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J Biol Chem, Vol. 273, Issue 44, 28878-28888, October 30, 1998
From the Abundant hyaluronan is present between epidermal
keratinocytes. However, virtually nothing is known regarding its
organization in the limited extracellular space between these cells. We
have used metabolic labeling with [3H]glucosamine
and [35S]sulfate and a hyaluronan-specific biotinylated
probe to study the metabolism of hyaluronan and its localization in
monolayer cultures of a rat epidermal keratinocyte cell line.
Hyaluronan (~20 fg/cell) was present on the apical and lateral
surfaces of the cells in two nearly equal pools, either in patches
(~160/cell) or diffusely spread. The hyaluronan in the patches is
bound to CD44 as indicated by co-localization with an antibody to CD44, and by displacement with hyaluronan decasaccharides as well as with an
antibody that blocks hyaluronan binding to CD44. The inability of
hyaluronan oligomers shorter than 10 monosaccharides to displace hyaluronan suggests that CD44 dimerization or cooperative interactions are required for tight binding. The diffuse hyaluronan pool is likely
bound to hyaluronan synthase during its biosynthesis.
Hyaluronan is well known as a constituent of connective tissue
extracellular matrices, but more recent studies have also demonstrated its abundance in stratified squamous epithelia including the epidermis (1-3). In contrast with connective tissue extracellular matrices that
contain mixtures of collagens, fibronectins, other multiadhesive glycoproteins, proteoglycans, and hyaluronan, hyaluronan is the only
known extracellular matrix macromolecule present in high concentration,
~2 mg/ml, in the small extracellular space between adjacent
epithelial cells (keratinocytes) that form the epidermis (4, 5).
Additionally, studies of human skin organ cultures have shown that the
hyaluronan within the epidermis is rapidly turned over (6), an
observation that suggests that the epidermis possesses efficient
mechanisms to catabolize hyaluronan that are closely coordinated with
its synthesis.
Although the coating of keratinocytes by hyaluronan is not generally
appreciated, it is widely known that cell types of mesodermal origin
(7), including fibroblasts (8), chondrocytes (9, 10), and mesothelial
cells (11) display surface coats, often several micrometers in
thickness, visualized indirectly as a domain excluding particles
such as red blood cells. These coats 1) are removed by digestion with
highly specific hyaluronidase, 2) can be stabilized by the
serum-derived protein inter- Extracellular hyaluronan is often anchored to CD44, a ubiquitous,
abundant, and structurally variable plasma membrane receptor that has a
hyaluronan binding domain (17). Smaller amounts of hyaluronan may bind
to RHAMM, a receptor involved in cell motility and cell transformation
through hyaluronan-dependent signaling involving tyrosine
phosphorylation (18). In addition, some cell-surface hyaluronan appears
to remain tethered to hyaluronan synthase, while biosynthesis
continues on the cytoplasmic side of the plasma membrane
(19-21). Variations in CD44 polypeptide sequence (22, 23),
glycosylation (22, 24-27), and oligomerization (28, 29) all influence
its avidity for binding hyaluronan. Still, current understanding of the
mechanisms of hyaluronan binding to CD44 and other receptors on normal
adherent cells, and the role that these interactions have in hyaluronan
organization and catabolism, remains far from complete.
We have developed a rat keratinocyte model for epidermal
differentiation that is suitable to explore these issues. This
epidermal keratinocyte cell line is unique in its ability to reform an
epidermis essentially identical to native tissue in the absence of
feeder cells when cultured at an air-liquid interface (30) and exhibits a nearly complete set of morphological differentiation markers (31,
32). These cells can also be maintained in a continuous growth phase
when regularly trypsinized and cultured as conventional monolayers on
plastic dishes. In this report, the essentially monolayer character of
recently subcultivated rat keratinocytes enabled us to quantitate and
characterize the hyaluronan bound on keratinocyte cell surfaces in ways
that are not technically feasible in complete epidermis covered with an
impermeable stratum corneum. Our results show that about half of the
hyaluronan is bound on the apical and lateral keratinocyte surfaces to
CD44 and about half remains bound, most likely to hyaluronan synthase. Furthermore, hyaluronan oligosaccharides with at least five repeat units (decasaccharides) are required to displace hyaluronan from CD44.
Cell Culture--
A newborn rat epidermal keratinocyte
(REK)1 cell line was
developed by MacCallum and Lillie (30) from neonatal rat epidermal cells originally isolated by Baden and Kubilus (31). REKs were cultured
in Dulbecco's minimum essential medium (low glucose, Life
Technologies, Inc.) with 10% fetal bovine serum (HyClone, Logan, UT)
at 37 °C in a humidified atmosphere containing 5% CO2. Cells were passaged when confluent using 0.05% trypsin (w/v), 0.02%
EDTA (w/v) in phosphate-buffered saline (PBS) (Life Technologies, Inc.). When seeded at 10,000 cells/cm2, the cells grew to
confluency in about 2 days and formed multilayers 2-4 cells thick
after 4-6 additional days of culture. For biochemical assays and
radiolabeling, the cells were grown close to confluency in 6-well
plates and incubated in the presence of 20 and 100 µCi/ml [3H]glucosamine and [35S]sulfate (Amersham
Pharmacia Biotech, Little Chalfont, UK), respectively. For microscopic
studies, the cells were plated in 8-well chamber slides (Lab-Tek and
Nalge, Naperville, IL), 10,000-30,000 cells/well, precoated for 30 min
at 37 °C with fetal bovine serum. For ELISA-like assays of
cell-associated hyaluronan in situ, the cells were grown on
Costar Transwell inserts (polycarbonate, 0.4-µm pore size, 4.5 cm2, 6-well plate, Costar Corp., Cambridge, MA) seeded at
10,000 cells/cm2.
