1 Department of Anatomy, University of Kuopio, 70211 Kuopio, Finland
2 Department of Biomedical Engineering, Lerner Research Institute, Cleveland
Clinic Foundation, Cleveland, Ohio 44195, USA
3 Department of Clinical Microbiology, University of Kuopio, 70211 Kuopio,
Finland
* Author for correspondence (e-mail: raija.tammi{at}uku.fi)
Accepted 26 June 2002
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Summary |
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Key words: Hyaluronan, Hyaluronan synthase 2, Keratinocyte, Migration, Adhesion plaques, Vinculin, CD44
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Introduction |
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Hyaluronan is synthesized at the inner face of the plasma membrane by
hyaluronan synthases (Has). The enzymes act by alternative addition of
glucuronic acid and N-acetylglucosamine from their UDP-sugars to the growing
hyaluronan chain, which is simultaneously extruded through the membrane into
the extracellular space. Three isoenzymes have been identified in vertebrates,
designated as Has1, Has2 and Has3 (for a review, see
Weigel et al., 1997), each
with distinct kinetic properties and product size
(Brinck and Heldin, 1999
;
Itano et al., 1999
). The first
reports on the roles of the different isoenzymes have shown the importance of
Has2 in the maintenance of cartilage matrix
(Nishida et al., 1999
) and
ovulation (Salustri et al.,
1999
). Furthermore, developing mouse embryos deficient in Has2
activity die during gestation in utero, whereas those lacking Has1 and Has3
show no major defects (Camenisch et al.,
2000
). All three Has types are expressed in skin keratinocytes
(Pienimäki et al., 2001
;
Sugiyama et al., 1998
).
Hyaluronan has long been associated with stimulated migration of cultured
cells (Schor et al., 1989),
and extensive clinical data suggest that hyaluronan enhances spreading of
epithelial cancers (Anttila et al.,
2000
; Auvinen et al.,
2000
; Ropponen et al.,
1998
). Hyaluronan may contribute to cell migration as a structural
component of the extracellular space, creating a highly hydrated, elastic
matrix that may help cell movement by facilitating detachment and providing
space for migration (Tammi et al.,
2002
). In addition, hyaluronan probably controls the locomotion of
many cell types by interacting with its receptors such as CD44
(Bourguignon et al., 2000
;
Ladeda et al., 1998
;
Lewis et al., 2001
;
Thomas et al., 1992
) and
RHAMM/IHABP (Akiyama et al.,
2001
; Assmann et al.,
1999
; Hofmann et al.,
1998
; Savani et al.,
2001
; Turley et al.,
1991
). A variety of signaling pathways have been reported to
associate with RHAMM (Hall et al.,
1996
; Zhang et al.,
1998
), and CD44 (Bourguignon et
al., 2000
; Bourguignon et al.,
2001
; Lewis et al.,
2001
; Li et al.,
2001
; Ohta et al.,
1997
; Okamoto et al.,
2001
), which can account for the changes in migration. Still, the
exact role of the hyaluronan ligand as a triggering or regulatory agent in the
locomotion signaling has remained obscure. For instance, CD44 seems to have a
relatively low affinity for hyaluronan, requiring clustering or
oligomerization of CD44 for stable binding and implying the requirement for a
size of hyaluronan sufficient to occupy multiple receptors
(Lesley et al., 2000
).
Signaling may thus depend on the size distribution of hyaluronan and the way
hyaluronan is presented to the cell surface. Furthermore, the bulky hyaluronan
may non-specifically mask or block other cell surface interactions when bound
to its receptors or when it is being extruded through the plasma membrane
during its synthesis.
The contribution of endogenous hyaluronan synthesis to migration has been
confirmed by Has gene transfections. However, it turned out that
overexpressed Has1 and Has2 enhance migration in melanoma cells
(Ichikawa et al., 1999) as
does Has2 in mesothelioma cells (Li and
Heldin, 2001
), whereas Has1, Has2 and particularly Has3 inhibit
the migration of CHO cells (Brinck and
Heldin, 1999
). Likewise, exogenous hyaluronan added in fibroblast
cultures induced, inhibited or did not affect migration, depending on the
tissue origin of the fibroblasts (Andreutti
et al., 1999
). In keratinocytes, upregulated Has2 and hyaluronan
synthesis by epidermal growth factor correlated with higher migratory activity
(Pienimäki et al., 2001
).
Obviously the influences on locomotion caused by increased hyaluronan and
hyaluronan synthesis rate depend on the cellular background. Whether or not
specific inhibition of endogenous Has expression is associated with changes in
cell motility has not been studied. Further, whether or not soluble, exogenous
hyaluronan surrounding the cell and that synthesized by the cell itself have
similar effects on cell behaviour is also unknown.
