From the Department of Anatomy, University of Kuopio, FIN-70211
Kuopio, Finland and the Department of Biomedical
Engineering, Connective Tissue Biology Section, Lerner Research
Institute, Cleveland, Ohio 44195
Received for publication, August 21, 2000, and in revised form, February 26, 2001
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ABSTRACT |
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Hyaluronan is an abundant and rapidly
turned over matrix molecule between the vital cell layers of the
epidermis. In this study, epidermal growth factor (EGF) induced
a coat of hyaluronan and a 3-5-fold increase in its rate of synthesis
in a rat epidermal keratinocyte cell line that has retained its ability
for differentiation. EGF also increased hyaluronan in perinuclear
vesicles, suggesting concurrent enhancement in its endocytosis.
Cell-associated hyaluronan was most abundant in elongated cells that
were stimulated to migrate by EGF, as determined in vitro
in a wound healing assay. Large fluctuations in the pool size of
UDP-N-acetylglucosamine, the metabolic precursor of
hyaluronan, correlated with medium glucose concentrations but not with
EGF. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed no
increase in hyaluronan synthases 1 and 3 (Has1 and Has3), whereas Has2
mRNA increased 2-3-fold in less than 2 h following the
introduction of EGF, as estimated by quantitative RT-PCR with a
truncated Has2 mRNA internal standard. The average level of Has2
mRNA increased from ~6 copies/cell in cultures before change of
fresh medium, up to ~54 copies/cell after 6 h in EGF-containing
medium. A control medium with 10% serum caused a maximum level of
~21 copies/cell at 6 h. The change in the Has2 mRNA
levels and the stimulation of hyaluronan synthesis followed a similar
temporal pattern, reaching a maximum level at 6 h and declining
toward 24 h, a finding in line with a predominantly Has2-dependent hyaluronan synthesis and its transcriptional regulation.
Hyaluronan is a large glycosaminoglycan found in the extracellular
space of most animal tissues. It forms a loose, highly hydrated,
gel-like matrix that contributes to the maintenance of the
extracellular space and facilitates nutrient diffusion. Furthermore, hyaluronan is involved in cell proliferation and differentiation, produces an environment favorable for migration (1),
and stimulates cell locomotion (2, 3). Elevated tissue levels of
hyaluronan occur during embryonic growth of tissues and organs (1),
wound healing (4, 5), inflammation (6), and invasion of certain cancers
(7-10).
In skin epidermis, the narrow extracellular space surrounding
keratinocytes contains a high concentration of hyaluronan (11, 12), as
do other stratifying squamous epithelia (13, 14). The half-life of
labeled epidermal hyaluronan in human skin organ culture is ~1 day
(15), indicating fast local turnover by keratinocytes. The importance
of the strikingly high concentration and turnover of hyaluronan in the
multilayered squamous epithelia is not completely understood, but we
have hypothesized that the former is necessary to maintain an
extracellular space for the nutritional needs of the more superficial
cell layers, whereas the latter allows the dramatic modulation of cell
shape that occurs during differentiation and for the high migratory
potential of keratinocytes that is activated, e.g. in wound
healing (16).
Unlike other glycosaminoglycans, hyaluronan is synthesized at the inner
surface of the plasma membrane by hyaluronan synthase (Has)1 and is extruded
through the plasma membrane into the extracellular space simultaneously
with the ongoing synthesis (for review, see Ref. 17). Currently, three
different Has genes have been identified in mammalian cells:
Has1 (18, 19), Has2 (20-22), and Has3
(23). The three Has genes are highly homologous but
appear to differ from each other in kinetic properties and product size
(24, 25). Limited data are available on the factors that regulate the
expression level and enzymatic activity of the different Has enzymes in
various cells and tissues (20, 26-29), but a number of studies have
suggested that the overall synthesis rate of hyaluronan is stimulated
by some growth factors and cytokines (30-38).
Epidermal growth factor (EGF) is one of the most powerful agents that
influences the behavior of keratinocytes. EGF transmits its information
through the EGF receptor (EGF-R), which belongs to the erbB receptor
tyrosine kinase family (39, 40). Signaling through EGF-R regulates many
cellular processes, including cell adhesion, expression of
matrix-degrading proteinases, and cell locomotion; these phenomena are
all important in skin wound healing. Keratinocyte EGF-R
expression is transiently elevated 5-7-fold within 2 days after
wounding and returns nearly to baseline within 4 days (41). It is also
important for keratinocyte proliferation and migration during
reepithelization (for review, see Ref. 42). Exogenously added EGF and
overexpression of EGF-R result in enhanced ligand-mediated migration of
keratinocytes and faster reepithelialization (43).
Aberrant expression or activation of the EGF-R is common and has been
proposed to have a role in epithelial tumor progression (44, 45). As in
wound healing, the EGF-R may provide an important contribution to the
migratory and invasive potential of carcinomas. The migration induced
by EGF requires the actin binding domain of EGF-R (46), and recent
studies have shown an important role of matrix metalloproteinases
(MMPs) in keratinocyte migration as well as in their ability to invade
other tissues (47, 48).