Culture "Compartments" Defined--
Cells were grown in
6-well plates (9.6 cm2/well) in 1-2 ml of medium and
subsequently washed with 2 × 250 µl of Earle's balanced salt
solution (EBSS, Life Technologies, Inc.). The combined medium and
washes were designated as "medium." The cell layer was trypsinized in 0.5 ml of 0.025% trypsin (w/v) and 0.02% EDTA (w/v) (Sigma). After
removing the cells, each plate was washed with 0.25% (w/v) soybean
trypsin inhibitor (Sigma) in 100 µl of EBSS and then with 200 µl of
EBSS. The cell suspension, combined with the washes, was centrifuged,
and the supernatant designated as "trypsinate" or "cell-surface
hyaluronan." The cell pellet was suspended in 200 µl of EBSS and
designated as the "intracellular" hyaluronan fraction.
Purification of Radiolabeled Hyaluronan--
Carrier (4 µg of
hyaluronan in 40 µl, Healon®, Amersham Pharmacia Biotech, Uppsala,
Sweden) and 4 volumes of ethanol saturated with NaCl were added to each
medium, trypsinate, and intracellular sample. After 1 h at
Superdex Chromatography--
Each purified sample was dissolved
in 50 µl of 50 mM sodium acetate, pH 6.7, and digested
for 3 h at 37 °C with 25 milliunits of chondroitinase ABC and 1 milliunit of Streptococcus hyaluronidase (both from
Seikagaku Kogyo Co., Tokyo, Japan), and 10-45 µl were injected onto
a 1 × 30 cm Superdex Peptide column (Pharmacia) and eluted at 0.5 ml/min with 0.1 M NH4HCO3. The
eluent was monitored at 232 nm, and aliquots of the 250-µl fractions
were counted for 3H and 35S. Undigested
glycosaminoglycans, consisting mainly of heparan sulfate, eluted near
the void volume, whereas disulfated, monosulfated, and non-sulfated
disaccharides were eluted in this order as separate peaks before the
total volume of the column. The carrier hyaluronan produced a
disaccharide peak at 232 nm which was used to monitor the recovery
(about 80%) and correct for any losses in purification.
Ion Exchange Chromatography--
The radiolabeled disaccharide
peaks from Superdex chromatography were dried, redissolved in water,
and their identities confirmed by ion exchange chromatography. The
non-sulfated disaccharides and monosulfated chondroitin disaccharides
were fractionated on 4 × 50-mm Carbopak MA1 and PA1 columns
(Dionex, Sunnyvale, CA) eluted isocratically with 20 and 500 mM sodium phosphate, pH 7.0, respectively. The disulfated
disaccharides were chromatographed on a 0.4-1.0 M LiCl
gradient on the PA1 column. More than 85% of the radioactivity in the
dried peaks coeluted with standards (Seikagaku) in the expected
positions.
Ion exchange chromatography on Carbopak MA1 showed that digests of
samples from these keratinocytes did not contain Chemical Quantitation of Hyaluronan with Double
Labeling--
Incorporation of label from
35SO4 provides a measure of the chemical
quantity of the chondroitin sulfate synthesized during the labeling
period. The [3H]glucosamine incorporated into the same
disaccharides provides an estimate of the effective specific activity
of the N-acetylglucosamine precursor pool and hence the
chemical content of newly synthesized hyaluronan, as described in
detail previously (33). The accuracy of hyaluronan content calculated
from the double labeling technique was confirmed as follows: 1) by
pooling media and trypsinates for parallel, ELISA-like,
hyaluronan-competitive binding assays (5), and 2) by determination of
unlabeled disaccharides in chondroitinase and hyaluronidase digests
without added carrier hyaluronan, at 232 nm on the Carbopak MA1 column
as described above.
Preparation of Hyaluronan Oligosaccharides--
Commercial
hyaluronan from human umbilical cord (Sigma) was dissolved in 0.1 M sodium acetate, 0.1 M NaCl, pH 6.0. Bovine testicular hyaluronidase (~6000 units/mg, Calbiochem) was incubated with the hyaluronan at 37 °C until the size distribution peaked at
10-20 monosaccharide units, as determined by elution on an analytical
Superdex 75 gel filtration column (Pharmacia). Preparative fractionation of the oligosaccharides was done on a 5 × 95-cm column of Superdex 30 (Pharmacia), eluted with 0.1 M
NH4HCO3. The fractions corresponding to the
peak of each oligosaccharide were pooled, lyophilized twice, dissolved
in the appropriate medium, and filtered sterile. Ion exchange high
pressure liquid chromatography of aliquots of the purified
oligosaccharide peaks on a 9 × 250-mm Propak PA1 column (Dionex),
eluted with an NH4HCO3 gradient, showed 3%
cross-contamination of the HA6 with HA8 but no
detectable HA10, and 9 and 7% cross-contaminations of the
HA10 with HA8 and HA12, respectively. HA8 had a 3% cross-contamination from both
HA6 and HA10.
The Biotinylated Hyaluronan Probe--
The hyaluronan-specific
probe, biotinylated hyaluronan binding complex (bHABC), was purified
from a 4 M guanidine HCl extract of bovine articular
cartilage, dialyzed, and digested with trypsin, as described previously
(34). The ternary complex with the G1 domain of aggrecan,
the link protein, and hyaluronan was purified using hydroxyapatite
chromatography and gel filtration on Sephacryl S-1000 (Pharmacia),
followed by biotinylation. Hyaluronan and excess biotin were then
removed from the complex by dissociative gel filtration on Sephacryl
S-400 (Pharmacia) (34). In SDS-polyacrylamide gel electrophoresis
analysis, the probe exhibited biotinylated G1 domain of
aggrecan and biotinylated link protein as the only silver-stained and
biotinylated proteins.
