báo cáo khoa học: Factor XII binding to endothelial cells depends on caveolae ppt

Factor XII binding to endothelial cells depends on caveolae Inger Schousboe 1 , Peter Thomsen 2 and Bo van Deurs 2 1 Department of Medical Biochemistry & Genetics, and 2 Structural Cell Biology Unit, Department of Medical Anatomi, The Panum Institute, University of Copenhagen, Denmark It is now generally accepted t hat factor XII (FXII) binds to cellular surfaces in the vascular s ystem. One of t he suggested receptors of this binding is the glyco sylphosphatidylinositol- anchored urokinase-like plasminogen activator (u-PAR) harbored in caveolae/lipid rafts. However, binding of FXII to human umbilical vein endothelial cells (HUVEC) has never been shown to be localized to these specialized mem- brane structures. Using microscopical techniques, we here report that FXII binds to specific patches of the HUVEC plasma membrane with a high density of caveolae. Further investigations of FXII binding to caveolae were performed by sucrose density-gradient centrifugations. This showed that the majority of FXII, chemically cross-linked to HUVEC, could be identified in the same fractions of the gradient as caveolin-1, a marker of caveolae, while the majority of u-PAR was identified in noncaveolae lipid rafts. Accordingly, cholesterol-depleted cells were found to bind significantly reduced amounts of FXII. These ob servations, combined with the presence of a minority of u-PAR in caveolae co ncomitant with F XII binding, indicate that FXII binding to u-PAR may be secondary and depends upon the structural elements within caveolae. Thus, FXII bindin g to HUVEC depends on intact caveolae on the cellular surface. Keywords: factor XII; HUVEC; lipid rafts; c aveolae; u-PAR. Factor XII (FXII) is a zymogen present in plasma at a concentration o f  350 n M . A t local increases i n the Zn 2+ concentration above the normal plasma level of 25 l M , FXII binds to endothelial cells along with plasma prekallikrein. The latter becomes attached to the cells via complex formation with high-molecular-weight kininogen (HK) [1]. Binding of FXII, as well as plasma prekallik- rein in co mplex with HK, initiates in vitro areciprocal activation of FXII and PK, which experimentally can activate plasminogen, and the coagulation and comple- ment systems. So far it h as not been possible to s how that FXII has an impact in any of these systems in vivo, and its biological role remains elusive. By affinity chromatography and antibody inhibition, receptors for HK have been identifi ed as c ytokeratin 1 [2], urokinase- like plasminogen activator (u-PAR) [3], and the receptor for the globular head of the subunit C1q in complement C1 (gC1qR, a lso known a s p 33) [4,5]. T he binding of FXII to immobilized, purified gC1qR in competition with HK tent atively suggested that FXII and HK share the gC1qR as a common receptor [1,5]. Incubation of FXII with prekallikrein and HK in the presence of gC1qR leads to an FXII-dependent conversion of prekallikrein to kallikrein. The observation that the same conversion was observed when gC1qR was exchanged with cytokeratin 1, suggested that c ytokeratin-1 could also be a receptor for FXII [6]. To analyze whether these putative receptors accounted for the binding of FXII to human umbilical vein endothelial cells (HUVEC), it was recently shown that fluorescein isothiocyanate (FITC)-labeled FXII inter- acts with the multiprotein assemply of not only gC1qR and cytokeratin 1, but also u-PAR [ 7]. u-PAR is a glycosylphosphatidylinositol (GPI)-linked glycoprotein, which plays a central role in the regulation of pericellular proteolysis and participates in events leading to cell activation. It is harbored in caveolae/lipid rafts [8]. This prompted us to analyze, by microscopical techniques and sucrose gradient centrifugation, whether FXII binding to HUVEC is dependent on caveolae/lipid rafts. Materials and methods Activated FXII (FXIIa) was obtained as a lyophilized powder from Enzyme Research Laboratories 1 (Leiden, the Netherlands). It was s tored at 4 °C until dissolved a nd then stored in