(Received for publication, October 23, 1995; and in revised form, February 23, 1996)
From the
Previous studies established that uterine epithelial cells and
cell lines express cell surface heparin/heparan sulfate (HP/HS)-binding
proteins (Wilson, O., Jacobs, A. L., Stewart, S., and Carson, D.
D.(1990) J. Cell. Physiol. 143, 60-67; Raboudi, N.,
Julian, J., Rohde, L. H., and Carson, D. D.(1992) J. Biol. Chem. 267, 11930-11939). The accompanying paper (Liu, S., Smith,
S. E., Julian, J., Rohde, L. H., Karin, N. J., and Carson, D. D.(1996) J. Biol. Chem. 271, 11817-11823) describes the cloning
of a full-length cDNA corresponding to a candidate cell surface HP/HS
interacting protein, HIP, expressed by a variety of human epithelia. A
synthetic peptide was synthesized corresponding to an amino acid
sequence predicted from the cDNA sequence and used to prepare a rabbit
polyclonal antibody. This antibody reacted with a protein with an
apparent M of 24,000 by SDS-polyacrylamide gel
electrophoresis that was highly enriched in the 100,000
g particulate fraction of RL95 cells. This molecular weight is
similar to that of the protein expressed by 3T3 cells transfected with
HIP cDNA. HIP was solubilized from this particulate fraction with NaCl
concentrations
0.8 M demonstrating a peripheral
association consistent with the lack of a membrane spanning domain in
the predicted cDNA sequence. HIP was not released by heparinase
digestion suggesting that the association is not via membrane-bound HS
proteoglycans. NaCl-solubilized HIP bound to heparin-agarose in
physiological saline and eluted with NaCl concentrations of 0.75 M and above. Furthermore, incubation of
I-HP with
transblots of the NaCl-solubilized HIP preparations separated by
two-dimensional gel electrophoresis demonstrated direct binding of HP
to HIP. Indirect immunofluorescence studies demonstrated that HIP is
expressed on the surfaces of intact RL95 cells. Binding of HIP
antibodies to RL95 cell surfaces at 4 °C was saturable and blocked
by preincubation with the peptide antigen. Single cell suspensions of
RL95 cells formed large aggregates when incubated with antibodies
directed against HIP but not irrelevant antibodies. Finally, indirect
immunofluorescence studies demonstrate that HIP is expressed in both
lumenal and glandular epithelium of normal human endometrium throughout
the menstrual cycle. In addition, HIP expression increases in the
predecidual cells of post-ovulatory day 13-15 stroma.
Collectively, these data indicate that HIP is a membrane-associated
HP-binding protein expressed on the surface of normal human uterine
epithelia and uterine epithelial cell lines.
Heparan sulfate proteoglycans (HSPGs) ()located
either on cell surfaces or in extracellular matrices are found in
nearly all mammalian
tissues(1, 2, 3, 4, 5) .
Functionally, HSPGs and a variety of HP/HS-binding proteins have been
shown to participate in a diverse range of biological processes such as
cell attachment, growth factor binding, cell proliferation, migration,
morphogenesis, and viral
pathogenicity(6, 7, 8) . Several lines of
evidence indicate that HSPGs play an important role during the initial
attachment of the apical plasma membrane of trophectodermal cells of
the blastocyst to the apical plasma membrane of the uterine epithelium.