Assay of Cell Associated (in Situ) Hyaluronan--
Keratinocytes
were seeded at 10,000 cells/cm2 and grown to confluence on
Costar Transwell® polycarbonate 0.4-µm pore membrane inserts.
Standardized 0.55-cm2 discs were punched out of the
membrane. The cell layers on the discs were fixed and incubated with
bHABC as described below for histological staining. After washing, the
discs were incubated for 30 min at 37 °C with 100 µl of
streptavidin peroxidase (Vector, Inc, Burlingame, CA), 1:10,000
dilution in 150 mM NaCl and 20 mM Tris acetate,
pH 8.2, washed 3 × for 5 min with the phosphate buffer, and 100 µl of 50 mM phosphate/citrate buffer, pH 5.0 (Sigma), containing 2 mg/ml o-phenylenediamine (Sigma) and 0.025%
H2O2 (v/v), was added. After 30 min at
37 °C, aliquots from the incubation solutions were read at 405 nm.
The linearity of the assay was confirmed by a concentration series of
hyaluronan (0.01-100 ng/100 µl) cross-linked with
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl, (Sigma) onto
Covalink® plates (Nunc, Roskilde, Denmark). Background absorbance
(15-20% of total), obtained using the bHABC probe, pretreated with
hyaluronan oligosaccharides, was subtracted. Streptomyces
hyaluronidase digestion removed 95-100% of the specific absorbance,
i.e. produced the same values as the oligosaccharide-treated probe, indicating the specificity of the reaction.
The standards chondroitin, chondroitin 4-sulfate (chondroitin sulfate
A), chondroitin 6-sulfate (chondroitin sulfate C), and heparan sulfate
were purchased from Seikagaku.
Localization of Hyaluronan--
Each cell layer to be analyzed
was washed with Dulbecco's PBS (D-PBS) (Life Technologies,
Inc.) and fixed at room temperature for 20 min either in 2%
paraformaldehyde (v/v) and 0.5% glutaraldehyde (v/v) in
D-PBS for electron microscopy and light microscopy with peroxidase detection or in paraformaldehyde alone for fluorescence microscopy. After fixation, the cells were washed 3 times for 2 min
each with D-PBS and then blocked in 1% BSA (w/v)
containing 0.3% Triton X-100 (v/v) and 50 mM glycine in
D-PBS, for 30 min at 37 °C.
For light microscopy, the bHABC probe, diluted to 2-10 µg/ml in 3%
BSA (w/v), was added to the fixed cells, and incubated overnight at
4 °C. The slides were then washed and treated with avidin-biotin
peroxidase (ABC standard kit, Vector) for 1 h. The color was
developed using DAB and H2O2 (kit from Vector)
according to the manufacturer's instructions. Counterstaining was done
with hematoxylin for 2 min before mounting either in Chrystalmount® or
DPX® (Biomeda, Foster City, CA).
For electron microscopy, fixation, incubation with bHABC, blocking, and
washes were done as for light microscopy. The samples were then
incubated with streptavidin/gold (LM grade, Amersham Corp.) 1:40
dilution for 1 h. After washing, the samples were postfixed in 1%
glutaraldehyde (v/v) for 20 min and washed 5 times for 1 min with
distilled water. Silver enhancement was done according to the
manufacturer's instructions (IntenSE M, Amersham Corp.). The cells
were dehydrated in graded ethanol and embedded in Spurr's resin. Thin
sections were cut, placed onto Formvar-coated copper grids, stained
with uranyl acetate, and viewed with a JEOL 1200 EX microscope.
The specificity of the staining was controlled by pre-digesting the
fixed cultures with Streptomyces hyaluronidase (100 turbidity reducing units/ml, 50 mM sodium acetate buffer,
pH 5.0, 3 h at 37 °C) in the presence of protease inhibitors
(4) or by preincubating the bHABC probe with HA oligosaccharides
(length ~20 monosaccharides, 3 µg/1 µg bHABC) to reveal possible
nonspecific binding of the probe.
Image Analysis of Cell Associated Hyaluronan--
The cells were
grown on 8-well chamber slides and stained for hyaluronan with DAB as a
chromogen, as described above, but without hematoxylin counterstaining.
A Leitz BK II microscope with 16 ×/0.45 numerical aperture objective
(Leitz, Wetzlar, Germany) was connected with a 12-bit digital camera
(Photometrics CH 200, Tucson, AZ) equipped with a KAF 1400 CCD detector
(Eastman Kodac Co.). The optimum wavelength for DAB was found at 543 nm
using an interference filter (Schott, Wiesbaden, Germany). Camera
control and image analysis were done with IPLab software (Signal
Analytics, Vienna, VA). The settings of the microscope and camera were
kept constant during the study. Spatial resolution of the system was 1.43 µm/pixel. During system calibration and measuring, each image of
interest was corrected using a flat fielding algorithm. The imaging
system was calibrated with neutral density filters (Schott) in the
range of 0 to 3 OD to convert the pixel density values into the linear,
standardized scale of OD. Multiple fields (10-20) (731 × 841 µm) were systematically sampled from each well. Area-integrated mean
OD values, including both DAB-positive and background intensities, were
calculated for each whole digitized area, excluding possible artifact
areas. In addition, DAB-positive staining areas were estimated from
binary images with a cut-off at an OD value of 0.13. Based on the
positive area data and the sum of the pixels that fulfilled the
positivity criteria, the mean area-integrated OD values for the
DAB-positive material were calculated accordingly, and those data were
used for the presentation of the results.