aliquots at )80 °C. FXII was purchased from Haematologic Technologies 2 (Essex Junction, VT, USA), as a high-concentration solution in 50% (v/v) glycerol, and stored at )20 °C as recommended by the manufacturer. It migrated as a single band, with an M r of 80 000, on reducing SDS/PAGE. SDS polyacryl- amide gels (4–12%) were from Invitrogen (Taa-Stru ¨ p, Denmark). All dilutions of FXII were performed in siliconized test tubes and excess dilutions were d iscarded. Correspondence to I. Schousboe, Department of Medical Biochemistry & Genetics, The Panum Institute, University of Copenhagen, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark. Fax: + 45 35367980, Tel.: + 45 35327800, E-mail: schousboe@imbg.ku.dk Abbreviations:DTSSP,3,3¢-dithio-bis(succinimidylpropionate); FXII, Factor XII; FXIIa, activated FXII; GPI, glycosylphosphatidylino- sitol; HK, high-molecular-mass kininogen; HRP, horseradish peroxidase; HUVEC, human umbilical vein endothelial cells; MbCD, methyl-b-cyclodextrin; u-PAR, urokinase-like plasminogen activator receptor. Enzyme: activated factor XII (EC. 3.4.21.38). (Received 2 December 2003, revised 5 May 2004, accepted 27 May 2004) Eur. J. Biochem. 271, 2998–3005 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04229.x Poly(vinylidene difluoride) membranes were obtained from Amersham Biosciences AB, and the chemiluminis- cence enhancer (SuperSignalÒ West Femto Maximum Sensitivity Substrate) and the crosslinking reagent 3,3¢- dithio-bis(succinimidylpropionate) ( DTSSP) were obtained from Pierce. Methyl-b-cyclodextrin ( MbCD) was from Sigma. The primary antibodies used were affinity-purified goat anti-(human FXII) IgG from Affinity Biologicals Corp. 3 (Ancaster, Ontario, Canada), affinity-purified rab- bit anti-(caveolin-1) I gG from Transduction Laboratories 4 (San Diego, CA, USA), mouse anti-(human CD31) immunoglobulin, clone JC70A (M0823), from Dako- Cytomation 5 (Glostrup, Denmark), and protein G-purified rabbit anti-(u-PAR) immunoglobulin (399R) from American Diagnostics 6 (Greenwich, CT, USA). For immunofluorescence, the secondary antibodies were Alexa 488-conjugated chicken anti-(goat IgG) and TRITC- conjugated swine anti-rabbit IgG from Transduction Laboratories. For Western blotting, the s econdary anti- bodies were horseradish peroxidase (HRP)-conjugated swine anti-rabbit 7 immunoglobulins (P 0217) and b iotin- ylated rabbit anti-goat immunoglobulins (E 0466) and biotinylated goat anti-rabbit immunoglobulins (E 0432) from DakoCytomation. Stabilized HRP-conjugated goat anti-(mouse IgG) Ig and Restore TM Western blot stripping buffer were from Pierce. All the biotinylated antibodies had been solid-phase absorbed to minimize cross-reactions with human immunoglobulins and fetal bovine serum. HRP-conjugated streptavidin (P 0397) was from D akoCytomation and molecular m ass m ark- ers from Bio-Rad. H- D -Pro-Phe-Arg-para-nitroaniline (S-2302) was from Chromogenix (Milan, Italy) 8 .All other reagents were of the purest grade commercially available. Endothelial cell culture Cryopreserved primary cultures of HUVEC (Clonetics 9 ,San Diego, CA, USA) were subcultured as described previously [9]. Seven/eight generation c ells (third passage) were used throughout the experiments. F or gradient centrifugation the cells were plated in 75 cm 2 flasks, and for activity measure- ments the cells were plated in 12-well microtiter plates. Microscopic analysis w as performed on cells plated on Labtek 4-chamber slides (Nalgene Nunc International Corp.) at a d ensity of 10 4 cellsÆcm )2 . The mediu m was changed on day 3 and the cells used on days 4 or 5. To eliminate any influence from FXII present in t he complete medium, cells were exposed to serum-free medium (medium lacking fetal bovine s erum) 10 , prior to the experimental application, when indicated. Cholesterol depletion and cholesterol determination Cells were grown in serum-free medium for 3 h and subsequently in the same m edium containing 1% (w/v) MbCD for 30 min or the period of time indicated. The cholesterol concentration in lysates of native and c holes- terol-depleted cells was