In mice, HSPGs are expressed on the cell surfaces of two-cell stage and
post-implantation stage embryos(9) . Furthermore, blastocyst
attachment to laminin, fibronectin, and isolated mouse uterine
epithelial cells is inhibited by HP. Embryo attachment also is
inhibited by the treatment of embryos with HP/HS lyases or inhibitors
of proteoglycan biosynthesis(10, 11) . Immunological
studies of murine embryo implantation sites indicated that the core
protein of the basement membrane HSPG, perlecan, and HP/HS chains are
located between the apical cell surfaces of trophectodermal cells and
uterine epithelial cells during the peri-implantation
stage(12) . Expression of perlecan on the external
trophectodermal surface correlates with acquisition of attachment
competence in vitro as well. Externally disposed H/HS-binding
sites have been described on the cell surface of mouse uterine
epithelial cells(13) . Furthermore, using a heterologous cell
adhesion assay, we demonstrated that HP/HS-like glycosaminoglycans
participate in the initial attachment between two human cell lines, JAR
and RL95, used to mimic the initial attachment of the human embryonic
trophectoderm to human uterine epithelial cells,
respectively(14) . As is the case for mouse uterine epithelia,
the human uterine epithelial cell line, RL95, has specific, high
affinity cell surface HP/HS-binding sites, which are sensitive to mild
trypsin digestion of intact cells. Three tryptic peptides that retained
HP/HS binding specificity were isolated from such trypsinates and
partially amino-terminal sequenced (15) . In the accompanying
paper(36) , the full-length cDNA sequence to one of these
proteins, named HIP for HP/HS interacting protein, was obtained and
shown to encode a cell surface protein with an M
of 24,000 when expressed in transfected 3T3 cells. HIP is
expressed in a cell type-specific fashion by many human cell lines,
particularly those of epithelial origin.
In the current study, we have generated and characterized a rabbit antibody to a synthetic peptide designed from a predicted 16-amino acid sequence of HIP. These studies demonstrate that HIP is a peripheral membrane protein that directly binds HP and is expressed on the surfaces of normal human uterine epithelia and many uterine epithelial cell lines.
Subcellular fractionation was used as an initial step to partially
purify HIP for subsequent analytical studies. Fractionation of RL95
cells and subsequent Western blot analysis determined that HIP was most
highly enriched in the 100,000 g pellet; however, HIP
was detected in other particulate fractions as well (Fig. 1).
Lower molecular weight components immunologically related to HIP were
detected in the 1000
g/20 min and 10,000
g/20 min particulate fraction. These components were presumed
to be partially degraded forms of HIP. In contrast, HIP appeared to be
quantitatively depleted from the 100,000
g soluble
fraction. A similar distribution of HIP was observed in JAR and HEC-1a
cells, human trophoblastic and uterine adenocarcinoma cell lines,
respectively (data not shown). The high speed particulate fraction was
used further as the most convenient source of HIP.
Figure 1:
HIP is enriched in the 100,000 g particulate fraction. Subcellular fractions were prepared
from RL95 cells and analyzed by SDS-PAGE and Western blotting as
described under ``Experimental Procedures.'' The Western blot
was probed with antibody to HIP. Approximately 50 µg of protein was
added per lane. Lane 1, total RL95 homogenate; lane
2, 1000
g/10-min supernatant; lane 3,
10,000
g/20-min supernatant; lane 4, 100,000
g/1.0-h supernatant; lane 5, 100,000
g/4-h supernatant; lane 6, 1000
g/10-min pellet; lane 7, 10,000
g/20-min pellet; lane 8, 100,000
g/1.0-h pellet; lane 9, 100,000
g/4-h pellet. The migration position of intact HIP is
indicated to the left and of the molecular mass marker
proteins (in kDa) to the right.
Figure 2:
HIP is eluted from the particulate
fraction with high salt. Six pellets of the 100,000 g particulate fraction, 80 to 100 µg of protein each, were
collected, and each pellet was subjected to salt extraction as
described under ``Experimental Procedures.'' Half of each
extract or pellet was used for protein determination, and the other
half was used for SDS-PAGE and Western blot analysis as described under
``Experimental Procedures.'' Lanes marked Pel. and Sup. represent 100,000
g
/60-min
pellets and supernatants obtained after NaCl extraction at the molar
concentration indicated at the top, respectively. The lane
marked Con. was the control pellet not extracted with NaCl.
The arrow marks the position of HIP. The migration positions
of molecular mass marker proteins are indicated (in kDa) to the left of the figure. Note that HIP is only partially eluted
with 0.4 M NaCl and quantitatively eluted with NaCl
concentrations of 0.8 M or higher.
Figure 3:
HIP is not secreted or released from
RL95 cells. Serum-free RL95 cell-conditioned medium from a 24-h
incubation was collected and centrifuged at 100,000 g for 1 h. The 100,000
g/60-min supernatant was
trichloroacetic acid-precipitated, and equal portions of all fractions
were analyzed for the presence of HIP by Western blotting as described
under ``Experimental Procedures.'' Lane 1, RL95 cell
homogenate; lane 2, 100,000
g pellet from
conditioned medium; lane 3, the 100,000
g supernatant from conditioned medium.