Localization of CD44--
The cells were fixed and blocked with
BSA as described above and then incubated overnight at 4 °C with the
OX 50 antibody (Biosource Inc., Camarillo, CA) at 1:100 dilution in 3%
BSA. After washing, the cells were incubated for 1 h with
biotinylated anti-mouse secondary antibody (Vector) diluted to 1:100 in
1% BSA. After washing, they were treated with avidin-biotin
peroxidase, developed with DAB, counterstained with hematoxylin, and
mounted in Chrystalmount® or DPX®, as described above for bHABC.
Negative controls included isotype-specific control serum (Sigma) and
omission of the primary antibody.
For double staining of hyaluronan and CD44, the cells were fixed in 2%
paraformaldehyde, blocked as described above, and then incubated
overnight at 4 °C with the OX 50 antibody (1:100 dilution) and bHABC
(3 µg/ml) in 3% BSA. After washing, the cells were incubated for
1 h with fluorescein isothiocyanate-labeled anti-mouse secondary antibody (Boehringer GMBH, Mannheim, Germany, 1:50) and Texas Red-labeled streptavidin (Vector) 1:1000 in 1% BSA, washed, and mounted in Vectashield (Vector).
Modification of Hyaluronan Binding with CD44
Antibodies--
Recently confluent or near-confluent cultures of REKs
were incubated in the presence of monoclonal antibodies against CD44: 1M7 (Zymed Laboratories Inc., San Francisco, CA, and a
gift from Dr. W. Knudson, Chicago), Ox 50 (Biosource Int., Camarillo,
CA), and Hermes 3 (a gift from Dr. Sirpa Jalkanen, Turku) and with non-immune IgG (Sigma) before staining for hyaluronan as described above.
Steady State Content of [3H]Hyaluronan on Cell
Surface--
Recently confluent cultures of rat keratinocytes were
labeled for times between 6 and 24 h with
[3H]glucosamine and [35S]sulfate. The
3H-labeled hyaluronan was determined for medium, cell
surface (trypsinate), and intracellular compartments, and the values
were used to determine the mass of [3H]hyaluronan with
the double label method. As shown in Fig.
1, the content of newly synthesized
hyaluronan in the trypsinate (i.e. on the cell surface)
reached a plateau value by ~18 h. The [3H]hyaluronan in
the medium continued to increase linearly, after an initial lag, during
24 h indicating continued synthesis of hyaluronan throughout. The
intracellular [3H]hyaluronan (i.e. the cell
pellet after trypsin digestion) remained constant and low (~4% of
the total at 24 h). The kinetics to steady state content of
[3H]hyaluronan in the trypsinates indicates that the
half-life of a newly synthesized hyaluronan molecule on the cell
surface is only ~8 h and the equilibrium content is ~200
pg/104 cells (or ~20 fg/cell). The loss of
[3H]hyaluronan from the cell surface reflects loss to the
medium and some internalization for catabolism.
Cell-surface Hyaluronan Is Partially Displaced by Hyaluronan
Decasaccharides--
Confluent cultures of keratinocytes were
incubated at 37 °C for 2 h with hyaluronan oligosaccharides of
different sizes, and the displacement of cell-surface hyaluronan was
determined using the in situ hyaluronan assay described
under "Experimental Procedures." Oligosaccharides HA10
(Fig. 2a) or larger (not
shown), when used at 0.3-1.0 mg/ml, reduced cell layer-associated
hyaluronan in this ELISA assay to 45-56% of controls. In contrast,
HA6 had no effect (Fig. 2a). Related
glycosaminoglycans, such as chondroitin and chondroitin 6-sulfate, did
not displace hyaluronan even at high concentrations (Fig.
2a). The oligosaccharide HA8 and heparan sulfate
were also without effect (data not shown).
A nearly identical displacement of cell-surface
[3H]hyaluronan (to 53-55% of controls) was found in
cultures labeled for 24 h with [3H]glucosamine in
the presence of HA14 oligosaccharides (Fig. 2b). Again, HA6 oligosaccharides had no effect. This confirmed
that HA6 was unable to displace endogenous hyaluronan and
that 3H-labeled cell-surface hyaluronan had equilibrated
with the total content of hyaluronan on cell surfaces. Increasing
oligosaccharide size from 10 to 18 monosaccharides did not
significantly increase the release of cell-surface hyaluronan (data not
shown). Hyaluronan molecules on the surfaces of the keratinocytes,
therefore, are present in two different pools, one with the
characteristics of a specific receptor (displaced by
HA Hyaluronan Is Present in the Form of Patches on the
Keratinocyte--
Localization of hyaluronan with the biotinylated
probe (bHABC) in confluent cultures revealed dense patches decorating
the cell-cell contact areas, as well as spots and linear deposits on
the apical surfaces (Fig. 3a).
In contrast, the underside of the keratinocytes, facing the plastic or
glass surface (position determined by the focal plane), was almost
always devoid of hyaluronan. Digestion with either
Streptomyces hyaluronidase (Fig. 3b) or trypsin
(Fig. 3c) completely removed the cell-surface signal. An
intracellular, granular hyaluronan signal was present in some of the
cells (Fig. 3b). The ability of trypsin to release all cell-surface hyaluronan was also confirmed by the absence of any hyaluronan degradation products in the hyaluronidase containing incubation medium of cultures that had been first treated with trypsin
(data not shown).