Whereas both increased hyaluronan and EGF signaling have been observed
in migrating cells and in wound healing, neither activation of
Has nor production of hyaluronan in response to EGF has been investigated in keratinocytes. In this study, we establish that EGF
specifically increases the mRNA level of Has2, resulting in enhanced synthesis of hyaluronan that correlates with enhanced keratinocyte migration in a wounding assay. Interestingly, a large proportion of the newly synthesized hyaluronan of EGF-treated cells
resides in intracellular vesicle-like structures, suggesting that a
significant proportion of Has2-directed hyaluronan is endocytosed immediately and recycled back into the cell.
Cell Culture--
A newborn rat epidermal keratinocyte (REK)
cell line was developed by MacCallum and Lillie (49) from neonatal rat
epidermal cells originally isolated by Baden and Kubilus (50). REKs
were cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc., Grand Island, NY) with 10% fetal bovine serum
(HyClone, Logan, UT), streptomycin (50 µg/ml), penicillin (50 units/ml) and 1-2 mM L-glutamine (all from PAA
Laboratories, Linz, Austria) at 37 °C in a humidified atmosphere
containing 5% CO2. Cells were trypsinized when they
reached confluency using 0.02% EDTA (w/v), 0.025% trypsin (w/v)
(Sigma). For biochemical assays and radiolabeling, the cells were grown
close to confluency in 6-well plates and incubated in the presence of
20 µCi/ml of [3H]glucosamine, and 100-200 µCi/ml
[35S]sulfate (Amersham Pharmacia Biotech). The depletion
of glucose in the culture medium by the metabolic acitivity of the
cells was estimated from 10-µl aliquots, derivatized with the
fluorophore 2-aminoacridone (AMAC) and separated and quantitated by
electrophoresis on polyacrylamide slab gels essentially as described
(51) using mannose as an internal standard.
Isolation of Secreted and Cell-associated
Glycosaminoglycans--
Cells were grown in 6-well plates (9.6 cm2/well) in 1 ml of medium and subsequently washed with
400 µl of Hank's solution (HyClone). For each culture, the medium
and wash were combined and designated as "medium." Each cell layer
was trypsinized, the resulting suspension removed, and the well was
washed with 250 µl of minimum essential medium. Each cell suspension,
combined with the washes was centrifuged. The resulting supernatant and
two subsequent 250-µl washes of the cell pellet with serum-free
medium were combined and designated as "trypsinate." The resulting
cell pellet was designated as the "intracellular" fraction.
Purification of Radiolabeled Hyaluronan--
Carrier (6 µg of
hyaluronan, Healon®, Amersham Pharmacia Biotech) was added to each
sample to improve the recovery of radiolabeled hyaluronan during the
purification procedures and gel filtration. Each cell fraction was
suspended in 500 µl of 50 mM sodium acetate containing 5 mM EDTA and 5 mM cysteine, pH 6. Cysteine and
EDTA were added into each of the medium and trypsinate fractions (5 mM final concentrations). Each sample was treated with
papain (Sigma) (200 µg/ml final concentration) at 60 °C for
1.5 h. Papain was inactivated in a boiling water bath (10 min).
After cooling, cetylpyridinium chloride (1% in water, 1.2 ml) was
added to each sample followed by incubation at room temperature for 10 min. After centrifugation at 13,000 × g for 15 min,
each supernatant was carefully removed by aspiration and discarded.
Samples were washed with 1 ml of water, centrifuged, and the
supernatants were discarded as above. Each cetylpyridinium chloride
precipitate was dissolved in 50 µl of 4 M guanidine HCl,
and 900 µl of absolute ethanol was added. Samples were kept at
Chemical Quantitation of Hyaluronan with Double
Labeling--
Each purified 30-µl sample was mixed with 4.5 µl of
0.5 M ammonium acetate, pH 7.0, 5 µl of 25 milliunits
chondroitinase ABC, and 5 µl of 1 milliunit of
Streptococcus hyaluronidase followed by incubation for
3 h at 37 °C (both enzymes from Seikagaku Kogyo Co., Tokyo,
Japan). Each digest was analyzed on a 1 × 30-cm Superdex Peptide
column (Amersham Pharmacia Biotech) 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 non-sulfated 3H-labeled disaccharide peak of
samples from these keratinocytes indicated the content of hyaluronan
(52). The carrier hyaluronan produced a disaccharide peak at 232 nm
that was used to monitor the recovery and to calculate corrections for
any losses in purification.
Incorporation of 35SO4 provides a measure of
the amount of the chondroitin/dermatan sulfate synthesized during the
labeling period. The [3H]galactosamine, derived from
[3H]glucosamine, incorporated into the same
chondroitin/dermatan sulfate disaccharides provides an estimate of the
effective specific activity of the UDP-N-acetylhexosamine
precursor pool and hence can be used to determine the chemical content
of newly synthesized 3H-labeled hyaluronan, as described in
detail previously (53, 54).
RNA Isolation and Northern Blot--
Keratinocytes were cultured
in ~28 cm2 dishes until confluency and scraped into
TRIzol®-reagent (Life Technologies, Inc.) for total RNA isolation
according to the instructions of the manufacturer. RNA was dissolved in
a small amount of distilled H2O and quantitated with a
spectrophotometer at 260 nm.