determined spectrophotometri- cally by a cholesterol/peroxidase assay [ 10]. Protein concentration was determin ed by the m ethod of Bradford [11]. Binding of FXII to cells The culture medium was aspirated and the cells were washed twice over a period of 20 min with Locke’s buffer (154 m M NaCl, 5.6 m M KCl, 3.6 m M NaHCO 3 ,2.3m M CaCl 2 ,1.0m M MgCl 2 ,5.6m M glucose, 5 m M Hepes, pH 7.4) containing 15 l M ZnCl 2 (wash buffer) followed by a 15 min incubation in 0.1% (w/v) gelatin in wash buffer (block buffer). As judged visually by microscopic inspection, the washing procedure did not remove cells from the surface, or change the morphology of the cells. The block buffer was aspirated and the c ells incubated at room temperature with FXII or FXIIa diluted at least 300-fold in block buffer, giving a final concentration of 100–300 n M . After 60 min of incubation, the cells were placed on ice, the incubation medium above the cells was aspirated and the cells were washed continuously for 5 s in a mild stream of ice-cold wash buffer a nd subsequently handled as described below. Amidolytic activity of cell-bound FXIIa As it has previously been shown that FXIIa binds to the cells in a manner identical to FXII [9], binding of FXIIa was used to quantify relatively the amount of FXII bound to cholesterol-depleted cells. Normal and cholesterol-depleted cells in 12-well microtiter plates were incubated with FXIIa, as described above, and analyzed for the amidolytic activity of cell-bound FXIIa, as previously described [12]. To each well was added 600 lLof0.8m M S-2302 in 50 m M Tris, 12 m M NaCl, 10 m M EDTA, pH 7.8. From previous experiments it was known that FXIIa cleavage of S-2302 on the surface of HUVEC was linear, with time, for at least 4 h. After this period of time, the reaction was s topped by acidification and the absorbtion read at 405 n M . Electron microscopy HUVEC cells were fixed with 2% (v/v) formaldehyde and 0.1% (v/v) glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2. The cells were washed, scraped off t he flasks, pelleted, and postfixed with O sO 4 , contrasted en bloc with 1% (w/v) uranyl acetate, dehydrated in a graded series of ethanols, an d embedded in Epon. Sections were examined in a Philips CM 100 electron microscope (Philips, Eindh- oven, the Netherlands). Immunofluorescence microscopy For immunofluorescence microscopy of cell-bound FXII and its colocalization with caveolin-1, the cells were fixed with )20 °C methanol subsequent to washing after incuba- tion with FXII. Methanol was chosen as a fixative because preliminary results had shown that glutaraldehyde fixation destroyed the immunogenicity of FXII. The fixed cells were blocked in block buffer [5% (w/v) goat serum (DakoCyto- mation) in NaCl/P i (PBS)] and incubated with primary and fluorescent secondary antibodies. The primary antibodies were a mixture of goat anti-(human FXII) IgG (diluted 1 : 50) and rabbit anti-(caveolin-1) IgG (diluted 1 : 400). The secondary antibodies were a mixture of Alexa 488- conjugated chicken anti-(goat IgG) Ig (Molecular Probes) Ó FEBS 2004 FXII binding to caveolae (Eur. J. Biochem. 271) 2999 (diluted 1 : 400) a nd TRITC-conjugated pig anti-(rabbit IgG) Ig (DakoCytomation) (diluted 1 : 50), r espectively. The cells were mounted with prolong anti-fade medium from Molecular Probes. Specificity analyses of the anti- bodies showed no reaction of the secondary antibodies with cells incubated in the absence of the primary antibodies. Confocal microscopy The microscope used was a Zeiss LSM 510 confocal microscope. The objectives were a plan-neofluor ·20/0.5, a C-apochromate ·40/1.2 W corr, and a Plan apochromat ·100/1.4 oil Iris lens. The 488 nm line from an Argon laser, and the 543 and 633 lines from two Helium/Neon lasers, were used for excitation. Sucrose gradient centrifugation Protein separation on a sucrose gradient was performed as described by Spisni et al. [13]. Briefly, one 75 cm 2 flask containing a confluent layer of HUVEC was incubated with FXII, and FXII adhered to the cell surface was crosslinked to the receptor with D TSSP, as