(Received for publication, November 21, 1995)
From the
High endothelial venules (HEV) are specialized plump postcapillary venules in lymphoid tissues that support high levels of lymphocyte extravasation from the blood. We have recently identified a novel human transcript, expressed to high levels in HEV, that encodes a secreted, acidic protein closely related to the anti-adhesive extracellular matrix protein known as BM-40, osteonectin, and SPARC (secreted protein acidic and rich in cysteine). Here, we show that this protein, designated hevin, is associated with basal, lateral, and apical surfaces of HEV cells, and unlike MECA-79 antigen, is not expressed on the underlying basement membrane. In contrast to fibronectin or other adhesive extracellular matrix proteins, purified hevin does not support endothelial cell adhesion in vitro. Moreover, addition of soluble exogenous hevin inhibits attachment and spreading of endothelial cells on fibronectin substrates. Hevin-treated cells do not form focal adhesions and exhibit a rounded morphology. Together, these results suggest that hevin is an abundant extracellular protein that modulates high endothelial cell adhesion to the basement membrane.
Lymphocytes continuously recirculate between the blood and
lymphatic systems, thereby providing an effective immune surveillance
for foreign invaders(1) . In lymphoid organs, high numbers of
lymphocytes leave the blood by recognizing and migrating through
specialized postcapillary venules called high endothelial venules (HEV) ()(for a review, see (2) ). The endothelial cells of
HEV are called high endothelial cells by reference to their typical
plump, almost cuboidal, morphology, very different from the flat
appearance of endothelial cells that line other vessels. Another
important feature of HEV endothelium is the expression of sialomucin
counter-receptors for lymphocyte L-selectin (3, 4, 5) , that are important in the initial
step of lymphocyte binding to HEV and are decorated with sulfated
oligosaccharides recognized by L-selectin and the HEV-specific
mAb MECA-79(6, 7, 8) .
Although the molecular mechanisms involved in the induction and maintenance of the specialized morphology and phenotype of HEV have not yet been identified, local microenvironmental factors, such as extracellular matrix (ECM) molecules and cytokines associated with the immune response, are likely to play an important role(2) . The ECM is an important component of the cellular environment, which plays a key role in the modulation of cell shape(9) , cell differentiation, and tissue-specific gene expression(10) . Endothelial cells are separated from adjacent connective tissue by a specialized sheet of ECM, known as the basement membrane, that contains adhesive ECM proteins such as laminin, collagen IV, and fibronectin. The ECM composition of the basement membrane has been shown to influence both endothelial cell morphology and differentiation(11, 12) .
Two antiadhesive proteins
of the ECM, thrombospondin (TSP) and SPARC (BM-40), have been shown to
modulate the adhesion of endothelial cells to ECM and
substratum(13) . TSP substrates support the attachment of some
endothelial cells but not cell spreading or formation of stable
cell-substrate adhesion plaques or focal adhesions, while soluble TSP
inhibits focal adhesion formation in cells seeded on fibronectin
substrates(14) . SPARC is an acidic
Ca-binding glycoprotein (15) that regulates
endothelial cell shape and barrier function (16) by inhibiting
cell spreading (17) and modulating focal adhesion
disassembly(18) . Although the precise mechanisms by which
SPARC regulates endothelial cell adhesion are not well understood, a
Ca
-binding EF hand, located in the carboxyl-terminal
part of the protein, has been shown to have both antispreading (19) and focal adhesion-labilizing activity(20) .
Furthermore, this region binds to collagen IV(21) .
