© 2000 American Heart Association, Inc.
Vascular Biology |
Assembly of Multimeric von Willebrand Factor Directs Sorting of P-Selectin
From the Department of Biochemistry (C.H., H.P), Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands; the Department of Medicine and Pharmacology (A.G., M.D., I.R.P.), Section of Molecular Genetics, University of Sheffield, Sheffield, UK; and the Department of Blood Coagulation (H.P.d.L., H.-J.M.B., J.A.v.M.), CLB, Sanquin Blood Supply Foundation, Amsterdam, Netherlands.
Correspondence to Dr H. Pannekoek, Academic Medical Center, Department of Biochemistry (K1-159), Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail h.pannekoek{at}amc.uva.nl
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Abstract—We designed a model system to study the role of von Willebrand factor (vWF) in the sorting of P-selectin and the biogenesis of Weibel-Palade body (WPB)–like organelles. For that purpose, a human epithelial cell line (T24) that synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis and storage organelles, was transfected with full-length vWF cDNA or a deletion mutant thereof. Stable transfectants of T24 with full-length vWF cDNA revealed the generation of WPB-like organelles as demonstrated by colocalization of vWF and P-selectin with double-labeling immunofluorescence. In contrast, T24 cells transfected with vWF delD’D3 cDNA, encoding a mutant that is unable to form vWF multimers, displayed only perinuclear vWF staining, whereas no indication was found for the presence of WPB-like organelles. The contents of the organelles in full-length vWF cDNA–transfected T24 cells were released on activation of the protein kinase C pathway, similar to the situation with genuine endothelial cells. The expression of vWF did not affect the biosynthesis of P-selectin, as deduced from the observation that untransfected and vWF cDNA–transfected T24 cells contained the same amount of P-selectin mRNA. We propose that the biosynthesis of multimeric vWF directs the generation of WPB-like organelles, as evidenced by the sequestering and anchoring of P-selectin into these storage granules.
Key Words: von Willebrand factor • P-selectin • Weibel-Palade bodies • protein sorting
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Introduction |
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Multimeric von Willebrand factor (vWF) is an obligatory glycoprotein for the formation of a hemostatic plug. It connects the platelet to the subendothelium that is exposed on injury of the vessel wall. vWF is synthesized by megakaryocytes and vascular endothelial cells. Endothelial cells display 2 secretory routes for newly synthesized vWF, namely, a constitutive and a regulated pathway.1 Constitutively secreted vWF is rapidly transported in secretory vesicles toward the plasma membrane. This material consists of vWF dimers and low-molecular-weight multimers. Another portion of the newly synthesized vWF is encountered in specific storage organelles, denoted Weibel-Palade bodies (WPBs). These organelles fuse with the plasma membrane on activation of the protein kinase C signal transduction pathway, followed by release of their contents (regulated secretion).2 3 vWF routed by this pathway consists of high-molecular-weight multimers that are
2 orders of
magnitude more effective in mediating adhesion of platelets to the
injured vessel wall than are vWF dimers or low-molecular-weight
multimers.4
The WPBs are considered to be endothelial
cell–specific, rod-shaped, electron-dense storage
organelles.5 Apart from vWF multimers and the vWF
propolypeptide,6 7 8 the WPB contains a number of other
proteins, namely CD63, a transmembrane glycoprotein that is
also present in lysosomal membranes of many cell
types9 10 ; interleukin-811 ; tissue
plasminogen activator12 ; and the
transmembrane receptor P-selectin (also called GMP-140 or
PADGEM).13 14 P-selectin acts as an "anchor" for the
adhesion of leukocytes to endothelial cells. Before
that action, the protein kinase C signal transduction pathway must be
activated, leading to fusion of WPBs with the plasma membrane
and exposure of P-selectin on the cell surface. After guiding the
adhesion of leukocytes, which may ultimately transmigrate to the
subendothelium to combat local
inflammation,15 P-selectin is endocytosed and recycled to
the membrane of WPBs. Under physiological
conditions, vWF and P-selectin are synthesized in the same cell types
(endothelial cells and megakaryocytes) and sorted to
the same organelles (endothelial WPBs and platelet
-granules). However, under pathophysiological
conditions, other cell types are found to express P-selectin. Notably,
it has been reported that epithelial cells from breast
carcinomas,16 from paranasal chronic sinusitis
mucosa,17 and from glomerulonephritis
biopsies18 express P-selectin. This property has been
exploited in this study to develop a model system to assess the role of
vWF in the sorting of P-selectin and the biogenesis of WPB-like
organelles.