described above. To avoid disruption of caveolae/lipid rafts during lyses of the cells, this and the following procedures were performed on ice, unless stated otherwise. After scraping off and collecting the cells by centrifugation, the cell pellet was homogenized by trituration (an 0.80 · 80 mm ne edle) and the cells were lysed for 30 min in 1 mL of ice-cold 25 m M P i , 150 m M NaCl, 5 m M EDTA (PNE)/TX-buffer [PNE buffer 11 containing 1% (v/v) Triton X-100, 0.1 m M phenyl- methanesulfonyl fluoride and 0.22 mgÆmL )1 leupeptin). Nuclei and cell debris were removed by centrifugation (5000 g, 5 min). The supernatant was mixed (1 : 1) with 80% (w/v) sucrose in PNE buffer and placed at the bottom of an ultracentrifuge tube. A linear sucrose gradient [5–35% (w/v) in TNE buffer) was layered on top of the lysate. The gradient was centrifuged for 20 h at 200 000 g (4 °C) in an SW 40 Ti Beckman rotor. Fractions of 600 lLwere withdrawn from the bottom of the gradient and mixed 1 : 1 (v/v) with 40% ice-cold trichloroacetic acid. After 2 h of incubation (at 4 °C), precipitated protein was c ollected by centrifugation, the supernatant aspirated and the precipitate washed in 1 mL of ether/ethanol (1 : 1, v /v). Precipitated proteins were disso lved by boiling for 5 min in reducing Laemmli buffer. SDS/PAGE and immunoblotting For Western blot analysis, proteins were separated on 4–12% SDS/polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes according to stand- ard procedures. Standard samples of caveolin-1 and FXII and molecular mass markers were run simultaneously. After incubation for 1 h with NaCl/Tris (TBS) block buffer [50 m M Tris, 0.15 m M NaCl, pH 8.0, containing 0.1% (v/v) Tween 20 and 0.1% (w/v) BSA], the membranes were probed with goat anti-FXII immunoglobulin (diluted 1 : 10 000)/biotinylated rabbit anti-goat 12 immunoglobulins (diluted 1 : 10 000)/HRP-conjugated streptavidin (diluted 1 : 10 000) and rabbit anti-(caveolin-1) immunoglobulins (diluted 1 : 10 000)/HRP-conjugated swine anti-rabbit immunoglobulin (diluted 1 : 10 000), respectively. The blot probed with antibodies against FXII was subsequently stripped u sing Restore TM Western b lot stripping buffer, used according to the manufacturer’s instructions, and probed against rabbit anti-(u-PAR) immunoglobulin (diluted 1 : 10 000)/HRP-conjugated swine anti-rabbit Ig (diluted 1 : 10 000) or mAb CD31 (diluted 1 : 1000)/HRP- conjugated goat anti-(mouse IgG) Ig (diluted 1 : 10 000). Dilutions of antibodies were performed in 1% nonfat skim milk in TBS block buffer. Detection was carried out using the chemiluminescence enhancer, SuperSignalÒ West Femto Maximum Sensitivity Substrate, as recommended by th e manufacturer, and the results were monitored on a Las Chemiluminator. The intensity of the bands was measured using the Image Gauge, quant menu. Statistics Analysis of variance ( ANOVA ) with the post hoc Student’s t-test was u sed to determine the s tatistical significance of difference between sample groups. Results FXII binds to HUVEC caveolae Caveolae, 50–100 nm invaginations of the plasma mem- brane, are a subset of sphingolipid- and cholesterol-enriched lipid rafts, characterized by the presence of t he protein caveolin [14]. Caveolae are abundant in endothelial cells [15]. In agreement with this, HUVEC contained numerous caveolae, as revealed by electron microscopy (Fig. 1A,B). Interestingly, the caveolae were not evenly distributed in the plasma membrane of these cells, but appeared regionally at very high densities, separated b y caveolae-free membrane segments. This concentration of caveolae at certain regions of the HUVEC surface was also visualized by confocal microscopy using an antibo dy against caveolin-1. Thus, an intense immunofluorescence signal was obtained in certain stretches of the plasma membrane (Fig. 2A). Double- immunofluorescence labeling for caveolin-1 and bound FXII showed a very high degree of co-localization (Fig. 2B,C), indicating t hat bound FXII was associated with caveolae. The integrity of caveolae depends on a certain level of plasma membrane cholesterol, and