Figure 4:
HIP binds tightly to heparin-agarose. The
0.8 M NaCl extract of a 100,000 g/60-min
particulate fraction was subjected to heparin-agarose chromatography as
described under ``Experimental Procedures.'' A portion of the
sample was used for direct Western blot analyses (Ext.), and
the remainder was diluted to 0.15 M NaCl before incubation
with heparin-agarose. HIP was serially eluted batchwise from
heparin-agarose with buffers containing increasing concentrations of
NaCl as indicated at the top of the figure. Each eluate was
trichloroacetic acid-precipitated and analyzed by Western blot
analyses. Each lane (0.15-2.0) represents the total
material obtained in each eluate.
Figure 5:
HIP directly binds I-heparin. The 100,000
g particulate
fractions were isolated and analyzed by two-dimensional non-equilibrium
gel electrophoresis as described under ``Experimental
Procedures.'' Duplicate gels were used for silver staining or
transfer to nitrocellulose and Western blotting as described under
``Experimental Procedures.'' Panel A, silver-stained
gel; panel B, Western blot from panel C subjected to
I-HP overlay; panel C, Western blot used in panel B probed with anti-HIP. The arrow indicates the
spot corresponding to HIP in all panels. The migration positions of
molecular mass standards are indicated to the right, and the
positions of pI standards are indicated at the bottom.
Figure 6:
Binding of anti-HIP to intact RL95 cells
is saturable and specific. A, monolayers of RL95 cells were
grown in a 24-well tissue culture plate to 90% confluency. Cells were
incubated at 4 °C for 45 min with anti-HIP () or nonimmune
rabbit IgG (
) as described under ``Experimental
Procedures.'' The data represent the average ± S.E. of
duplicate determinations. The triangles represent the average
± S.E. obtained for specific binding (anti-HIP binding) minus
the average binding obtained with nonimmune rabbit IgG (nonspecific).
Binding is both specific and saturable between 5 and 10 µg of
anti-HIP/ml. B, in a similar experiment 25 µg of anti-HIP
or nonimmune rabbit IgG was preincubated without (stripedboxes) or with (openboxes) 100 µl
of peptide affinity matrix for 2 h before incubation with RL95 cells.
The data are the averages ± S.E. for duplicate determinations in
each case.
Figure 7: Anti-HIP reacts with the surfaces of intact cells. HEC-1a (25) cells were grown on glass coverslips to 50% confluency, fixed with paraformaldehyde, and stained as described under ``Experimental Procedures.'' Antibodies to type I cytokeratins were used to stain cells that either were not (panel A) or were (panel B) methanol-permeabilized. Note the lack of staining of the nonpermeabilized cells demonstrating the integrity of the plasma membrane. Nonpermeabilized cells were stained with affinity purified anti-HIP (panel C) or no primary antibody (panel D). Apparent variations in staining intensity between cells are due to differences in focal depths or multi-layering of cells.
It was further reasoned that if HIP was on RL95 cell surfaces then non-fixed, single cell suspensions of living RL95 cells could be aggregated by anti-HIP. As shown in Fig. 8, incubation of single cell suspensions of RL95 cells with anti-HIP greatly enhanced cell aggregation. Parallel controls, including PBS, PBS containing 0.02% sodium azide and an antibody to the cytoplasmic tail of the mucin, MUC1(24) , did not enhance RL95 cell-cell aggregation. Collectively, these data strongly indicate that HIP is located on the extracellular surface of the plasma membrane of human uterine epithelial cell lines.
Figure 8: Aggregation of RL95 cells by anti-HIP. RL95 cells were harvested intact from tissue culture plates with 10 mM EDTA in PBS. Cells were incubated for 3 h in the presence or absence of antibodies and photographed. Panel A represents time 0. Panels B-D represent 3 h of incubation. Panel B shows cells incubated with buffer only. Panel C shows cells incubated with antibodies generated to the cytoplasmic domain of the Muc-1 mucin (24) and represents another negative control. Panel D shows cells incubated with anti-HIP. In panels B and C, some cell-cell aggregation is noted after 3 h of incubation; however, panel D indicates that this aggregation is greatly enhanced by the presence of anti-HIP. Samples were photographed with a Nikon Diaphot inverted microscope using inverted phase microscopy.