The requirements for routing vWF to the WPBs are subject to discussion. Initially, it was proposed that storage of vWF was operational only in cells harboring a regulated, secretory pathway and that the propolypeptide of vWF acted as a sorting signal to direct vWF multimers to the storage organelles.19 However, full-length vWF cDNA–transfected heterologous cells, which are not known to harbor a regulated secretory pathway, all synthesize and store multimeric vWF in WPB-like organelles.20 21 The ability of heterologous, transfected cells to generate these organelles was correlated with the formation of multimers, because nonmultimerizing vWF deletion mutants are not stored in these granules. Taking these reports together, we proposed that the generation of these organelles is not an exclusive property of endothelial cells but that the biogenesis is triggered by the synthesis and assembly of multimeric vWF.20 21 Direct evidence for this hypothesis would be provided if we could demonstrate that sorting of a WPB-resident protein occurs exclusively in the presence of vWF synthesis and assembly of multimers. As a WPB-resident protein, P-selectin was chosen on the basis of observations that several epithelial cells express this protein under pathophysiological conditions.16 17 18 Accordingly, we found that the epithelial cell–like human cell line T2422 expresses P-selectin mRNA (vide infra), and therefore, we used these cells to design a model system to address the hypothesis outlined above. These cells lack endogenous vWF mRNA and are devoid of storage granules. Transfection of T24 cells with full-length vWF cDNA revealed that multimeric vWF triggers the biogenesis and assembly of WPB-like storage organelles, as evidenced by the sorting of P-selectin into these granules.
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Methods |
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Tissue Culture and Transfection
T24 cells22 (human bladder carcinoma cells; ECACC No. 85061107) were maintained in medium 199 supplemented with 2 mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL Fungizone, and 10% (vol/vol) heat-inactivated fetal calf serum (Gibco Laboratories). T24 cells were stably transfected with plasmid pSVL-vWF containing full-length vWF cDNA.20 The pSVL vector carries neomycin resistance that allows for selection during 10 days in culture medium containing 400 µg/mL neomycin (G418); 36 clones were later chosen, of which 19 survived. These clones were screened by immunofluorescence with the use of a rabbit anti-human vWF polyclonal antibody. Eleven clones expressed vWF antigen, of which T24-vWF-10 and T24-vWF-12 were chosen for further analysis. Plasmid pSVL-vWF delD’D3 was used for transient transfection.18 The composition of these plasmids has been described previously.23 24
RNA Isolation and Northern Blotting
Total RNA of T24 cells and of stably vWF cDNA–transfected cells
was isolated with TRIzol reagent according to the manufacturer’s
instructions (Gibco BRL). Northern blotting of 10 µg of RNA was done
as described.25 A 482-bp HindIII (position
2235)–BamHI (position 2717) vWF cDNA fragment (GenBank
accession number X04385), radiolabeled with use of the random-primer
DNA labeling system (Gibco BRL) and
[-32P]dATP, was used as a probe for DNA-RNA
hybridization. Unincorporated radioactivity was discarded by Sephadex
G-50 chromatography. After hybridization, radioactive
probes were removed by incubating the blots twice for 20 minutes at
90°C in 0.1x SSC, 0.1% SDS, and 1% sodium pyrophosphate.
Subsequently, the blots were reused for hybridization with a
radiolabeled 478-bp XhoI (position 561)–NcoI
(position 1039) cDNA fragment of P-selectin (GenBank No. U02297), and
after stripping the blot by washing as indicated above, the blots were
reused for hybridization with a radiolabeled 84-bp EcoRI
(position 517)–SalI (position 601) cDNA fragment of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
GenBank No. M17851) for calibration of the amount of RNA applied.