cholesterol depletion achieved by incubating cells with M bCD makes caveolae disappear and allows the released caveolin to become diffusely distributed in the plasma membrane and to be internalized [16, 17]. To a nalyze further the apparent association of FXII binding with HUVEC caveolae, cells were therefore c holesterol-depleted by incubation with MbCD. Incubation of HUVEC with MbCD [1% (w/v), 60 min] caused a reduction in cellular cholesterol to 20% of the level found in untreated cells. By electron microscopy it could be shown t hat this treatment of HUVEC resulted in an almost complete removal of caveolae. Thus, the patches with high concentrations of caveolae were never seen in cholesterol-depleted cells and, only rarely, could single, caveolae-like structures be identified (Fig. 1 C). This was further confirmed by confocal microscopy (Fig. 2C,E). The impact of MbCD treatment o n the binding of FXII was 3000 I. Schousboe et al. (Eur. J. Biochem. 271) Ó FEBS 2004 analyzed a t vary ing MbCD concentration and treatment periods. By confocal microscopy, only little and diffusely distributed FXII could be identified on HUVEC after 60 min of incubation with 1% (w/v) MbCD (Fig. 2D). The ability of MbCD-treated cells to bind FXII was analyzed quantitatively by measuring the amidolytic activity of FXIIa, w hich binds to the cells in a m anner i dentical to that of FXII [9]. This way it w as shown that progressively decreasing amounts o f FXIIa adhered to t he cells with increasing periods of exposure to 1% (w/v) MbCD. The amidolytic activity of FXII a in the wells exposed for a longer period of time than 30 min remained constant, but significantly lower (P<0.001) than the activity measured in the wells not exposed to MbCD (Fig. 3). No changes in cell number or in protein content were identified during the treatment. FXIIa binding associated with the Triton X-100 insoluble low-density fraction A common means of identifying proteins associated with caveolae is to investigate the detergent i nsolubility at 4 °C. To examine whether the FXII/receptor complex might be insoluble in cold Trito n X-100, FXII was crosslinked to the receptor by t he cleavable disulfide cross-linking reagent, DTSSP. This prevented dissociation of FXII from the receptor during the subsequent solubilization and ultracen- trifugation. To minimize contamination of the surface of the plastic with nonspecifically bound FXII, the cells were scraped off before solubilization. Figure 4 shows a Western blot of an SDS polyacrylamide gel of lysates of native and MbCD-treated cells incubated with block buffe r i n t he presence or absence o f FXII and subsequently exposed to DTSSP. While no visible bands were observed in the lysates of cells incubated in the absence of FXII, FXII was present in the reduced lysates of c ells incubated with F XII, regardless of wh ether these were native or treated with MbCD. The intensity of t he anti-FXII reacting band in the lysates f rom MbCD-treated cells was, however, apparently weaker than that from native cells. To confirm that this was not caused by analytical variations, the blot was stripped and probed with anti-CD31. CD31 is a marker protein of endothelial cells. It appeared as a clear band in reduced samples and as a smear in nonreduced samples of lysates of cells treated with DTSSP. M easuring the intensity of the anti-FXII and the anti-CD31 reacting bands in the reduced samples on the same blot showed, in three individual experiments, that the FXII/CD31 in lysates of cells treated with MbCD was 50 ± 10% of that measured in lysates of native cells. The invisibility of FXII in nonreduced samples Fig. 1. Caveolae in human umbilical vein endothelial cells (HUVEC) cells, detected by electron microscopy. Caveolae in control cells are shown in (A) and (B). (A) A section perpendicular to the plasma membrane, revealing the typical shape of these structures, whereas (B) is a tangential section better showing the high density of caveolae. CP, clathrin-coated pit. (C) Section through a cholesterol-depleted cell in which the arrow indicates a single, caveolae-like structure. Bar: 200 nm. Ó FEBS 2004 FXII binding to caveolae (Eur. J. Biochem. 