The cell-surface patches were rather uniform in size ranging from 0.8 to 2.4 µm (1.1 ± 0.03 µm, mean ± S.E.). The estimated number of the patches per keratinocyte was 160 ± 20 (mean ± S.E., 6 cultures).
All experiments included control cultures probed with bHABC
preincubated with hyaluronan oligosaccharides (HA~20),
which combine with the specific binding sites in the probe and prevent its binding to hyaluronan. No signal was detected in these controls, indicating the specificity of the reaction (Fig. 3d).
The Hyaluronan Patches Are Displaced by Hyaluronan
Decasaccharides--
Since hyaluronan in the trypsinate was present in
two different pools, one displaced by HA
The hyaluronan not displaced appeared evenly distributed on the plasma
membrane and gave a signal of very low contrast (Fig. 4, f
and g), unlike the patchy pattern in controls (Figs.
3a and 4a). Interestingly, a stronger signal was
observed in plasma membrane areas with slender cytoplasmic extensions
(Fig. 4g), possible loci of hyaluronan synthase.
Ultrastructural Localization of Hyaluronan in
Keratinocytes--
Gold-labeled streptavidin (1-nm gold particles) was
applied on fixed and permeabilized cultures previously incubated with bHABC, and the signal was subsequently enhanced with silver (Fig. 3e). Controls using the bHABC probe presaturated with
hyaluronan oligosaccharides (HA~20) showed very sparse
labeling and no specific localization (Fig. 3f), similar to
cultures digested with Streptomyces hyaluronidase prior to
the staining (data not shown). The silver grains formed clusters in the
extracellular pouches between adjacent keratinocytes, corresponding to
the patches observed in light microscopy. The label was often
associated with the microvilli (Fig. 3e) and most
conspicuous around the cytoplasmic extensions between neighboring cells
in cells treated with HA Reappearance of the Hyaluronan Patches after Hyaluronidase
Treatment--
Confluent keratinocyte cultures were treated with
Streptomyces hyaluronidase to remove cell-surface hyaluronan
specifically. The cultures were then washed and chased for 2-8 h.
After a 2-h chase, new hyaluronan was present on cell surfaces, with a
distribution resembling that in untreated cultures, but with smaller
and less intensely stained patches (Fig.
5a). With increasing chase
time, the patches increased in size and signal intensity (shown at
8 h chase, Fig. 5b). Hyaluronan distribution was not
affected by 8 h chase in the absence of serum (Fig.
5c), suggesting that inter- Co-localization of CD44 and Hyaluronan--
An antibody specific
for CD44 (OX50) was used with a biotinylated secondary antibody to
localize CD44 in a confluent culture (Fig.
6a). The CD44 signal resembled
the staining pattern of hyaluronan, being more intense at cell-cell
contact areas and forming small patches on the apical cell surfaces.
Cultures probed with control antiserum or without the primary antibody
were negative (data not shown). Double staining with bHABC and
anti-CD44 using red and green fluorochromes, respectively, showed
colocalization on the apical cell surfaces (Fig. 6, b and
c). However, images focused at a lower level, corresponding
to lateral plasma membrane domains (Fig. 6, d-f) showed
areas where CD44 was present, but hyaluronan was absent. This occurred
particularly in subconfluent cultures at the leading edges of the
growing keratinocyte colonies (Figs. 6, d-f).
An Antibody to CD44 That Blocks the Hyaluronan Binding Reduces
Cell-surface Hyaluronan and Inhibits Patch Formation--
Nearly
confluent cultures were incubated for 8 h with IM7 antibody, which
partially inhibits binding of hyaluronan to CD44 in rat chondrocytes
(10, 35), as well as inducing the shedding of CD44 in mice (36). A
reduction in cell-surface hyaluronan staining was observed at 8 h
(Fig. 7b) compared with
untreated control (Fig. 7a). Control cultures incubated in
the presence of non-immune IgG (not shown), or the antibody Hermes 3 specific for human, but not rat CD44 (Fig. 7f), showed an
intense signal for hyaluronan, indistinguishable from untreated
cultures (Fig. 7a). In cultures treated for 8 h with
increasing concentrations of IM7, image analyses of the hyaluronan
staining showed a dose-dependent decrease in hyaluronan
signal, with a maximum reduction of optical density ~30% at 120 µg/ml (Fig. 8).
The CD44 Antibody OX50 Increases Binding with HA10
Specificity--
Nearly confluent REK cultures were incubated with
OX50 anti-CD44 antibody for different times and then stained with bHABC (6 h cultures are shown in Fig. 7, c-e). The amount of
endogenous hyaluronan on keratinocyte surfaces was considerably
increased by incubation with this monoclonal antibody, as compared with non-treated (7a) or Hermes 3-treated cultures (Fig.
7f). Other CD44 antibodies that enhance hyaluronan binding
have been described previously (28, 37). Image analyses indicated that
the enhanced binding was detectable 1 h after adding the antibody
and reached its full effect by 4 h (Fig.
9a).
We tested the specificity of this effect by treating cultures with OX50
in the presence of HA6 and HA10 for 6 h.
As shown in Figs. 7d and 9b, HA10
reduced keratinocyte-surface hyaluronan to approximately half of that
in control cultures. In contrast, OX50 treated cultures showed
hyaluronan levels higher than controls in the presence of
HA6 (Figs. 7e and 9b). These results
indicate that enhanced hyaluronan binding to CD44 induced by OX50
exhibits specificity for HA Two Pools of Hyaluronan on Keratinocyte Surface--
The steady
state content of hyaluronan, bound primarily on the apical and lateral
cell surfaces of the rat keratinocytes in nearly confluent monolayer
cultures, was determined to be ~20 fg per cell by independent
chemical and double radiolabeling assays. Of this, ~50% is bound to
the cell-surface receptor, CD44, as indicated by the following:
(a) colocalization of hyaluronan and the anti-CD44 OX50
antibody, and (b) the ability of anti-CD44 IM7 antibody,
which inhibits hyaluronan binding to CD44, to displace hyaluronan from
the "patches." The amount, ~10 fg per cell, bound to CD44
corresponds to 900-3000 hyaluronan molecules if they average 2-7
million Da in molecular mass (19, 38). Thus, each of the ~160 patches
observed per cell would contain 6-19 molecules of bound hyaluronan.