Immunofluorescence
Cells were grown until they reached confluence on gelatin-coated
glass coverslips. Then the cells were washed twice with 10 mmol/L
sodium phosphate (pH 7.4), 0.14 mol/L NaCl (PBS) and subsequently fixed
for 10 minutes at room temperature with methanol. After fixation, the
cells were washed twice with PBS and incubated for 1 hour with 1 of the
following: (1) a mixture of 3 murine anti-human vWF monoclonal
antibodies (CLB-RAg 201, CLB-RAg 35, and CLB-RAg 21; all from CLB,
Amsterdam, Netherlands); (2) a rabbit anti-human vWF polyclonal
antibody (Dako-Patts, Glostrup, Denmark); (3) a rabbit anti-human
cathepsin D polyclonal antiserum (kindly donated by Dr J.M.F.G. Aerts,
Department of Biochemistry, Academic Medical Center, Amsterdam,
Netherlands); (4) a mixture of 3 murine anti-human P-selectin
monoclonal antibodies: F1.18 (a kind gift of Dr H.K. Nieuwenhuis,
Academic Hospital Utrecht, Utrecht, Netherlands),
C2 (a kind gift of Dr P. Modderman, CLB, Amsterdam, Netherlands), and
CD62 (Pharmingen, San Diego, Calif); or (5) a combination of the
antibodies listed above in PBS supplemented with 3% (Organon Teknika).
Subsequently, the coverslips were washed twice with PBS and incubated
for 1 hour with a cy-3 (Amersham/Pharmacia Biotech)–labeled
conjugate of goat anti-mouse or goat anti-rabbit immunoglobulins,
FITC-conjugated goat anti-mouse or goat anti-rabbit immunoglobulins
(Jackson Immuno Research Laboratory), or both in PBS supplemented with
3% BSA and 0.1% (vol/vol) Tween-20. Finally, the coverslips were
washed 3 times, prepared for microscopy by embedding in mounting
medium, and analyzed under a fluorescence microscope
(Axioplan 2, Zeiss). For single- and double-labeling experiments, the
appropriate filters were used to visualize exclusively cy-3–labeled
antigens (red) or FITC-labeled antigens (green).
Determination of vWF Antigen
The release of vWF antigen in the conditioned medium of cultured
T24-vWF cells by secretagogues was measured by ELISA essentially as
described previously.26
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Results |
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Design of a Model System to Study Sorting of P-Selectin
It has been reported that neoplastic epithelial cells may express nonepithelial markers, such as the endothelial cell–specific protein P-selectin.16 Accordingly, we show here by Northern blotting analysis that human T24 carcinoma cells, which display an epithelial cell–like morphology,22 synthesize P-selectin mRNA but do not produce vWF mRNA, another endothelial cell marker (Figure 1A
, lane 3). However, vWF mRNA
synthesis was readily achieved by stable transfection with full-length
vWF cDNA (Figure 1A, lanes 1 and 2). Northern blot
analysis of RNA preparations of 2 independent clones,
T24-vWF-10 and T24-vWF-12, revealed comparable amounts of vWF mRNA, as
determined by PhosphorImager analysis, by using GAPDH
mRNA as a standard for equal loading (Figure 1B). It should be
noted that P-selectin mRNA synthesis is not affected by vWF expression,
because synthesis of the former is similar in transfected and
untransfected cells (Figure 1B). Transfected T24 cells were
further explored as a model system to study vWF-directed sorting of
P-selectin. To that end, we analyzed the fate of vWF and
P-selectin proteins in these transfected cells.
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Immunofluorescence Analysis of vWF
cDNA–Transfected T24 Cells
Double-labeling immunofluorescence with both
anti–P-selectin antibodies and anti-vWF polyclonal antibodies was
performed to establish the presence and localization of vWF and
P-selectin antigens. Nontransfected T24 cells showed no staining with
anti-vWF polyclonal antibodies (Figure 2a). A homogeneous, faint
background staining was consistently observed with
anti–P-selectin antibodies in nontransfected T24 cells, indicative of
cytoplasmic P-selectin (Figure 2d). Interestingly, stably
transfected T24-vWF cells displayed a punctate staining pattern that
was identical regardless of whether or not anti-vWF antibodies or
anti–P-selectin antibodies were used (Figures 2b and 2e).
Besides these punctate entities, it is noteworthy that no intracellular
background staining was observed in T24-vWF cells with either 1 of
these antibody preparations, in contrast to what was observed in
untransfected cells with anti–P-selectin antibodies.