271) 3001 indicates a poor anti-FXII immunoglobulin reactivity of FXII crosslinked to the receptor, or formation of very high M r complexes of FXII. The solubility, in cold Triton X-100, of the FXII/receptor in lysates o f c ells was a nalyzed by sucrose gradient centrifugation. Figure 5A shows a Western b lot of a reducing SDS polyacrylamide gel of every second fraction of the gradient consisting of 20 fractions. An 80 kDa molecular mass anti-FXII immunoglobulin-reacting band was present in the soluble fractions (representing one of three fractions), with the density of a 40% sucrose solution, as well as in the higher density fractions of the floating fractions w ith sucrose d ensities from 40 to 2 3%. W ith decreasing intensity throughout the gradient, all of t he undiluted fractions stained heavily when probed with anti- caveolin-1 (results not shown). This indicated that caveolae were distributed throughout the entire gradient. The intensity of these bands, however, d ecreased with decreasing density of t he gradient. T his was evidenced by probing for caveolin in samples d iluted fivefold, which convincingly showed the presence of t he highes t c oncentration o f caveolin-1 in the h igher density of the floating fractions. FXII was thus identified in the same fractions as those containing the highest concentrations of caveolin-1. This indicates that FXII originating from the disulfide FXII/ receptor complex was harbored in caveolae. As a possible receptor for FXII, the localization of u-PAR (M r 50 000– 60 000) was analyzed. In accordance with the presence of large vesicles rich in GPI-anchored proteins (lipid rafts), as well as smaller caveolar vesicles lacking GPI-anchored proteins in Triton X-100 insoluble membranes [18], u-PAR could be i dentified mainly in the very-low-density part of the Fig. 2. Colocalization of cell-bound factor XII (FXII) and caveolin-1. Human umbilical vein endothelial cells (HUVEC) were grown in serum-free medium for 4 h. Then, medium in half of the wells was exchanged with the same medium containing methyl-b-cyclodextrin (MbCD) and the incubation was continued for another 60 min. The cells were then washed, incubated with 100 n M FXII, and subsequently fixed with methanol. The fixed cells were first incubated with a mixture of goat anti-FXII (1 : 50) and rabbit anti- (caveolin-1) (1 : 200) and second with a mixture of Alexa 488-conjugated chicken anti-(goat IgG) Ig (1 : 400) and TRITC- labeled swine anti-(rabbit IgG) Ig (1 : 50). (B) and (D) (green) show FXII, and (A), (C) and (E) (red) show caveolin-1. The panels are representative photomicrographs of at least three independent experiments. Bar: 20 lm. 3002 I. Schousboe et al. (Eur. J. Biochem. 271) Ó FEBS 2004 gradient (Fig . 5A). U pon treatment w ith MbCD, the density of caveolae, as well as lipid rafts, increased. As a result of this, the presence of caveolin-1 and FXII d isap- peared from their upper density fractions, while u-PAR was skewed towards the higher density fractions (Fig. 5B). The weakly stained 60- and 70 kDa anti-FXII reacting bands, noticeable throughout the gradients, were present with varying intensity in all of the three experiments performed and were considered to be artifacts. Discussion By a combination of microscopy and gradient centrifuga- tion, the present study shows that HUVEC-bound FXII localizes to caveolae, and that this localization is dependent upon the structural integrity of these elements in the plasma membrane. It is generally accepted that FXII binds to HUVEC and that this binding is receptor mediated. Investigation of this binding, by immunofluorescence microscopy of monolayers of HUVEC incubated with FXII, indicated, in contrast to a previous study [7], that FXII does not bind diffusely to the cells but to specific patches in the plasma membrane corresponding to caveolae-rich domains. The reasons for the discrepancy between this and the former study may be several. In the present study, native FXII was incubated with morphologically intact HUVEC before