The remaining molecules, ~50% of the total, are not bound to CD44 as
indicated by the inability of HA
This residual hyaluronan could be bound by one or more of several
established alternative mechanisms. Hyaluronan may remain bound on
hyaluronan synthase (20, 39, 40), bound to matrix components like type
VI collagen (41, 42) and aggregating proteoglycans (43-45). However,
type VI collagen and aggrecan are not expressed in epidermis, and no
proteoglycans with the properties of versican were detected in our rat
epidermal keratinocytes when labeled with [3H]glucosamine
and
35SO4.2
Therefore, association with hyaluronan synthase remains the most likely
tether of the hyaluronan that is not bound to CD44. The fact that the
endogenous hyaluronan in this compartment is not accessible to
extracellular HA Hyaluronan Receptors on Keratinocytes--
The contribution of
RHAMM to the immobilization of endogenous hyaluronan has not been
established in quantitative terms but is probably minor and transient
(46). Our unpublished findings indicate that a RHAMM antibody known to
block hyaluronan binding does not influence the signal intensity of the
keratinocyte hyaluronan patches. The inability of hexasaccharides to
displace the hyaluronan patches should rule out the involvement of the
novel hyaluronan receptor Cdc37, identified by the antibody IVd4, from
which hyaluronan can be displaced by HA6 (47, 48).
In addition to localizing CD44 on rat keratinocytes and blocking HA
binding with anti-CD44 antibody IM7 in this study, further evidence for
CD44 as the main hyaluronan receptor on keratinocytes comes from the
fact that epican, the major CD44 variant on epidermal keratinocytes
(with variant exons v3-v10), binds hyaluronan and that this binding is
blocked by an antibody to CD44/epican (49). Moreover, a transgenic
mouse with selective suppression of CD44 gene expression in epidermis,
shows a 75% reduction in endogenous hyaluronan on the surface of
cultured keratinocytes derived from this mouse (50). Collectively,
these data indicate that CD44 is the major receptor for hyaluronan in
epidermal keratinocytes.
Molecular Basis of the HA10 Specificity of Epidermal
CD44--
Our observation that HA
However, high affinity binding of hyaluronan to CD44 appears to require
an amino acid (Lys-68) on the outer edge (52) and an established
hyaluronan-binding peptide (amino acids 150-162) completely outside of
the link module (56-58). Thus, a larger binding site with greater
affinity for hyaluronan than the current one requiring HA6
may exist. One possibility is that CD44 molecules on keratinocytes can
achieve close enough proximity such that the HA binding domains of two
adjacent molecules form a cooperative site effectively converting them
to a functional site with two link modules equivalent to that in the
link protein, which requires HA Regulation of CD44 Affinity for Hyaluronan--
The increased
hyaluronan binding induced by OX50 antibody observed in our study
suggests that CD44 molecules that weakly bind hyaluronan can be made
more efficient. Our results make it unlikely that highly mobile CD44,
free to dimerize or change conformation, binds additional hyaluronan
from the medium compartment. Such binding would be inconsistent with
the gradual increase of CD44-bound hyaluronan observed following the
addition of the OX50 antibody, an observation more consistent with the
possibility that newly synthesized hyaluronan, or CD44, or both are
required for the increased hyaluronan present on the cell surface.
Hyaluronan is thus present on the surface of rat epidermal
keratinocytes in two different forms as follows: one that can be displaced by hyaluronan oligomers HA We thank Dr. Mikko Lammi for helpful
suggestions on the manuscript; Päivi Perttula for technical help
in the chromatographic assays of hyaluronan; and Eija Rahunen and Arja
Venäläinen for preparing the light and electron microscopic
specimens.
*
This work was supported by Biotechnology Project Funds from
the University of Kuopio and by the Cleveland Clinic Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Anatomy,
University of Kuopio, P. O. 1627, FIN-70211 Kuopio, Finland. Tel.:
358-17-163009; Fax: 358-17-163032; E-mail: rtammi{at}messi.uku.fi.
The abbreviations used are:
REK, rat epidermal
keratinocyte; EBSS, Earle's balanced salt solution; bHABC, biotinylated hyaluronan binding complex; ELISA, enzyme-linked
immunosorbent assay; BSA, bovine serum albumin; PBS, phosphate-buffered
saline; D-PBS, Dulbecco's phosphate-buffered saline; DAB, 3,3'-diaminobenzidine; HA, hyaluronan.
2
R. Tammi, J.-P. Pienimäki, and M. Tammi,
unpublished data.