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T24 cells were also transfected with plasmid pSVL-vWF delD’D3. This
plasmid encodes a deletion mutant of vWF that is able to dimerize but
is unable to multimerize owing to the lack of the D’ and D3
domains of vWF.23 As shown before, the absence of
multimerization coincided with the lack of WPB-like vWF storage
organelles in several different transfected cell
types.21 23 Double labeling of T24-vWF delD’D3 cells with
both anti-vWF and anti–P-selectin antibodies revealed a perinuclear
vWF staining, representing the presence of vWF in the rough
endoplasmic reticulum and in the Golgi apparatus (Figure 2c
). However, with the anti–P-selectin antibodies, only faint
cytoplasmic staining was observed in T24-vWF delD’D3 cells, similar to
that seen in untransfected T24 cells (Figure 2f). Taking all of
these observations into consideration, we concluded that the expression
of full-length vWF cDNA leads to the formation of multimers,
irrespective of the transfected cell type
studied,19 20 23 24 and directs the biogenesis of
organelles as visualized by a characteristic punctate staining pattern.
Moreover, expression of multimeric vWF causes sequestering of
P-selectin into these punctate entities, tentatively denoted as storage
organelles.
Release of Stored Proteins by Activation of the Protein Kinase
C Pathway
We verified whether the typical feature of WPBs of
endothelial cells to release their contents on
activation of the protein kinase C signal transduction pathway could
also be attributed to the granules in transfected T24-vWF cells. For
that purpose, T24-vWF cells were incubated for 1 hour with
2x10-6 mol/L phorbol dibutyrate (PDB),
subsequently washed to remove the PDB, and allowed to recover for 24
hours. This agonist was chosen because of its property to be removed
during washing of the cells, in contrast to the more frequently used
phorbol myristate acetate (PMA). The cells were
analyzed by immunofluorescence before
activation, at 1 hour after activation, and after a recovery period of
24 hours (Figures 3a through 3c).
Alternatively, cells were incubated for 1 hour with
1x10-7 mol/L of the potent agonist PMA
dissolved in 0.1% (vol/vol) DMSO or with 0.1% (vol/vol) DMSO alone
and processed as indicated above. In this case, PMA was chosen because
it is considered a more potent agonist than PDB. The vWF released from
activated T24-vWF cells and recovered from the conditioned
medium was determined by ELISA. The conditioned medium of cells treated
for 1 hour with 0.1% (vol/vol) DMSO alone contained 0.34±0.03 mU vWF
per 1.5 mL, whereas cells activated for 1 hour with
1x10-7 mol/L PMA in DMSO contained 0.89±0.19
mU vWF per 1.5 mL of conditioned medium. These data and the
immunofluorescence observations are
consistent with the interpretation that the organelles of
T24-vWF cells share essential features with genuine
endothelial cell WPBs. Notably, the punctate vWF
pattern initially disappeared on PDB (Figure 3b) or PMA
treatment (concomitant with the appearance of vWF in the conditioned
medium) and reappeared after an adequate recovery period (Figure 3c). Furthermore, it is worth mentioning that we attempted to
observe the appearance of P-selectin at the surface of T24-vWF cells
after PDB treatment but failed to detect P-selectin antigen by
immunofluorescence analysis (see Methods),
possibly owing to its transient exposure and rapid internalization
(data not shown).
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To exclude the possibility that the observed storage organelles in
T24-vWF cells were lysosomal structures, double-labeling
immunofluorescence was performed with a mixture of
murine anti-human vWF monoclonal antibodies and a rabbit anti-human
cathepsin D polyclonal antiserum. This experiment unambiguously showed
that the intracellular localization of the typical lysosomal marker
cathepsin D was distinct from that of vWF (Figure 4).
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Discussion |
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The routing of P-selectin has been studied in several transfected cell types. These studies were performed in the absence of vWF synthesis.27 Specifically, murine pituitary AtT-20 cells, which have a regulated sorting pathway, and nonregulated Chinese hamster ovary cells were transfected with P-selectin cDNA. In P-selectin cDNA–transfected AtT-20 cells, P-selectin is routed to adrenocorticotropic hormone–containing storage granules, whereas in transfected Chinese hamster ovary cells, P-selectin is not stored but instead is exposed on the plasma membrane.27 From those observations and from the data reported in the current study, one may conclude that in the absence of vWF synthesis and multimeric assembly, P-selectin requires the presence of a regulated secretory pathway to be routed to storage granules. In the absence of both multimeric vWF assembly and a regulated secretory pathway, the fate of P-selectin is difficult to predict, because in P-selectin cDNA–transfected Chinese hamster ovary cells, the receptor is apparently exposed on the surface, whereas endogenously synthesized P-selectin in T24 cells is presumably present in the cytoplasm and not exposed on the surface, as deduced from the faint cytoplasmic staining of untransfected T24 cells (Figure 2d
).