RNA was analyzed by electrophoresis on 1% formaldehyde/agarose gels
and transferred onto Hybond-NTM nylon membranes (Amersham Pharmacia
Biotech). A Has2-specific probe (1200 base pairs) was obtained from
human mRNA by RT-PCR using the primers 5'-GAAACAGCCCCAGCCAAAGAC-3' and 5'-CTCCCCCAACACCTCCAACC-3' and labeled with
[ RT-PCR with Has1, Has2, Has3, and GAPDH Primers--
For
RT-PCR, keratinocyte RNA was isolated with the TRIzol®-reagent. Equal
amounts were measured with spectrophotometer and were DNase-treated.
The RT-PCR reactions were done with the RNA PCR Core Kit (PerkinElmer
Life Sciences, Branchburg, NJ). To obtain rat Has1- and Has3-specific
primers, cDNA sequences were amplified from rat keratinocyte RNA
with mouse Has1 and Has3 specific primers using RT-PCR. The PCR
products were cloned into a pSport1 (Life Technologies, Inc.) plasmid
and sequenced. Primers for Has1 and Has3 were 5'-GCTCTATGGGGCGTTCCTC-3'
and 5'-CACACATAAGTGGCAGGGTCC-3', 5'-ACTCTGCATCGCTGCCTACC-3' and
5'-ACATGACTTCACGCTTGCCC-3', respectively. Rat Has2- and GAPDH-specific
primers (5'-TCGGAACCACACTGTTTGGAGTG-3'and 5'-CCAGATGTAAGTGACTGATTTGTCCC-3'; and 5'-TGATGCTGGTGCTGAGTATG-3' and
5'-GGTGGAAGAATGGGAGTT GC-3') were designed from
GenBankTM/EBI sequences AF008201 and M17701, respectively.
For quantitation of Has2 mRNA, a shortened (internal standard) Has2
cDNA containing the primer binding sites identical to those in the
wild-type Has2 cDNA was prepared by PCR. A poly(T)8
sequence was tagged into this cDNA at its 3'-end through an
appropriately designed Has2-specific downstream primer. Thus after
in vitro transcription treatment, the shortened Has2 RNA
strand contained a poly(A)8 tail. The shortened Has2 cRNA
was purified with FastTrackTM mRNA isolation kit (Invitrogen BV,
Leek, The Netherlands), dissolved in a small amount of H2O and quantitated with a spectrophotometer at 260 nm. RT-PCR was done
with constant amounts of the wild type and different concentrations of
the shortened Has2 RNAs. The resulting products were run on an agarose
gel, digitized by a BioDocIITM Video Documentation System (Biometra,
Göttingen, Germany), and quantitated by EtBr fluorescence by
using Image software (Wayne Rashband, NIH, Bethesda).
Assay of Keratinocyte Migration and
Proliferation--
Keratinocytes were cultured until they just reached
confluency. Two lines (~1-mm wide) crossing each other at right
angles were drawn with a 250-µl disposable pipette tip. The migration of keratinocytes to the cleared area was inspected under a microscope. The areas covered with cells were measured before and 18 h after the treatment. The change in the area was counted in pixels using the
NIH Image software and converted to mean migration distance (µm) of
the cell front.
To determine the proliferation rate, about 20,000 REKs were seeded in
400 µl of medium into the wells of a 24-well plate, and grown for 3 days. Then medium was changed and supplemented with 0-200 ng/ml EGF.
For each EGF concentration, 2 wells were harvested by trypsin digestion
after 24 and 48 h. The cells were then collected by
centrifugation, resuspended, and counted in a hemocytometer.
Microscopic Detection of Hyaluronan--
Each cell layer to be
analyzed was washed with Hank's balanced salt solution (HyClone) and
fixed at room temperature for 20 min in phosphate-buffered saline with
2% paraformaldehyde (v/v) for fluorescence microscopy. For electron
microscopy and light microscopy with peroxidase detection, a fixative
with 2% paraformaldehyde (v/v) and 0.5% glutaraldehyde (v/v) were
used. After fixation, the cells were washed three times for 2 min each
with 0.1 M sodium phosphate buffer, pH 7.4, and then
blocked in 1% bovine serum albumin (w/v) containing 0.1% Triton X-100
(v/v) in the same buffer for 30 min at room temperature.
Hyaluronan staining was done with a specific probe, biotinylated
hyaluronan binding complex (bHABC), purified from a 4 M
guanidine-HCl extract of bovine articular cartilage after dialysis and
trypsin digestion, as described previously (52). The probe is a
purified mixture of biotinylated G1 domain of aggrecan and link protein.