Hyaluronan Bound to CD44 on Keratinocytes Is Displaced by
Hyaluronan Decasaccharides and Not Hexasaccharides*
§¶,
,,, and§
Department of Anatomy, University of Kuopio,
FIN-70211 Kuopio, Finland, the § Department of Biomedical
Engineering, Connective Tissue Biology Section, Cleveland Clinic
Research Institute, Cleveland, Ohio 44195, and the Department
of Anatomy and Cell Biology, University of Michigan,
Ann Arbor, Michigan 48109
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-trypsin inhibitor which interacts with
hyaluronan (12-14), and 3) can be increased in size and reinforced by
proteoglycans that bind specifically to hyaluronan (15, 16).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
20 °C, precipitates were centrifuged for 15 min at 2500 × g. Each precipitate was suspended in 200 µl of 50 mM sodium acetate, pH 5.8, containing 5 mM
cysteine HCl and 5 mM EDTA to which 50 µg of papain
(Sigma) in 50 µl of water was added followed by incubation at
60 °C for 4 h. The samples were heated at 100 °C for 10 min,
centrifuged at 13,000 × g for 15 min, and supernatants
containing hyaluronan and other glycosaminoglycans recovered.
Cetylpyridinium chloride (1% in 20 mM NaCl, 4 volumes) was
added to each supernatant followed by incubation for 1 h at room
temperature. After centrifugation at 13,000 × g for 15 min, each supernatant was carefully removed by aspiration. Each
precipitate was washed with 1 ml of H2O, centrifuged, and
the supernatant discarded as above. Each cetylpyridinium chloride
precipitate was dissolved in 50 µl of 4 M guanidine HCl,
and 900 µl of ethanol, saturated with NaCl, was added. After 1 h
at 20 °C, each sample was centrifuged, and the supernatant removed
as described above.
Di-0S, the
nonsulfated disaccharide from chondroitin sulfate. Hence the nonsulfated peak in the Superdex eluents contained only Di-HA and
indicated the content of hyaluronan.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (22K):
[in a new window]
Fig. 1.
Accumulation of [3H]hyaluronan
in different compartments during 24 h labeling with
[3H]glucosamine. Confluent cultures of keratinocytes
were incubated in medium with [3H]glucosamine and
[35S]sulfate for times between 6 and 24 h. The
hyaluronan content, calculated from the double label method, was
determined in the medium, trypsinate, and intracellular compartments as
described under "Experimental Procedures." The content of newly
synthesized hyaluronan at the end of the indicated labeling periods is
shown. The bars show the range of duplicate cultures.
View larger version (17K):
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Fig. 2.
Hyaluronan displacement from the cell surface
of keratinocytes by hyaluronan oligosaccharides. a,
confluent keratinocyte cultures were incubated for 2 h with
purified hyaluronan hexasaccharides (HA6), decasaccharides
(HA10), chondroitin 6-sulfate (C6S), chondroitin
(C), and the hyaluronan content in cell layers were
determined with the in situ ELISA-like assay described under
"Experimental Procedures." b, cultures were labeled for
24 h with [3H]glucosamine in the presence of
HA6 or HA14 oligosaccharides.
[3H]Hyaluronan content in the trypsinates were determined
as described under "Experimental Procedures" and are expressed as a
percentage of the control cultures. The bars show the range
of duplicate cultures.
10-oligosaccharides, but not with other
glycosaminoglycans), and one not displaced and likely still attached to
hyaluronan synthase.
View larger version (138K):
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Fig. 3.
Localization of hyaluronan on
keratinocytes. Confluent REK cultures were fixed and stained for
hyaluronan using the bHABC probe (a, b, c, and e)
or bHABC preincubated with hyaluronan oligosaccharides
HA~20 (d and f). The
bHABC was visualized using the ABC-peroxidase technique with DAB
chromogen (a, b, c, and d) or with gold-labeled
streptavidin, intensified with silver (e and f).
The untreated cultures (a, d, e, and f) were
fixed after a brief wash with the buffer, whereas b was
incubated for 45 min with Streptomyces hyaluronidase and
c for 5 min with trypsin at room temperature, prior to the
fixation. Magnification bar in d represents 10 µm for a, b, c, and d and 1 µm for
e and f. See text for descriptions.
10
oligosaccharides, and the other not (Fig. 2), we tested whether the
patches belonged to one or both of these pools. Cultures incubated for
2-4 h at 37 °C with 1 mg/ml HA4 or HA6 (not
shown) appeared identical to controls (Fig.
4a). The culture containing
HA8 showed only a slight reduction in the hyaluronan signal
(Fig. 4c), while that with HA10 resulted in a
complete loss of the patches (Fig. 4, f and g).
Cultures incubated with chondroitin 4-sulfate and heparan sulfate did
not differ from controls (Fig. 4, d and e), and
only a slight reduction was observed with chondroitin (Fig.
4b). These findings are consistent with those in the
biochemical assays (Fig. 2) and indicate that the patches represent
only the pool displaced by HA10 oligosaccharides.
View larger version (152K):
[in a new window]
Fig. 4.
Effects of hyaluronan oligosaccharides and
other glycosaminoglycans on displacement of hyaluronan from
keratinocyte cell surfaces. Confluent REK cultures were fixed and
stained for hyaluronan using the bHABC and ABC-peroxidase method
(a-g) or the gold-labeled streptavidin and silver
enhancement method (h). A control culture fixed after a
brief wash with the buffer is presented in a, whereas those
in b-h were incubated for 2 h in the presence of 1 mg/ml of the following substances: chondroitin (b),
HA8 (c), chondroitin 4-sulfate
(d), heparan sulfate (e), and
HA10 (f, g, and h).
Magnification bar in f represents 10 µm for
a-f and 1 µm for h. The bar in
g represents 10 µm. See text for descriptions.
10 oligosaccharides (Fig. 4,
g and h). The oligosaccharide-resistant signal
was very close to plasma membranes, as if hyaluronan was tightly
associated with the cell surface (Fig. 4h).
-trypsin inhibitor is not
required (12-14). The presence of HA6 did not prevent the
formation of new patches during the chase (Fig. 5d), whereas
HA10 completely inhibited their reappearance (Fig.
5e).