Thus, although there is a wealth of evidence for the importance of
hyaluronan synthesis in cell proliferation and migration, few details of the
mode of action are available. The aim of this study was to modulate endogenous
hyaluronan synthesis by upregulation and downregulation of Has2 in
keratinocytes and to examine the consequences in terms of their proliferative
and migratory activities, cell adhesion and morphology and to compare those to
results obtained by addition of exogenous hyaluronan, removing endogenous cell
surface hyaluronan and competing for hyaluronan binding to surface receptors.
For this we used a non-transformed cell line that can differentiate in a
manner closely resembling epidermal keratinocytes in vivo
(Tammi et al., 2000) and
established clones stably transfected with constitutively active Has2
gene constructs in sense and antisense orientations. Our results demonstrate
the importance of the rate of Has2-dependent hyaluronan synthesis for
keratinocyte motility, spreading and adhesion in vitro.
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Materials and Methods |
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Cell culture
A newborn rat epidermal keratinocyte (REK) cell line
(Baden and Kubilus, 1983) was
cultured in minimum essential medium, (MEM, Life technologies Ltd, Paisley,
Scotland) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT),
4 mM glutamine (Sigma, St. Louis, MO) and 50 µg/ml streptomycin sulfate and
50 U/ml penicillin (Sigma). Keratinocytes were passaged twice a week at a 1:5
split ratio using 0.05% trypsin (w/v), 0.02% EDTA (w/v) in phosphate-buffered
saline (PBS, Reagena Ltd, Kuopio, Finland).
Transfections
24 hours after plating on a 35 mm dish, 150,000 REKs were transfected
according to the manufacturer's instructions with 3 µl FuGENETM 6
transfection reagent (Boehringer-Mannheim, Mannheim, Germany) combined with 1
µg of plasmid DNA. The next day, the transfected cells were trypsinized,
seeded on a 90 mm dish, and grown with 500 µg/ml G418
(Calbiochem-Novabiochem Corp., La Jolla, CA). The new selection medium was
changed every 3-4 days until separate colonies about 0.5 cm in diameter were
found. Individual colonies were trypsinized with sterile cloning cylinders,
seeded into 24-well plates and grown to sufficient numbers for the
experiments. The transfected genes were maintained by keeping G418
continuously in the culture medium at 250 µg/ml except during the
experiments. The cell lines were designated as follows: W, wildtype; M,
mock-transfection (pCl-neo without insert); A, antisense; and S, sense cell
lines. The cell lines were verified to be mycoplasma negative.
Southern blotting
Confluent cultures were trypsinized and lysed in 10 mM Tris, pH 8.0, 0.1 mM
EDTA, 0.3 M Na-acetate, 1% SDS, then digested with proteinase K (Sigma) and
RNAse A. DNA was extracted with phenol and chloroform, precipitated with
ethanol, digested with EcoRI (MBI), electrophoresed on a 0.9% agarose
gel and transferred by capillary blotting to a nylon membrane
(Sambrook et al., 1989). The
membrane was probed with a radiolabeled Has2-specific cDNA probe (1200 bp),
which was amplified from the human chondrosarcoma cell line (HCS 2/8)
(Takigawa et al., 1989
) using
human Has2-specific primers 5'-GAA ACA GCC CCA GCC AAA GAC-3' and
5'-CTC CCC CAA CAC CTC CAA CC-3'. The intensities of the bands
were measured with a CCD camera and analyzed by NIH Image® software. The
number of Has2 gene construct copies in each analysis was estimated
by comparing the intensity of the construct band to that of the endogenous
Has2 gene.
Hyaluronan disaccharide analysis
Media (400 µl) from keratinocytes grown on 35 mm plates with 1% FBS was
analyzed for secreted hyaluronan levels. Healon® (Amersham Pharmacia
Biotech, Uppsala, Sweden) was used as a standard. The samples were boiled for
10 minutes to denature proteins and digested with 40 µl of proteinase K
(Sigma, 600 µg/ml in 100 mM ammonium acetate, pH 6.5) for 1.5 hours at
60°C. After proteinase K inactivation by boiling for 10 minutes, 50 µl
of 50% trichloroacetic acid was added and the samples centrifuged for 15
minutes at 13,000 g. Each supernatant was dialyzed overnight
against water and evaporated to dryness after the addition of 0.5 nmol mannose
as an internal standard. Each sample was dissolved in 100 mM ammonium acetate,
pH 6.5 and digested for 3 hours at 37°C with 2 mU of
Streptococcus hyaluronidase (Seikagaku Kogyo Corp., Tokyo, Japan).