However, it should be emphasized that under
physiological conditions, the expression of
P-selectin invariably occurs in the same cell types as that of vWF (ie,
endothelial cells and megakaryocytes). Moreover, in
both endothelial cells and megakaryocytes, P-selectin
and vWF are routed and stored in the same compartment, and we speculate
that cosorting of these proteins in the same cells is obligatorily
linked. In this study, we have provided further evidence for our proposal that the biogenesis of WPBs in endothelial cells or of WPB-like organelles in vWF cDNA–transfected heterologous cells is dependent on the synthesis and assembly of multimeric vWF.20 The major finding reported here is that the synthesis and assembly of multimeric vWF trigger sequestering of P-selectin into WPB-like storage organelles. In our view, these observations provide strong support for the hypothesis outlined before. Furthermore, our findings are reminiscent of the helper function of chromogranin B in the selective sorting of intermediates of a peptide hormone precursor.28 29 In those studies, it was demonstrated that overexpression of chromogranin B redirects these intermediates toward the regulated secretory pathway of AtT-20 cells. These observations can be explained by the tendency of chromogranin B to aggregate in the lumen of the trans-Golgi network, thereby enabling a selected set of proteins with a reduced aggregation capacity to be sorted to the same compartment of these neuroendocrine cells. Similarly, we propose that the aggregation (ie, multimerization) of vWF in the trans-Golgi network of endothelial cells or of heterologous vWF-transfected cells may have a helper function in sorting P-selectin, analogous to that of chromogranin B in the neuroendocrine cell line AtT-20. Finally, our findings on sequestering of P-selectin by multimeric vWF are also reminiscent of the data acquired by recent vWF and factor VIII coexpression studies in transfected AtT-20 cells and in bovine aortic endothelial cells.30 In both cell types, colocalization of factor VIII and vWF was observed in storage granules, whereas no storage of factor VIII could be detected in the absence of vWF synthesis. In those studies, it was elegantly shown that intracellular vWF–mediated trafficking of factor VIII was due to actual binding of factor VIII to vWF in the trans-Golgi network. Whether P-selectin also binds to multimeric vWF before assembly into storage granules remains to be established.
We speculate that the sequestering of P-selectin by multimeric vWF will have consequences for the pathology of atherosclerosis, as deduced from data obtained with experimental animals. First, studies with P-selectin–knockout mice have demonstrated that leukocyte rolling and extravasation are severely compromised compared with those processes in wild-type mice,31 providing support for an essential function of this receptor in early leukocyte recruitment at sites of inflammation. In this respect, it might be expected that the absence of P-selectin would protect against atherosclerosis.15 Indeed, this expectation has been borne out by the results of studies on the initiation and progression of atherosclerosis in P-selectin–knockout mice compared with wild-type mice, which were eventually crossed into an atherogenic apoE-knockout genetic background.32 33 Clearly, the absence of P-selectin protected against atherosclerosis, presumably by delaying the formation of fatty streaks. Second, it has been reported that pigs with von Willebrand disease, similar to human type 3 von Willebrand disease (ie, no vWF antigen), are relatively protected against diet-induced atherosclerosis.34 35 This finding has been attributed to the lack of vWF-mediated platelet adhesion to the subendothelium as an important contributing event to atherogenesis. In view of the findings on the sequestering and subsequent storage of P-selectin by multimeric vWF, we suggest that the protection from atherosclerosis in vWF-deficient pigs may be accounted for by the absence of vWF and the subsequent deficient availability of P-selectin.
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Acknowledgments |
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This work was supported by the Netherlands Heart Foundation (grant 91.126).
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Footnotes |
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Part of this work has been presented at the XVth Congress of the International Society on Thrombosis and Haemostasis, July 1997, Florence, Italy.
Received September 17, 1999; accepted January 18, 2000.
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