For regular transmitted light microscopy, the bHABC probe, diluted to 5 µg/ml in 3% bovine serum albumin (w/v), was added to the fixed cells
and was incubated overnight at 4 °C. After washing, avidin-biotin
peroxidase (ABC-standard kit, Vector Laboratories, Inc., Burlingame,
CA) was added for 1 h. The color was developed using
3,3'-diaminobenzidine (DAB) (0.05%) and H2O2
(0.03%). Counterstaining was done with hematoxylin for 2 min before
mounting in Aquamount (BDH Laboratory Supplies, Poole, England). For
confocal analysis Texas Red-labeled streptavidin (dilution 1:1000) was
used instead of ABC. The cells were mounted with Vectashield (Vector),
and viewed with a PerkinElmer UltraVIEW confocal microscope
(Wallac-LSR, Oxford, UK). For a side view of the cells, fixation,
incubation with bHABC, and the DAB reaction were done as above. The
cells were then dehydrated in graded ethanol and embedded in Spurr's resin. Semi-thin sections were cut perpendicular to the cell layer and
stained with toluidine blue. Ultrathin sections were stained with
uranyl acetate and lead citrate and viewed with a Jeol EX1200 electron microscope.
The specificity of the staining was controlled by predigesting 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
(12) or by preincubating the bHABC probe with hyaluronan
oligosaccharides (length ~20 monosaccharides, 3 µg/1 µg bHABC) to
reveal possible nonspecific binding of the probe.
The optical densities of the DAB-stained cultures were measured as
described previously (52) using a Leitz BK II microscope with a × 16 /0.45 N.A. objective (Leitz, Wetzlar, Germany) and connected
with a 12-bit digital camera (Photometrics CH 200, Tucson, AZ).
Area-integrated mean O.D. values, including both DAB-positive and
background intensities, were calculated for each whole digitized area.
In addition, DAB-positive staining areas were estimated from binary
images with a cutoff at an O.D. value of 0.13. Based on the
DAB-positive area data and the sum of the pixels that fulfilled the
positivity criteria, the mean area-integrated O.D. values for the
DAB-positive material were calculated.
Has2 Antisense Cells--
The eukaryotic expression vector
pCl-neo (5474 base pairs, Promega, Madison, WI) was linearized with
SalI (MBI Fermentas, Vilnus, Lithuania), and a rat
Has2 full-length cDNA (4172 base pairs,
GenBankTM/EBI AF008201) was ligated into the multiple
cloning site of pCl-neo. After transformation, the plasmid sequences
were confirmed, and the REK cells were transfected with Has2 antisense
plasmids according to the manufacturer's instructions with FuGENETM 6 transfection reagent (Roche Molecular Biochemicals). Transfected cells
were cultured in the presence of 500 µg/ml of G418
(Calbiochem-Nevabiochem Corp., La Jolla, CA) until separate colonies
about 0.5 cm in diameter were found. The colonies were reseeded and
grown in the presence of 250 µg/ml G418 except during the
experiments. The presence of the Has2 antisense construct was verified
with Southern blotting.
Induction of Hyaluronan Secretion in Epidermal Keratinocytes
by EGF--
Confluent monolayer rat keratinocyte cultures were labeled
with [3H]glucosamine and [35S]sulfate for
6 h. The amounts of newly synthesized
[3H]hyaluronan,
[3H,35S]chondroitin sulfate, and
chondroitinase-resistant glycosaminoglycans (mainly heparan
sulfate) in medium were determined using the double label method
described under "Experimental Procedures." Hyaluronan synthesis
rate was highest when keratinocytes were cultured in medium containing
20 ng/ml EGF (Fig. 1). When the
concentration of EGF was further increased, the stimulation of
hyaluronan synthesis decreased somewhat but remained ~2-fold higher
than the basal level. The synthesis of other glycosaminoglycans
(heparan sulfate and chondroitin sulfate) was not altered appreciably
by EGF treatment. Subsequent experiments used the optimal
concentration of 20 ng/ml EGF.
Because serum also stimulates hyaluronan synthesis, we examined the
interactive effects of EGF and serum. In the control cultures, 6 h
after the medium change, 10% serum increased the rate of hyaluronan synthesis by ~70% when compared with cells cultured with 0.5% serum
(Fig. 2). In EGF-treated cultures, the
presence of 10% serum showed an approximately additive increase in
hyaluronan synthesis (compare the UDP-[3H]N-acetylhexosamine Specific Activity with
Time After Replacing the Medium--
Previous studies have shown that
it is important to correct for precursor dilution by endogenous
substrates, as indicated by the marked changes in specific activity of
[3H]glucosamine in newly synthesized glycosaminoglycans
(Fig. 3a), to correctly
interpret 3H incorporation with this precursor (53, 54).
The metabolic activity of the cells gradually depletes essential
nutrients in the growth medium, such as glucose (Fig. 3b)
and glutamine that are utilized in the intracellular synthesis of
glucosamine. As glucose was depleted, the proportion of the exogenous
radiolabeled [3H]glucosamine that contributes to the
intracellular pool, i.e. its specific activity, increased.
This was particularly pronounced after 12 h (Fig. 3a).
However, whereas the specific activity of the hexosamines (UDP-GlcNAc
and UDP-GalNAc are in an equilibrium in the precursor pool) increased
greatly in the glycosaminoglycans synthesized at later times after
medium change, EGF-treated and control cultures did not differ
significantly from each other (Fig. 3). This suggests that the supply
of UDP-GlcNAc was sufficient to meet the larger requirements created by
the EGF-enhanced hyaluronan synthesis.
Time Course of EGF-induced Hyaluronan Synthesis--
To monitor
the rate of hyaluronan synthesis at different times between 0 and
24 h following introduction of EGF, we used 3-h labeling windows
by adding small aliquots of the radiolabeled precursors into the medium
at the different times indicated in Fig.