The samples were dried under vacuum centrifugation, and 5 µl of 0.1 M
2-aminoacridone (AMAC, Lambda Fluoreszenztechnologie GmbH, Graz, Austria) in
3:17 (v:v) acetic acid:dimethylsulfoxide, and 5 µl of 1 M
NaBH3CN was added followed by incubation overnight at 37°C. The
AMAC-derivatized disaccharides were stored at -20°C until electrophoresis
as described previously (Calabro et al.,
2000), with the following modification: 30% PAGE gels were cast in
the laboratory in 100 mM Tris-borate buffer, pH 8.9, and the same buffer was
used as the running buffer. The intensities of hyaluronan disaccharide bands
derived from the hyaluronan standards, internal standards and samples were
digitized on a UV-light box using a CCD camera. Quantitative image processing
was done with NIH-Image®.
RT-PCR with Has2 and GAPDH primers
For RT-PCR, keratinocyte RNA was isolated with the RNeasy® Mini kit
(Qiagen GmbH, Hilden, Germany) and treated with DNAse. Equal quantities of the
RNA, measured with a spectrophotometer, were subjected to RT-PCR reactions
with the RNA PCR Core Kit (Perkin Elmer, Branchburg, NJ). Rat Has2 and GAPDH
specific primers, 5'-TCG GAA CCA CAC TGT TTG GAG TG-3' and
5'-CCA GAT GTA AGT GAC TGA TTT GTC CC-3', and 5'-TGA TGC TGG
TGC TGA GTA TG-3' and 5'-GGT GGA AGA ATG GGA GTT GC-3',
respectively, were designed from GenBank sequences AF008201 and M17701,
respectively. In the assay for Has2 expression, the primer specific for Has2
(sense) mRNA was used for reverse transcription to avoid amplification of the
possible Has2 antisense transcripts. The resulting products were run on an
agarose-gel and visualized by ethidium bromide fluorescence.
bHABC-staining and image analysis
Keratinocytes were seeded at 20,000 cells/well on eight-well chamber
slides precoated for 30 minutes at 37°C with FBS (Nalge Nunc, Naperville,
IL) and grown at 37°C for 48 hours. The slides were washed with 0.1 M
sodium phosphate buffer, pH 7.4 (PB), fixed at room temperature for 30 minutes
with 2% paraformaldehyde (v/v) and 0.5% glutaraldehyde (v/v) and washed
5x2 minutes with PB. Cells were permeabilized at room temperature with
0.3% Triton X-100 in 3% BSA and probed with 3 µg/ml of bHABC in 3% BSA
overnight at 4°C. After washing with PB, the slides were incubated with
avidinbiotin peroxidase (ABC standard kit, Vector Laboratories Inc.,
Burlingame, CA) for 1 hour, and the color was developed with 3,3'
diaminobenzidine (DAB) and H2O2 and mounted in
Supermount (BioGenex, San Ramon, CA), as described previously
(Tammi et al., 2001
). The
specificity of the staining for hyaluronan was controlled by removing
hyaluronan with Streptomyces hyaluronidase (Seikagaku Kogyo Corp.,
Tokyo, Japan), and the specificity of the bHABC probe was verified by
pretreating it with hyaluronan oligosaccharides (average size 20
monosaccharides).
The optical density measurements were done as described before
(Tammi et al., 1998). A Leitz
BK II microscope with a 16x objective with 0.45 numerical aperture
(Leitz, Wetzlar, Germany) was connected to a 12-bit digital camera
(Photometrics CH 200, Tucson, AZ) equipped with a KAF 1400 CCD detector
(Eastman Kodak Corp., New York, NY). Camera control and image analysis were
done with IPLab software (Signal Analytics Vienna, VA). Ten fields
(731x841 µm) beginning from a randomly selected corner were
systematically sampled along a line across each well, and area-integrated mean
optical density (OD) values, including both DAB-positive and background
intensities, were calculated for each whole digitized area. In addition,
DAB-positive areas were estimated from binary images with a cut-off at an OD
value of 0.13. On the basis of the positive area data and the sum of the pixel
values that fulfilled the positivity criteria, the mean area-integrated OD
values for the DAB-positive material were calculated.
For confocal analysis of hyaluronan localization, cells were fixed with 2% paraformaldehyde (v/v), permeabilized and treated with bHABC as described above, but instead of the ABC reagent, FITC-labeled avidin (1:500 dilution, 1 hour, Vector) was used as a reporter. After washing, cells were mounted in Vectashield (Vector).