Experiments also were performed to determine if HIP is expressed by other human uterine epithelial cell lines as well as normal human uterine epithelium in situ. As shown in Fig. 9, Western blots of several human uterine epithelial cell lines as well as human endometrium displayed a prominent band corresponding to the molecular weight of HIP. A 1.3-kilobase transcript is detected in all three cell lines by Northern analyses using HIP cDNA as a probe (36) .
Figure 9:
Expression of HIP by various human uterine
epithelial cell lines and human endometrium. Total protein extracts
(100 µg per lane) were obtained and analyzed by Western blotting
after SDS-PAGE as described under ``Experimental
Procedures.'' Lane 1, RL95 cells; lane 2,
Ishikawa cells; lane 3, HEC-1A cells; lane 4, normal
human endometrium (day 21 of menstrual cycle). Note the presence of a
predominant band with a M of 24,000 in all cases.
The migration positions of molecular mass markers are indicated to the right.
Figure 10: HIP is expressed by normal uterine epithelial cells. Frozen, methanol-fixed sections of human uterine endometrium were incubated with anti-HIP using the immunocytochemical protocol described under ``Experimental Procedures.'' All photographs were taken at a constant exposure time. Magnification is indicated on the figure. Photographs were taken of both glandular (gl; panels A, C, and E) and lumenal (lu; panels B, D, and F) epithelia in each case. The stages of the menstrual cycle presented are days 8 (panels A and B), 13 (panels C and D) and 21 (panels E and F). In all cases, HIP expression appears to be predominantly epithelial with glandular epithelium displaying variations in intensity of expression. Low reactivity is observed in areas of the sections containing stromal cells (St) that fail to react with anti-HIP.
Figure 11: HIP is expressed in predecidual cells. Sections of day 29 (panels A-C) or day 13 (panel D) endometrium were stained with antibodies to HIP (panel A), laminin (panels C and D), and factor VIII (panel B). On day 29, HIP is not only expressed by lumenal epithelial cells (lu) but also by vascular endothelium (ve), indicated by staining of a serial section with anti-factor VIII (panel B), and surrounding predecidual cells of the underlying stroma (panel A), indicated by the expression of laminin in the extracellular matrix (panel C). The position of the basal lamina (bm) in panels B-D are indicated by arrows. In contrast, stromal tissue of day 13 uteri displays laminin in basal lamina but not interstitial matrix (panel D). The position of stromal elements (st) and glandular epithelium (gl) in panel D are indicated. Magnification is indicated on the figure.
A number of studies described above have demonstrated that HSPGs are expressed on the surfaces of mouse blastocysts and human trophoblastic cell lines where they function in cell adhesion events. In these studies, it was further demonstrated that adhesive activity resides in the constituent HS chains of the HSPGs. Consistent with these observations, specific HP/HS-binding sites were identified on the surfaces of both mouse uterine epithelial cells and human uterine epithelial cell lines(13, 15) . HP/HS-binding sites have been described on the surfaces of a number of cell lines(28, 29, 30, 31) ; however, identification of these proteins has been elusive. N-CAM represents one well described cell surface HP/HS-binding protein (32) but is not expressed in the uterus. Recently, heparin-binding epidermal growth factor-like growth factor was identified at mouse implantation sites (33) and is one potential ligand for embryonic HSPGs. Several other candidate proteins have been described that display HP/HS-binding activity (34, 35) but have not been well characterized. In previous studies, we were able to obtain a partial amino-terminal sequence of several tryptic peptides derived from RL95 cell surfaces that retained HP/HS-binding activity. This sequence was used to obtain a full-length cDNA and predicted amino acid sequence of one of these proteins (36) . This protein is referred to as HIP. Inspection of the predicted amino acid sequence of HIP using several protein structure-predicting algorithms indicated regions likely to be antigenic and exposed on the exterior surface of the protein. One of these sequences was chosen for preparation of antibodies, and these antisera have been used in the present study.