4. In confluent keratinocyte cultures the
medium change alone caused a 2-3-fold increase of total hyaluronan
synthesis by 3-9 h (Fig. 4a). Nevertheless, EGF-treatment
showed an additional ~3-6-fold increase of newly synthesized total
hyaluronan above the control in the 3-9-h labeling windows (Fig.
4a). The stimulatory effect of EGF decreased thereafter, but
the total synthesis at 21-24 h was still more than twice that in the
control cultures (Fig. 4a).
Even if most of the hyaluronan synthesized in extended labeling periods
were found in the culture medium, a significant fraction (~50% at
max, 3-6 h) remained associated with the cell layer in the 3-h
labeling windows, either with the trypsinate (Fig. 4b) or
the intracellular (Fig. 4c) fractions. The newly synthesized hyaluronan in these fractions was substantially increased by EGF with
similar kinetics, peaking in the 3-9-h labeling windows. The amount of
newly synthesized trypsinate hyaluronan increased 2-3-fold in maximum
during the 3-9-h labeling windows (Fig. 4b). The highest
(6-10-fold) increase by EGF was detected in the newly synthesized
intracellular hyaluronan pool (Fig. 4c). The trypsinate hyaluronan represents molecules bound to the receptors, mainly CD44,
whereas the intracellular hyaluronan probably represents endocytosed
material destined for lysosomal degradation and perhaps those under
synthesis and still bound to the hyaluronan synthase (52).
Microscopic Assay of Total Cell-associated and Intracellular
Hyaluronan--
The EGF-induced increase of hyaluronan associated with
the cell layer was also shown by staining the keratinocyte cultures with the hyaluronan-specific probe (bHABC) (Fig.
5, a and b). This
visual impression was confirmed by determination of the total optical
densities as described under "Experimental Procedures," resulting
in higher values for EGF-treated cultures at all the time points (4-20
h) studied (data not shown). The proportion of intracellular hyaluronan
was specifically measured in cultures fixed and digested with
Streptomyces hyaluronidase to remove cell surface
hyaluronan, then permeabilized and stained with bHABC. The assay of
O.D. values for these stainings showed consistently higher
intracellular hyaluronan values already by 1 h after the addition
of EGF (Fig. 6).
Morphological Changes of Keratinocytes--
The keratinocytes were
quite flattened in subconfluent cultures and covered a large area of
the substratum (Fig. 5a). Shortly after introduction of EGF,
keratinocytes began to round up and form membrane ruffles and
microspikes (Fig. 5, b-e), which was followed by cell
elongation and the appearance of lamellipodia (Fig. 5, c and
e). These changes in morphology were apparent in a few of
the cells even after 1 h, and most cells showed the altered morphology after a 60-h EGF treatment.
Localization of Cell Surface Hyaluronan in EGF-treated
Keratinocytes--
The distribution of hyaluronan in control cultures
was similar to that described previously (52), with most of the
hyaluronan residing in plasma membrane patches (Figs. 5a and
7c). In EGF-treated cells, the hyaluronan signal intensity
was generally increased (Figs. 5, b, c, e and
7d). Hyaluronan covered the membrane ruffles and microspikes
in cells undergoing rounding (Fig. 5, b-e). In elongating
cells the midportion (around the nucleus) and the trailing edge showed
an intense hyaluronan signal (Fig. 5, c and e).
The lamellipodia, instead, appeared almost negative or showed only localized spots (Fig. 5, c and e,
arrows). The amount of hyaluronan that accumulated in
response to EGF treatment was sufficient to exclude sedimenting red
blood corpuscles on keratinocyte surfaces, a frequently used test of
hyaluronan coat formation and hyaluronan synthesis (Fig.
7, a and b). More
hyaluronan was also found on the underside of the EGF-treated cells
than in control cells as seen in Fig. 7, c and d
(arrows), and in confocal images (not shown). This was
particularly evident in the rounded, apparently migrating cells.
Electron microscopy of ultrathin vertical sections suggested that in
sites where hyaluronan was deposited under the cell, the distance
between plasma membrane and the substratum increased (Fig.
7e).
EGF-induced Changes in Intracellular Hyaluronan--
Intracellular
hyaluronan was specifically detected by removing cell surface
hyaluronan with Streptomyces hyaluronidase (Fig. 5f). The
intracellular localization was also confirmed by confocal analysis (not
shown) and in semi-thin vertical sections (Fig. 7,
c and d, arrowheads).
Hyaluronan signal was present in cytoplasmic structures conforming to
vesicles of various sizes and appeared to line their membrane (Figs. 5,
c and f, short arrows and 7, c and d, arrowheads). No nuclear
hyaluronan signal was found. The accumulation of intracellular
hyaluronan in response to EGF was most conspicuous in rounding cells,
whereas cells retaining a more flattened morphology contained less.
Stimulation of Migration but Not Proliferation by EGF
Treatment--
The cells became elongated in EGF-treated cultures
(Fig. 5), a common finding in cells with enhanced mobility. A
stimulation in the migration of the keratinocytes by EGF was confirmed
by artificial wounding of the cell layer and quantitation of the speed
at which the cells migrated into the cleared area (Fig. 8). The migratory activity peaked at the
same EGF concentration as for the synthesis of hyaluronan (compare
Figs. 1 and 8a), and corresponded to that reported earlier
(55).