For double staining of vinculin and hyaluronan, the anti-vinculin mAb (1:1000, Sigma) was added to the bHABC solution (5 µg/ml), and in the secondary step, Texas red-labeled anti-mouse secondary antibody (Vector, 1:50) and fluorescein isothiocyanate-labeled avidin (1:500) were used together. Micrographs were obtained with an Ultraview® confocal scanner (Perkin Elmer Life Sciences, Wallac-LSR, Oxford, UK) on a Nikon Eclipse TE300 microscope using a 100x oil immersion objective.
Measurement of adhesion plaques
Cells were seeded at 10,000 cells per well on eight-well chamber
slides, fixed after 24 hours and stained for vinculin as above. Using the
confocal microscope, the area of vinculin staining in a plane just above the
substratum was measured to estimate the number and size of adhesion plaques.
20 randomly selected fields per cell line were recorded using a 60x oil
immersion objective. The 12-bit greyscale images were linearly scaled to
eight-bit and filtered with an unsharp mask (radius 6, amount 170, threshold
60) using Adobe Photoshop 5 software (Adobe Systems, San Jose, CA). Further
processing was done with IPLab software (Scanalytics Inc, Fairfield, VA). Each
image was duplicated, and plaques without overlying diffuse fluorescence were
directly thresholded using a constant threshold value (image a). Since some of
the adhesion plaques did not have a constant intensity ratio with the
background owing to fluorescent structures above the focal plane, they were
separated from the background with impulse filtering (matrix 5x5 pixels;
each kernel has the value -1 except the central pixel, which has a value of
+24; division coefficient 5; post-filter offset 140) and thresholded using a
constant value (image b). Images a and b were combined digitally, and the
count and areas of individual plaques were measured automatically. Structures
smaller than eight pixels were excluded from analysis. Finally, the cell area
was segmented with the aid of a colored overlay superimposed on the original
image, and the cumulative areas of the plaques were related to cell numbers in
each field.
CD44 immunodetection
For the immunocytochemical localization, cultures on chamber slides were
fixed with 2% paraformaldehyde in PBS for 20 minutes at room temperature,
washed with PBS and incubated with the OX-50 antibody at 1:100 dilution
overnight at 4°C. After washes with PBS, the signal was visualized with
1:50 diluted Texas Red-labeled antimouse antibody (Vector) for 1 hour at room
temperature.
For FACS analysis, cells were detached with 0.02% EDTA in PBS, blocked with 1% BSA in PBS for 10 minutes and then sequentially incubated with OX-50 (1:50 dilution), biotinylated antimouse antibody (1:200) and FITC-avidin (1:1000) for 30 minutes. Cells were fixed with 1% paraformaldehyde for 20 minutes and analyzed in a fluorescence-activated cell sorter.
Proliferation
Cells were seeded in 24-well culture plates at 60,000 cells/well.
Fresh culture medium was added every day to ensure optimal growth conditions
for every cell line. Cells from duplicate wells were trypsinized and counted
with a hemocytometer after 4 hours to determine plating efficiency, and after
1, 2, 3, 4 and 5 days to determine the proliferation rate. The number of
detached cells in media was also counted following concentration by low-speed
centrifugation. Doubling times of the cells were determined at days 0-1, 1-2
and 2-3 (Darbre and King,
1984
) by calculating log2/m, in which m represents the slope of a
straight line determined by two successive time points in the growth curve
[the plot of log(cell number) against time].
The proliferation rates in the wounded cultures were studied using
bromodeoxyuridine (BrdU) labeling and detection kit I from Roche (Roche
Diagnostics, Mannheim, Germany). Cells were labeled with 10 µM BrdU for 2
hours, fixed in 70% ethanol in 50 mM glycine-HCl buffer, pH 2.0 for 20 hours
(Dorsch and Goff, 1996) and
immunostained with anti-BrdU antibody and FITC-labeled secondary antibody
according to the manufacturer's instructions. To visualize all nuclei,
propidium iodide (1 ng/ml) was included in the primary antibody solution. The
labelings were done 2, 6, 10, 16 and 22 hours after the wounding. The
specimens were photographed with a 20x objective on the confocal
microscope at 10 consecutive fields from the wound edge at 488 and 560 nm
wavelengths. The number of BrdU-positive cells and propidium-iodide-positive
nuclei were counted using the NIH Image® software.