View larger version (133K):
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Fig. 5.
Reappearance of hyaluronan after
hyaluronidase digestion. Confluent REK cultures were treated with
Streptomyces hyaluronidase for 45 min and chased for 2 h (a), and 8 h (b, c, d, and e). Fixed
cultures were then stained for hyaluronan using bHABC and the
ABC-peroxidase technique. Whereas the other cultures contained 10%
serum during the chase, c was chased in serum-free medium.
d 1 mg/ml of HA6 and in e 1 mg/ml
HA10 was added when starting the chase. Magnification
bar in d represents 10 µm. See text for
descriptions.
View larger version (81K):
[in a new window]
Fig. 6.
Localization of CD44 on keratinocytes.
a, confluent REK cultures were fixed and stained with an
antibody to CD44, followed by a biotinylated secondary antibody. The
ABC-peroxidase-DAB technique was used for detection. b-f, a
double staining procedure using a monoclonal CD44 antibody with
fluorescein isothiocyanate-labeled secondary antibody and bHABC with
Texas Red-labeled streptavidin are shown. b and
c, the image was focused on the apical cell surface, on top
of the nucleus with the same locations on cells indicated by the
arrows. d-f, the image was focused at the level
of lateral cell-cell contacts. The arrows in d-f
indicate the same sites in all three micrographs on cells at the edge
of a growing keratinocyte colony, with a signal from CD44 but not
hyaluronan. f, both CD44 and hyaluronan are shown in a
double exposure. Magnification bar in a
represents 5 µm. See text for descriptions.
View larger version (160K):
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Fig. 7.
Effect of the anti-CD44 antibodies IM7 and
OX50 on hyaluronan distribution on keratinocytes. Nearly confluent
REK cultures were incubated without additions (a), with IM7
(60 µg/ml) for 8 h (b), with OX50 for 6 h
(c-e) or with Hermes 3 for 6 h (f), prior
to fixation and bHABC staining. d and e,
hyaluronan oligosaccharides, HA10 and HA6,
respectively, were added (at 1 mg/ml) for the last 2 h of the OX50
treatment. Magnification bar in f represents 10 µm. See text for descriptions.
View larger version (15K):
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Fig. 8.
Concentration-dependent decrease
of hyaluronan on keratinocytes after incubation with IM7 antibody.
Confluent cultures in chamber slides were incubated in the presence of
0-125 µg/ml of the antibody for 8 h, stained with bHABC,
ABC-peroxidase, and DAB for hyaluronan (without hematoxylin
counterstaining), and assayed with microscopic image analysis. External
calibration was used to convert pixel gray values into the linear scale
of optical density, as described under "Experimental Procedures."
The bars show the range of two cultures. See text for
descriptions.
View larger version (33K):
[in a new window]
Fig. 9.
Time-dependent increase of
hyaluronan on keratinocytes incubated in the presence of the OX50
antibody. a, nearly confluent cultures were incubated
with OX50 (1:10 dilution) for 0-6 h, stained for hyaluronan with
bHABC, and subjected to image analysis. External calibration was used
to convert pixel gray values into the linear scale of optical density,
as described under "Experimental Procedures." b shows
the optical density in untreated cultures (Contr.), and in
cultures incubated for 6 h with OX50 either alone (OX),
or with HA10 (OX+HA10) or
HA6 (OX+HA6) for the last 2 h.
The bars show the range of two cultures. See text for
descriptions.
10.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
10 to displace them and
by a distinctly different distribution (diffuse pericellular location)
from the clusters or patches of hyaluronan that can be displaced by
these same oligosaccharides.
10 oligosaccharides fits well with the
current model for the hyaluronan synthase activity which includes an
intracellular catalytic (binding) domain and a narrow pore for the
extrusion of the growing chain into the extracellular space (21).
However, if this model is true, the release of all hyaluronan with
trypsin suggests that a protein exposed on the cell surface, perhaps a
part of synthase itself, is required to hold the growing polysaccharide
chain.
10 oligosaccharides are
required to displace hyaluronan from CD44 on keratinocytes is
particularly striking. CD44 belongs to the class of hyaladherins with a
domain called the "link module," recently modeled in three
dimensions from the TSG-6 member of the family (51) and adapted to CD44 (52). The proposed hyaluronan site on the link module of TSG-6 is
unlikely to require a length greater than HA6 (51). In
fact, many cells in which CD44 functions to bind a pericellular coat of
hyaluronan exhibit specificity for HA6 since they are
displaced from the cell surface by hexasaccharides (16, 53), whereas proteins such as link protein and the G1 domain of
aggrecan with two link modules in tandem (54, 55) show specificity for
HA10 (45).
10 for binding.
Dimerization has, in fact, been implicated in enhanced binding of
hyaluronan to CD44, which can occur naturally with rat CD44v4-v7 (29),
or can be induced experimentally with certain divalent antibodies to
CD44 (28), or by constructing CD44 with cysteine in the intramembranous
domain that forms disulfide cross-links (59). However, coordinate
action of CD44 with another cell-surface molecule cannot be ruled out
(60).
10 that is bound to
CD44 and the other that can be removed by subsequent hyaluronidase or
trypsin treatment and probably is tethered to hyaluronan synthase. The
hyaluronan bound to CD44 can be increased in amount by the use of a
monoclonal antibody that probably cross-links or clusters the CD44, as
has been shown by other CD44 antibodies and other cells. Having gained
some insight into the amount and organization of hyaluronan in these
epidermal keratinocytes, we now are studying the biosynthesis and
organization of hyaluronan in the completely reformed epidermis that
these cells make when cultured at an air-liquid interphase.
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
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Abstract
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Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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