We have recently characterized a novel human cDNA encoding an acidic putative calcium-binding protein, designated hevin, that exhibits 62% identity with SPARC over a region of 232 amino acids spanning more than four-fifths of the SPARC coding sequence(22) . The greatest difference between the proteins is in the highly acidic amino-terminal domain of hevin (26% glutamic acid and aspartic acid residues), which is considerably larger (432 residues) than the corresponding domain in SPARC (71 residues). In situ hybridization analysis revealed that hevin mRNA is expressed to high levels in HEV from human lymphoid tissues. Although hevin mRNA is detected only in HEV in tonsil and is absent from human umbilical vein endothelial cells, Northern blots show that hevin is expressed in other tissues, including brain and heart, in a pattern distinct from SPARC. In view of its strong homology with the antiadhesive ECM protein SPARC and its high expression in HEV, hevin is a good candidate for an ECM protein that may facilitate lymphocyte migration by modulating high endothelial cell adhesion and phenotype. In this study, we have investigated the effects of hevin on endothelial cell adhesion in vitro. We found that hevin is antiadhesive and inhibits both endothelial cell attachment and spreading on fibronectin substrates. We also show that hevin is associated with basal, lateral, and apical surfaces of high endothelial cells in vivo but not with the underlying basement membrane. Together, these results suggest that the function of hevin could be to modulate high endothelial cell adhesion to the basement membrane.
Figure 1: Immunofluorescence staining of human tonsil frozen sections with hevin-peptide antibodies. Frozen sections of human tonsils (8 µm, fixed with acetone) were double-stained with affinity-purified hevin-peptide antibodies (A, C, and E) and HEV-specific monoclonal antibody MECA-79 (B) or monoclonal antibody against human fibronectin (D and F) for 1 h in a moist chamber at room temperature and binding was detected with FITC- and Texas Red-labeled secondary antibodies. Bar, 20 µm.
Figure 2:
Expression and purification of hevin
protein. A, hevin protein secreted by CHO cells. CHO-hevin (lanes 1-6) or -control (lane 7) clonal
transfectants were labeled for 4 h with
[S]cysteine and methionine, and 25 µl of
conditioned media was subjected to 10% SDS-PAGE under reducing
conditions and fluorography. Hevin translated in vitro was
used as a control (lane 8). B, immunodetection of
hevin protein in CHO-hevin but not CHO-control transfectants. Media
conditioned by CHO-hevin (lane 1) or CHO-control (lane
2) transfectants (10 µl of serum-free conditioned media
concentrated 20-fold) and purified hevin protein (1 µg, lane
3) were fractionated by 10% SDS-PAGE under reducing conditions,
and proteins were immunoblotted with affinity-purified hevin-peptide
antibodies. C, purification of hevin. Conditioned media (10
µl of serum-free conditioned media concentrated 20-fold) from
CHO-hevin (lane 1) or CHO-control (lane 2)
transfectants and hevin protein purified from conditioned media by
anion-exchange chromatography (1 µg, lane 3) were
fractionated by 10% SDS-PAGE under reducing conditions and subjected to
silver staining.
Silver
staining after fractionation by SDS-PAGE under reducing conditions
revealed that the hevin protein is a major component secreted by
CHO-hevin (Fig. 2C, lane 1) but not by
CHO-control (lane 2) transfectants. To purify hevin from
conditioned media, we took advantage of the acidic isoelectric point
predicted for the hevin protein (pI = 4.5). After 3 days of
culture in media without serum, we collected supernatants from the
CHO-hevin transfectants and separated the hevin protein from other
components of the conditioned media by anion-exchange chromatography at
pH 6 on HighTrap Q. Under the conditions used, the hevin protein was
retained on the column and eluted as a single 130 kDa band almost
devoid of contaminants (Fig. 2C, lane 3).
Starting with 250 ml of conditioned media, this strategy allowed us to
obtain 3 mg of pure protein in an intact native form appropriate
for functional studies.
Hevin was tested for its ability to mediate
attachment of human and bovine endothelial cells relative to substrates
composed of ECM proteins, which are known to support endothelial cell
adhesion. Since it was not possible to obtain sufficient purified HEV
cells from tonsils for these studies, we used HUVEC as a source of
human endothelial cells. We also used CPAE cells, which are sensitive
to the antiadhesive effects of SPARC (16) and have been shown
to express heparin-like ligands for L-selectin(28) .