The cell number almost doubled by 24 h, independent of the
presence of EGF (2-200 ng/ml) (Fig. 8b). Further increases
at 48 h were also independent of EGF (2-20 ng/ml) with some
inhibition at the 200 ng/ml level. Thus, EGF had no significant effect
on the proliferation rate of the keratinocytes in conditions that showed the highest stimulation in hyaluronan synthesis and migration.
EGF-induced Increase in Hyaluronan Synthase 2 mRNA--
To
reveal changes in the expression of the different hyaluronan synthases
responsible for the enhanced hyaluronan synthesis by EGF, we estimated
the mRNA levels of rat Has1, Has2, and Has3 using RT-PCR. This
comparative analysis suggested that Has1 and Has3 mRNA levels were
not markedly changed by EGF treatment, nor contributed to the
hyaluronan synthesis stimulation (Fig.
9a). In contrast, Has2 level
was increased by EGF (Fig. 9a). However, the basal level of
Has2 mRNA in the keratinocytes was so low that the level of
increase in its two transcripts was difficult to estimate by Northern
blot (Fig. 9b). To approximate the increase in Has2 mRNA
level in keratinocytes, we used quantitative RT-PCR with an internal,
truncated cRNA standard, and compared the sample band densities with a
set of standards as shown in Fig. 9c. These analyses for
control keratinocyte cultures indicated a low copy number before the
change to fresh medium (~6/cell), a detectable increase of Has2
mRNA even after 1 h, a peak at 6 h (~54/cell), and a
decrease toward basal level by 24 h (Fig. 9d). Whereas
the level of HAS2 mRNA also increased in control cultures following change into fresh medium, the number of Has2 mRNA copies was 1.5-8 times higher in the EGF-treated cultures at all time points examined (Fig. 9d).
Migration Inhibition by Has2 Antisense Transfection--
The
correlation between migration stimulation and Has2
activation in the EGF-stimulated cells was further explored by
analyzing a variant of the present REK cell line with a constitutively
expressed Has2 antisense gene. This cell line has a reduced
expression level of Has2 because of the presence of a stably
transfected Has2 antisense gene and hence reduced synthesis
of hyaluronan (Fig. 10b).
The Has2 antisense cells showed a clearly reduced migration compared with its mock-transfected controls (Fig. 10a), indicating
that Has2 has an important role in the migration
process.
Has2 mRNA Levels--
Hyaluronan synthase mRNAs are
presumed to occur in low numbers but, as far as we know, there is no
published data on its copy numbers per cell, estimated with internal
RNA standards. Whereas the actual amounts, not corrected for recovery
in RNA isolation, may be slightly higher than those in Fig. 9, the data
demonstrate a 10-fold increase of Has2 mRNA level and the maximum
of at least ~54 copies/cell. This increase in the mRNA
corresponded to about 30-fold enhancement of hyaluronan production from
the basal synthesis rate. The changes in mRNA and hyaluronan
synthesis levels also showed a temporal correlation, strongly
suggesting a tight transcriptional regulation of hyaluronan synthesis.
According to our unpublished data,2 the peak level of Has2
mRNA in the cumulus oophorus cells during the preovulatory
hyaluronan synthesis reaches ~400 copies per cell, about eight times
that in EGF-treated keratinocytes. However, the corresponding
hyaluronan synthesis rates in the EGF-treated keratinocytes (~0.2
pg/cell/12 h) and cumulus cells (~3 pg/cell/12 h) (56), show a
similar ratio between the peak Has2 mRNA levels and hyaluronan production.
Regulation of Different Has Genes--
Whereas it is obvious that
the synthesis rate of hyaluronan in various cell types is controlled by
cytokines and growth factors, including EGF (30, 34), the contribution
of the three known Has genes in this regulation remains
uncertain. Like in keratinocytes, the hyaluronan synthesis of
preovulatory ovarian follicle is up-regulated by Has2 mRNA levels
(20) whereas Has1 and Has3 are not affected.2 Keratinocytes
were reported to up-regulate Has1 mRNA as a response to TGF Intracellular Hyaluronan in EGF-treated Keratinocytes--
The
rapid and marked intracellular accumulation of endogenous hyaluronan in
perinuclear vesicles by EGF treatment was quite striking and
unexpected, although it is known that intracellular hyaluronan staining
increases upon stimulation of quiescent fibroblasts (60). At least a
part of the intracellular hyaluronan resulted from enhanced
receptor-mediated uptake because our unpublished data show that the
EGF-enhanced intracellular hyaluronan staining is reduced by incubation
with hyaluronan decasaccharides, known to displace the CD44-bound
hyaluronan in the keratinocytes (52). The very short half-life of
hyaluronan in the epidermis of human skin organ cultures (15) and in
organotypic keratinocyte cultures of the present cells (53) also
support the idea that a proportion of hyaluronan was catabolized
shortly after synthesis. This rapid catabolism has not been
demonstrated previously in cell cultures. Whereas the biological
importance of the hyaluronan turnover in keratinocytes is not currently
known, it correlates with the enhanced motility of the cells as seen in
the wound healing assay and is likely involved in some aspect of this
process (61).