fixation, while in the previous study the cells were fixed before incubation with FXI I. Furthermore, the FXII bind ing was, in the former study, visualized by FITC labeling, whereby Lys residues (of possible importance f or the binding of FXII to HUVEC) might have been blocked. Caveolae are highly immobile plasma membrane invag- inations [17] abundant in the cellular membrane of endo- thelial cells [14]. They are enriched in glyc olipids and cholesterol. Depleting the cells for cholesterol by treatment with MbCD disrupts the integrity of caveolae. Cholesterol- deprived cells incubated with FXII showed microscopically only little binding of FXII. This confirms that FXII binds to a receptor harbored in caveolae. Previous analyses have shown that MbCD preferentially extracts cholesterol from the outside and partially solubilizes GPI-anchored and Fig. 3. Quantification of factor XII (FXII) bin ding to c ells preincubated with methyl- b-cyclodextrin (MbCD) f or varying p eriods of time. Human umbilical vein endothelial cells (HUVEC), grown in serum-free med- ium, were, for varying periods of time, pre-exposed to 1% (w/v) MbCD and subsequently incubated with 100 n M activated FXII (FXIIa) for 1 h. After washing, the cells were incubated with S-2302. Theordinateindicatestheabsorbancemeasuredat405 nmafter4 hof incubation. The concentration of protein was measured in the incu - bation mixture by the method of Br adford [11]. The activity wa s measured on three individual setups of cells and determined as t he mean value ± SD. The significance of variance of FXIIa bound to HUVEC, pre-exposed for varying periods of time to MbCD, was calculated by a n alysis of variance ( ANOVA ). Use of an asterisk 14 indicates a P-value of < 0.001 relative to the control (0 min incubation period), determined using the post-hoc Student’s t-test. Fig. 4. Western blots of factor XII (FXII) cross-linked to membranes of native and cholesterol-depleted cells. Oneofthreecultureflasksof human u mbilical vein endothelial cells (HUVEC), grown in serum-free medium for 15 h, was pre-exposed to 1% (v/w) methyl-b-cyclodextrin (MbCD) for 30 min. This flask (+MbCD +FXII) and a second (–MbCD +FXII) of the three were subsequently incubated with 100 n M FXII, while the third flask (–MbCD –FXII) was incubated with block buff er. Aft er this incubation, the cells were washed and incubated with DTSSP. After neutralization of the DTSSP, the cells were lysed. Western blots of reduced(+)andnonreduced(–)samples 15 were visualized by sequential incub ation with goat anti-FXII, bio tin- ylated rabbit anti-(goat IgG), horseradish peroxidase (HRP)-conju- gated streptavidin and Sup erSignalÒ West Femto Maximum Sensitivity Substrate (upper part of the figure). The blot was then stripped us ing R esto re TM Western blot s tripping bu ffer and in cu bated sequentially with mouse anti-(human CD31), HRP-conjugated goat anti-(mouse IgG) and SuperSignalÒ West Femto Maximum Sensi- tivity Substrate (lower part of the fi gure). Ó FEBS 2004 FXII binding to caveolae (Eur. J. Biochem. 271) 3003 transmembrane p roteins [18]. A s i t has been previously shown that no differentiation exists between the binding of FXII and FXIIa, the change in ability of M bCD-treated HUVEC to bind FXII was quantified by measuring the amount of bound FXIIa colorimetrically [9]. This showed that a significantly lower amount of FXII was bound to MbCD-treated cells than to untreate d cells. However, treatment with MbCD did not completely prevent the binding of FXII to the layer of cells in the well. In fact,  50% of the binding persisted after MbCD treatment. This was confirmed by density measurements of Western blots of FXII extracted from the cells. As no FXII was observed on the fluorescen ce microscopy of MbCD-treated cells incu- bated with FXII, a considerable amount of the cell-bound FXII might h ave been removed during t he extensive washing of the fixed cells accompanying the immunofluo- rescence staining. Binding of FXII to lipid-rich domains in the c ellular membrane was f urther verified by sucrose gradient centrif- ugation. While