Individuals wells of a 96-well tissue culture dish were incubated for
18 h at 4 °C with 10 µg/ml of each ECM molecule. Cells were
then added to each well in serum-free medium and allowed to attach for
3 h at 37 °C. To quantitate cell attachment, a colorimetric assay
was used(29) . The A, a measure of
crystal violet staining of cells bound to the substrate, correlates
well with the visual scoring and allows a quantitative analysis of cell
attachment (Fig. 3). We observed efficient attachment and
spreading of both HUVEC (Fig. 3A) and CPAE cells (Fig. 3B) on substrates coated with fibronectin (Fig. 3), collagen 1, or a basal lamina ECM preparation
containing entactin, collagen IV, and laminin (data not shown). We also
observed efficient attachment and spreading of HUVEC and CPAE on
substrates coated with tenascin, which is in agreement with other
studies that have revealed that tenascin is not anti-adhesive for
endothelial cells but promotes endothelial cell attachment mediated by
and
integrins(29) . In contrast, substrates coated with hevin
did not promote any attachment after 3 h of plating at 37 °C. Since
the adhesive activity of tenascin has previously been shown to depend
on the coating conditions(30) , we coated hevin on plastic at
different concentrations (1-20 µg/ml), different temperatures
(4 or 37 °C) and for varying periods of time (1, 2, or 18 h). Under
all conditions examined, including at 10 µg/ml and 4 °C for 18
h, which for tenascin allows maximal attachment of endothelial cells,
hevin had no adhesive activity (Fig. 3, A and B). The lack of adhesive activity of the native hevin protein
is not due to inefficient coating of hevin on plastic, since when
coated under the same conditions hevin inhibits attachment of CPAE
cells to plastic (see below). Finally, another anti-adhesive ECM
protein, TSP, was tested in the endothelial cell attachment assay. TSP
supported attachment of some HUVEC and CPAE cells but was not as
effective as fibronectin, collagen 1, basal lamina ECM, or tenascin
substrates (Fig. 3, A and B). Moreover, in
contrast to the other substrates, HUVEC and CPAE did not spread on TSP
substrates.
Figure 3:
Hevin substrates do not support
endothelial cell attachment and spreading. Adhesion of HUVEC (A) and CPAE cells (B) to substrates coated with BSA,
fibronectin, tenascin, hevin or TSP. BSA (1 mg/ml) and purified ECM
proteins (10 µg/ml) were coated overnight at 4 °C. 10 cells were added to each well in serum-free medium and allowed to
attach for 3 h at 37 °C, 5% CO
. Cell attachment was
quantitated by a colorimetric assay. The A
correlates with the number of cells bound to the substrate. Cell
attachment was also assessed using an inverted microscope, and
representative photomicrographs are shown of cells on each substrate.
Results are mean and standard deviations of triplicate
determinations.
Figure 4:
Hevin inhibits endothelial cell adhesion. A, CPAE adhesion to plastic; B, CPAE adhesion to
fibronectin; C, HUVEC adhesion to fibronectin; D,
HUVEC adhesion to a basal lamina ECM preparation containing entactin,
collagen IV, and laminin. E, ECV 304 cell adhesion to
fibronectin; F, effects of conditioned media from CHO-hevin or
-control transfectants on HUVEC adhesion to fibronectin; G,
effects of antiadhesive ECM proteins on HUVEC adhesion to fibronectin.
Soluble proteins were added together with 5 10
cells at the time of seeding on plates coated with 1 µg/ml
purified fibronectin or 2 µg/ml basal lamina ECM. Soluble proteins
included hevin (1-10 µg/ml), BSA (10-5000 µg/ml),
tenascin, SPARC, or thrombospondin (10 µg/ml) or conditioned media
from CHO-hevin and -control transfectants (10 µl of serum-free
conditioned media concentrated 20-fold and diluted 20-fold in the assay
to a final concentration of 1-fold). Cell attachment was quantitated by
a colorimetric assay. The A
correlates with the
number of cells remaining bound to the substrate. Results are means of
two independent experiments performed in triplicate with the larger of
the S.D. for each point from the two
experiments.
Strong adhesion of endothelial cells to fibronectin substrates develops over time because of increases in cell area (spreading) and the active formation of focal adhesions(31) . One mechanism by which hevin could inhibit endothelial cell adhesion to fibronectin is by interfering with these processes. To test for the first possibility, the effects of soluble hevin on endothelial cell spreading were analyzed. HUVEC that attached to fibronectin substrates in the presence of BSA started to spread after 1 h, and most of the cells were spread after 3 h at 37 °C (Fig. 5A). In contrast, cells that received soluble hevin at 10 µg/ml, retained a rounded morphology on fibronectin-coated plates and did not spread after 3 h at 37 °C (Fig. 5B). The antispreading effect of hevin was found to be specific and not due to a minor contaminant secreted by CHO cells, since HUVEC incubated in the presence of conditioned media from CHO-hevin transfectants exhibited a rounded morphology, while HUVEC plated in the presence of conditioned media from CHO-control transfectants underwent extensive spreading during a 3-h incubation period (data not shown).