Biological Implications--
The data in this study establish that
hyaluronan, a major extracellular matrix molecule in stratified
epithelia such as epidermis, is specifically increased in keratinocytes
by EGF and therefore likely contributes to such biological consequences
of EGF as stimulated keratinocyte migration after wound healing.
Whereas hyaluronan synthesis rates higher than that induced here by EGF
have been reported in other cells, it is obvious that the
Has2 regulation described here could induce rapid and
dramatic changes in the cellular environment of epidermal
keratinocytes, considering the small extracellular space where
hyaluronan accumulates. Indeed, transfection of Has2 antisense cDNA
into the present keratinocytes demonstrates that hyaluronan synthesis
is one of the factors that control the migration rate of the cells.
The ability of epidermal keratinocytes to rapidly cover an open wound
is a biologically crucial motility response of those cells. Healing of
skin wounds involves transient up-regulation of EGF receptors (41) and
is aided by EGF-like growth factors (43). Hyaluronan is abundant in the
frontline keratinocytes migrating into a wound (5). Furthermore,
hyaluronan synthesis and Has2 expression are elevated at the edges of
an in vitro wound in mesothelial cell cultures (59). Mice
with targeted inhibition of the epidermal hyaluronan receptor CD44 (62)
show delayed wound healing. Taken together, these studies indicate that
stimulated EGF receptor signaling and enhanced hyaluronan metabolism
are intimately connected with each other and with the epithelial wound healing process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C for 30 min and then centrifuged. Supernatants were discarded
as above, and pellets were dissolved in 30 µl of water.
-32P]dCTP by PCR. Hybridization was done by following
the ULTRAhybTM hybridization protocol for DNA probes to RNA blots
(Ambion, Austin, TX).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
[in a new window]
Fig. 1.
EGF concentration dependence of the synthesis
of hyaluronan, heparan sulfate, and chondroitin/dermatan sulfate in
keratinocyte cultures. Confluent monolayer cultures of
keratinocytes were incubated with [3H]glucosamine and
[35S]sulfate for 6 h in the presence of EGF between
0 and 200 ng/ml. The content of newly synthesized hyaluronan in the
medium was calculated with the double-label method as described under
"Experimental Procedures." The bars show the range of
duplicate cultures.
increases). This suggested that
factors in serum, such as IGF-1 and PDGF that might contribute to
increased synthesis, are additive with EGF and that stimulation by EGF
is obvious in all serum concentrations. Because the vitality of cells
may suffer in extended cultures with low serum concentration, we
decided to use 10% serum in later experiments.
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[in a new window]
Fig. 2.
The effect of serum concentration on
hyaluronan synthesis in keratinocyte cultures. Nearly confluent
cultures of keratinocytes were incubated for 6 h with 0.5, 3, and
10% serum, and with or without 20 ng/ml EGF. The double-label method
was used to calculate the content of newly synthesized hyaluronan in
the medium as described under "Experimental Procedures." shows
the incremental increase in hyaluronan synthesis when 10% serum was
present. The bars show the range of duplicate
cultures.
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Fig. 3.
The specific activity of GalNAc in
chondroitin sulfate synthesized by confluent keratinocyte
cultures. a, the specific activities were measured
every 6 h, each after a 6-h labeling period (horizontal
bars). Culture medium was changed at 0 h. The 0-h control
represents the specific activity in control cultures before the change
of medium, 2 days after the previous change. The specific activities
were calculated from the double-label data as described under
"Experimental Procedures." b, time course of the glucose
concentration in culture medium during an experiment similar to that in
a. The shaded area shows the level of glucose in the medium
before addition to the cultures (range of 3 assays). All cultures were
treated with or without EGF (20 ng/ml), and the vertical
bars show the range of duplicate cultures.
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Fig. 4.
The time course of hyaluronan synthesis after
the addition of EGF (20 ng/ml) in confluent keratinocyte cultures.
The 0-h time point represents control cultures before the change of
medium, 2 days after the previous change. The total amount of newly
synthesized hyaluronan (a) and that associated with either
the trypsinate (b) or the intracellular compartment
(c) are shown. The 3-h labeling periods are indicated by
horizontal bars. The vertical bars show the range
of duplicate cultures.
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Fig. 5.
Effect of EGF on the morphology and
hyaluronan distribution in keratinocyte cultures. A control
culture is shown in a and those treated with 20 ng/ml of EGF
for 6 h in b and d, 12 h in
f, and 18 h in c and e. The
asterisks in c and d show
hyaluronan-rich ruffles on EGF-treated keratinocytes. The long
arrows (c and e) show lamellipodia at the
leading edge of keratinocytes. In f, the culture was
digested with Streptomyces hyaluronidase to cleave off the
extracellular hyaluronan before staining. The short arrows
(c and f) point to hyaluronan-rich cytoplasmic
vesicles. The brown color indicates the localization of
hyaluronan, whereas the blue counterstain (hematoxylin)
shows cell nuclei. All cultures were grown on chamber slides,
fixed, and probed for hyaluronan by bHABC as described under
"Experimental Procedures." The bars represent 10 µm,
except 100 µM in a and b.