The predicted pI of HIP, >10, is consistent with its behavior on
isoelectric focusing gels. Alternatively, HIP may be
post-translationally modified. No consensus sites for glycosylation are
indicated by the predicted sequence; however, other modifications are
possible. Subcellular fractionation studies indicate that HIP is most
highly enriched in the high speed particulate fraction and is
quantitatively depleted from the high speed supernatant, i.e. cytosolic fraction. We have detected various plasma membrane
markers in this fraction including
Na/K
-ATPase and radioiodinated cell
surface components(
); however, rearrangement of peripheral
membrane components like HIP may occur during such fractionation making
interpretation of subcellular locale by this approach problematic. The
ability of NaCl to release HIP from the particulate fraction is
consistent with the lack of a potential membrane spanning domain in the
predicted sequence of HIP and demonstrates that HIP is a peripheral
membrane protein. Digestion of membranes with a mixture of HP/HS lyases
did not release HIP into the 100,000
g soluble
fraction. This suggests that HIP is not retained by membrane-bound
HSPGs. Therefore, it is possible that other membrane components bind
and retain HIP. Alternatively, it is possible that HIP binds to a
region of HS close to the protein core and protects HS from enzymatic
digestion. Characterization of the HIP-binding sites is necessary to
define the nature of the HIP-membrane interaction.
Several lines of
evidence indicate that HIP is displayed on cell surfaces. Antibodies to
this protein bind specifically and in a saturable manner to intact RL95
cells under conditions where endocytosis should not occur. Assuming a
1:1 stoichiometry of IgG binding to HIP and protein A to antibody, it
can be calculated that there is an average of approximately 1.5
10
molecules of HIP displayed on the surface of each RL95
cell. If each IgG binds to two HIP molecules and each protein A
tetramer binds four IgG molecules then this estimate may be as high as
1.2
10
HIP molecules per cell surface. In either
case, these numbers are well below the number of
[
H]HP-binding sites (9
10
)
previously determined for RL95 cells(15) . Consequently, even
given potential inaccuracies in both estimates, it seems that HIP can
only be one of multiple cell surface HP/HS-binding proteins displayed
on RL95 cell surfaces. It is possible that many HIP molecules are
occupied by HS at the cell surface and masked from antibody binding. HS
lyase pretreatment of cells did not expose additional anti-HIP-binding
sites(
); however, if, as discussed above, HIP binding
``protects'' HS chains from digestion then HS lyases might
not be expected to expose more HIP.
Antibodies to HIP also display
staining patterns on intact RL95 cells that are consistent with those
of cell surface components, e.g. enrichment at cell
peripheries and regions of cell-cell contact. Similar patterns of
immunoreactivity with anti-HIP are detected on human trophoblastic and
breast cancer cell lines. ()Furthermore, these same
antibodies specifically aggregate RL95 cells in suspension, a property
expected for antibodies reacting with epitopes displayed on the cell
surface. Experiments with an impermeant chemical cross-linking reagents
destroyed antibody reactivity with HIP, but larger cell associated
bands were not observed.
Thus, while in one sense these
experiments suggest a cell surface disposition of the protein, the
apparent destruction of the epitope confuses interpretation.
Collectively, these data strongly argue that at least a fraction of the
population of HIP is displayed on RL95 cell surfaces where these
proteins may directly participate in HP/HS binding.
HIP is detected in several human uterine epithelial cell lines and in human endometrium by Western blotting of total protein extracts. Moreover, anti-HIP strongly reacts with uterine epithelial cells in sections of human endometrium through post-ovulatory day 7 of the cycle. By post-ovulatory day 13, HIP is also detected in the predecidual cells of the uterine stroma. The HSPG, perlecan, is expressed by human decidual cells(26) . It is possible that HS chains of perlecan also serve as ligands for HIP in basal lamina and in the decidual extracellular matrix. In any event, these observations indicate that HIP is expressed by normal human endometrium. Potential functions could involve binding to basal lamina or intercellular HSPGs expressed by uterine epithelia or HSPGs expressed by blastocysts during implantation. The antibody described in the present studies does not react with mouse uterine components either by immunostaining or Western blotting. Current efforts are being placed toward generating probes to the mouse homologue so that the physiological role of this protein in the uterus can be more rigorously examined by molecular genetic approaches.