Figure 5: Hevin inhibits endothelial cell spreading and focal adhesion formation on fibronectin substrates. A, spreading of HUVEC for 4 h on fibronectin in the presence of 10 µg/ml BSA; B, hevin at a concentration of 10 µg/ml inhibits HUVEC spreading on fibronectin; C-F, localization of vinculin (C and D) and F-actin (E and F) in HUVEC treated with 10 µg/ml BSA (C and E) or hevin (D and F); G-J, localization of vinculin (G and H) and F-actin (I and J) in ECV 304 cells treated with 10 µg/ml BSA (G and I) or hevin (H and J) and allowed to spread for 16 h. BSA-treated HUVEC or ECV 304 cells have numerous actin-containing stress fibers and vinculin-containing plaques terminating at the end of these stress fibers. Hevin-treated cells exhibit a primarily diffuse distribution of vinculin and do not have prominent stress fibers. Bar, 25 µm.
To further characterize the anti-adhesive activities of the hevin protein, we analyzed the formation of focal adhesions in HUVEC seeded on fibronectin substrates in the presence or absence of hevin. In the absence of hevin, staining for vinculin revealed that HUVEC formed many focal adhesions, which were present both over the central cell body and at the edges of the cells (Fig. 5C). Double-staining for F-actin revealed prominent stress fibers that traversed the cell body (Fig. 5E). In contrast, there was no formation of focal adhesions in hevin-treated cells, and the staining for vinculin was diffuse throughout the cytoplasm (Fig. 5D). Double staining for F-actin revealed the absence of prominent actin-containing stress fibers (Fig. 5F). Inhibition of spreading and focal adhesion formation by soluble hevin was also observed with the HUVEC-derived cell line ECV 304. Although spreading and focal adhesion formation of ECV 304 cells on fibronectin substrates required longer time than for HUVEC, essentially identical results were obtained. BSA-treated cells contained prominent actin stress fibers terminating at vinculin-positive focal adhesions (Fig. 5, G and I). In contrast, hevin-treated ECV 304 cells failed to spread and did not form focal adhesions (Fig. 5, H and J). These results demonstrate that hevin is able to inhibit both endothelial cell spreading and focal adhesion formation on fibronectin substrates.
The results presented here, together with
our previous work(22) , clearly show that hevin is related to
SPARC both structurally and functionally. However, the two proteins are
likely to have distinct, rather than overlapping, physiological roles.
The tissue distribution of hevin mRNA is clearly different from that of
SPARC(22) . For example, hevin is expressed to high levels in
brain and low levels in placenta and testis, while SPARC has an
opposite expression pattern in these tissues. In lymphoid tissues, both
proteins are expressed; however, they have a different cellular
distribution. Hevin is expressed in HEV, while SPARC is not detected in
HEV but can be found in scattered cells of the tonsils. ()
We compared hevin with other proteins for inhibition of
endothelial cell adhesion. TSP slightly reduces the number of
endothelial cells attaching to a fibronectin substrate. These findings
are in agreement with previous studies that have revealed that TSP
inhibits both endothelial cell adhesion and focal adhesion formation on
fibronectin substrates(14, 32) . Since there are no
structural similarities between hevin and TSP, it is likely that these
two proteins exert their antiadhesive effects by different mechanisms.
Although the heparin-binding domain of TSP has been shown to have focal
adhesion labilizing activity(33) , the molecular mechanisms by
which TSP inhibits focal adhesion formation remain to be characterized.
Tenascin has also been shown to modulate focal adhesion formation in
endothelial cells seeded on fibronectin substrates(18) .
However, tenascin is primarily adhesive for endothelial cells, since
human endothelial cells have been shown to use
and
integrins to attach and spread on tenascin
substrates(29) .