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[in a new window]
Fig. 6.
The time course of the intracellular
hyaluronan accumulation in EGF-treated keratinocyte cultures. EGF
(20 ng/ml) was added in a small volume of buffer into confluent
cultures on chamber slides in which fresh medium was changed 2 days
earlier. All cultures were terminated simultaneously with the fixative
described under "Experimental Procedures." The time (0-6 h)
between the introduction of EGF and fixation of the culture is
indicated. The 0 min point represents cultures without EGF addition.
The fixed cultures were treated with Streptomyces
hyaluronidase to remove extracellular hyaluronan, then permeabilized
and stained for remaining intracellular hyaluronan. The mean optical
density (± S.E.) from 19 randomly selected fields is shown at each
point. Two experiments with similar results were done.
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[in a new window]
Fig. 7.
Coat formation and deposition of hyaluronan
under the cells. In control (a) and EGF-treated
cultures (b) a suspension of fixed red blood corpuscles was
allowed to sediment on keratinocytes to demonstrate indirectly any
hyaluronan coats surrounding the cells. Control (c) and
EGF-treated (20 ng/ml) cultures (d) were fixed and stained
for hyaluronan, then embedded in plastic and cut into vertical semithin
sections. The arrowheads in c and d
point to hyaluronan-rich vesicles. The arrows in c,
d, and e indicate hyaluronan located on the
keratinocyte plasma membrane under the cells. The electron microscopic
image of an EGF-treated sample (e) demonstrates a hyaluronan
deposit located between the keratinocyte plasma membane and the
substratum. All cultures were grown on chamber slides, fixed, and
probed for hyaluronan by bHABC as described under "Experimental
Procedures." The bars represent 10 µm except 0.5 µm in
e.
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Fig. 8.
Effect of EGF on keratinocyte migration and
proliferation. a, confluent keratinocyte cultures were
artificially wounded, and the migration distance of keratinocytes at
the wound edge after 24 h was measured as described under
"Experimental Procedures." b, the numbers of
keratinocytes after 24-48 h growth in the absence or presence of EGF
(2-200 ng/ml) were counted in a hemocytometer. The bars
show the range of duplicate cultures.
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Fig. 9.
Hyaluronan synthase mRNA expression in
EGF-treated cultures. a, total RNA isolated from equal
numbers of keratinocytes treated with EGF for 3 h (E)
and controls (C) were reverse transcribed and amplified with
35 PCR cycles for the different Has types and GAPDH, an internal
control. b, Northern blot analysis of Has2 mRNA at the
indicated time points in control (C) and EGF-treated
cultures (E). The two Has2 transcripts and GAPDH as a
loading control are indicated. c, an example of the standard
curves used for the determination of the Has2 mRNA copy numbers.
The points indicate band fluorescence intensity ratios of native Has2
mRNA (wt) and the truncated internal standard Has2 cRNA
(is). d, assay of Has2 mRNA at different time
points following change into EGF containing (unfilled
circles) and control medium (filled circles), utilizing
the above standardization. The average RNA copy numbers per cell were
calculated without an attempt to correct for the recovery in RNA
isolation.
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Fig. 10.
Effect of reduced Has2 expression on
keratinocyte migration. An antisense Has2 gene was
stably transfected into the keratinocytes as described under
"Experimental Procedures." The migration (a) and the
quantities of newly synthesized hyaluronan on cell surface
(trypsinate) and growth medium (b) were
determined in control cells containing an empty transfection vector
(C) and the antisense gene (AS). The
bars in a show the standard error of 8 wounding
assays in separate dishes, whereas those in b show the range
of duplicate dishes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, but
its functional importance, or the synthesis of hyaluronan were not
studied (57). Among the cytokines and growth factors TGF
seems
exceptional because Has2 is the main target when hyaluronan synthesis is up-regulated by TNF
and IFN
in renal tubular
epithelial cells (28), by IL-1 in orbital fibroblasts (27), by PDGF
(58) or wounding (59) in mesothelial cells, and by osteogenic protein 1 in chondrocytes (29).
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ACKNOWLEDGEMENTS |
---|
We thank Alpo Pelttari for the facilities of the Dept. of Electron Microscopy. Expert technical help by Arja Venäläinen, Riikka Tiihonen, and Päivi Perttula is gratefully acknowledged.
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FOOTNOTES |
---|
* This work was supported by Academy of Finland Grant 40807 (to M. T.), Finnish Cancer Foundation (to R. T.), EVO funds of Kuopio University Hospital (to M. T.), and Kuopio University Biotechnology Funds (to R. T. and M. T.).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. B. 1627, FIN-70211 Kuopio, Finland. Tel.: 358-17-163019; Fax: 358-17-163032; E-mail: markku.tammi@uku.fi.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M007601200
2 C. Fülöp, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: Has, hyaluronan synthase; EGF, epidermal growth factor; EGF-R, epidermal growth factor receptor; REK, rat epidermal keratinocyte; RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; bHABC, biotinylated hyaluronan binding complex; DAB, 3,3'-diaminobenzidine.
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