confocal microscopy and the activity measurements were performed on intact cells without crosslinking, the gradient centrifugations were performed on lysates of cells to which FXII had been chemically crosslinked to cellular proteins in its immediate vic inity by incubation with the cleavable membrane-impermeable DTSSP. Sucrose gradient centrifugation of cold Triton X-100 lysates of cells showed that FXII was present in the same fractions as the marker protein for caveolae, caveolin- 1. Both FXII and the majority of caveolin-1 were distributed in the soluble fraction, as well as in the higher density floating fractions of the gradient. As it has previously been suggested that F XII binds to the G PI- anchored receptor, u-PAR [7], and this receptor is organized in caveolae and lipid rafts on the cell surface [19], it was tempting to investigate this possibility f urther. We showed that u-PAR, in contrast to FXII, originating from the FXII/ receptor complex, appeared to be present in mainly the lower density of the floating fractions and thus not only in the fractions containing the highest concentration of caveolin I and FXII. In line with this, immunofluorescence microscopy of the cellular localization of u -PAR indicated that u-PAR was equally distributed on the plasma mem- brane surface (results not shown). The difference in fractional distribution of u-PAR and FXII in the sucrose gradient indicates that FXII binding to u-PAR may be secondary to FXII binding to caveolae. This is in accordance with previous findings showing that FXII binding to HUVEC could be inhibited by antibodies to u-PAR, but not by the much smaller ligands of u-PAR, urokinase-like 13 plasminogen activator (u-PA), and vitronec- tin [7]. It may also explain the biphasic nature of FXII binding to HUVEC [9]. The binding of FXII to caveolae indicates that FXII binds to a receptor predominantly localized in these specific structural elements of the cell membrane containing a subset of sphingolipids and cholesterol. Moreover, in addition to GPI-anchored proteins, caveolae harbor GPI-anchored proteoglycans (glypicans). Glypicans are highly sulfated by substitution with heparan sulfate and chondroitin sulfate [20], creating a binding site for po lycationic molecules [21]. Among several glucosaminoglycans, both heparan sulfate and chondroitin sulfate have been shown to enhance the rate of FXII activation in solution [22]. Combining the established knowledge about FXII binding to negatively charged surfaces in general, and proteoglycans in particular [23], with our present findings, we tentatively propose that Fig. 5. Analysis of Triton X-100 solubility b y sucrose gradient centri- fugation. Native and methyl-b-cyclodextrin (MbCD)-treated cells, to which factor X II (FXII) had been c ross-linked, were e xtracted with ice- cold Triton X-100 and subjected to su crose gradient centrifugation, as described in the Materials and methods. Every second fraction of the gradient 16 was analyzed for the distribution of FXII, caveolin-1 and urokinase-like plasminogen activator receptor (u-PAR) by reducing SDS/PAGE and Western blotting. The distribution of FXII and caveolin-1 was probed o n individual blots, while the distribution of u-PAR was probed on stripped blots (F ig. 4). (A) Results obtained using native cells; (B) results obtained with MbCD-treated cells. The distribution is representative of three individual experiments. 3004 I. Schousboe et al. (Eur. J. Biochem. 271) Ó FEBS 2004 FXII binds to a glypican in caveolae. Direct analysis of the detergent-solubilized FXII/receptor complex is ongoing but at the moment it must be concluded that the present investigation was unable to confirm previous studies [7], suggesting that u-PAR is the most likely receptor of FXII. Acknowledgements The expert te chnical assistance of Ms Mette Olsen a nd Birgit Harder is greatly appreciated. The work was supported by grants from the Danish Medical Research Council and the Novo Nordisk foundation. References 1. Reddigari, S.R., Shibayama,Y.,Brunnee,T.&Kaplan,A.P. 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