Determination of cell cycle phase by FACS analysis
An equal number of wild-type and Has2 antisense (A22) cells were plated on
a 90 mm dish. After 16 hours, cells were trypsinized, fixed with 70% ethanol
for 24 hours at 4°C and treated with RNAase (0.15 mg/ml, Sigma) for 3
hours at 37°C. Cells were incubated with propidium iodide (10 µg/ml,
Sigma) for 2 hours at 37°C, and DNA contents of individual cells were
analysed with a fluorescence-activated cell sorter.
Migration analysis
The transfected and control cells were seeded at 500,000 cells/35 mm
plates and grown until confluence. A cell-free area was introduced by scraping
the monolayer crosswise with a sterile 1 ml pipette tip, which cleared cells
from
1000 µm wide lanes. The cultures were then washed with Hank's
balanced salt solution (Euroclone Ltd, Pero, Italy), and fresh medium with 10%
FBS was added. The effects of exogenous high molecular mass hyaluronan
(Healon®, Pharmacia, Uppsala, Sweden) and of purified hyaluronan
decasaccharides (Tammi et al.,
1998
) on migration were studied in medium without FBS.
Streptomyces hyaluronidase was present at 1 U/ml in serum-free medium
during the migration experiments on cultures pretreated with 5 U/ml of the
same enzyme before wounding. The areas covered by the cells were measured
immediately after scraping and 24 hours later using an Olympus CK 2 inverted
phase contrast microscope, a Panasonic Wv-CD 130-L video camera and NIH
Image® software. The average distance the outermost cells had migrated was
calculated using the formula: (
b-
a)/2, where a is the area
covered by the cells at 0 hours and b is the area covered by the cells after
24 hours. The results (in pixels) were converted to micrometers.
Apoptosis
Keratinocytes were seeded at 20,000 cells/well on eight-well chamber
slides (Nalge), grown for 24 hours, washed with cold PB and stained with the
Annexin V-FITC Apoptosis Detection Kit (R&D Systems, Minneapolis, MN)
according to the manufacturer's instructions. The percentage of positive cells
was counted.
Spreading analysis of individual cells
Spreading rates were determined by measuring the areas occupied by
individual cells 3, 6, 9, and 24 hours after seeding at 20,000 cells/well
on eight-well chamber slides. Five images of each cell line were captured at
every time point using a CoolSNAP CCD camera (Photometrics) mounted on a Nikon
Eclipse TE300 inverted microscope with a 20x DIC objective. The areas of
each individual cell were measured using NIH Image® software. The number
of cells measured per image was 3-20, and the total cell number measured at
each time point was 21-96. For the presentation of general cell morphology,
images were obtained from individual cells 24 hours after seeding at
100,000 cells/well on two-well chambered cover glasses (Nunc), using a
60x immersion oil DIC objective.
Statistical analysis
The data were subjected to a one-way analysis of variance or paired
t-test (StatView 512+ Software, Abacus Concepts, Berkeley, CA).
Comparisons between the means of different cell lines were done using Fisher's
PLSD test.
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Results |
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Synthesis of hyaluronan
Cytochemical assays of hyaluronan attached to the REK cell layers confirmed
our previous findings (Tammi et al.,
1998) of a patchy distribution on cell surfaces and variation in
quantity between individual cells (Fig.
2A). The pattern of hyaluronan distribution on cells was similar
in the wild-type, mock, sense and antisense Has2 transfected cells, but the
general staining intensity of the sense and antisense cell lines appeared
higher and lower, respectively, than that of wild-type or mock transfected
cells (Fig. 2A). Interestingly,
the cultures of Has2 sense cells contained a greater proportion of motile
looking, spindle-shaped cells that were also strongly hyaluronan positive
(Fig. 2A). A set of cultures
from sense and antisense cell lines were assayed for optical density using
image analysis, which confirmed the difference in cell-associated hyaluronan
(Fig. 2B).
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The REKs grown in monolayer cultures synthesize hyaluronan at a rate that
depends on cell density, with lowdensity cultures producing more hyaluronan
per cell (Tammi et al., 2001).
Fig. 2C shows an experiment
where all antisense and sense cell lines were analysed at the same time and
hyaluronan secretion plotted against cell density. This and other similar
experiments indicated that in most sense cell lines the secretion of
hyaluronan into growth medium exceeded that in the antisense cell lines. The
average production of hyaluronan was very similar between the mock-transfected
lines M1, M2, and M3 (15% maximum difference) and 12-25% lower than in the
parental wild-type cells (data not shown). Taken together, there were two
clear differences: (1) cell-associated hyaluronan
(Fig. 2A) is much greater in
sense than antisense cells with Mock and Wt at an intermediate level, (2) a
linear correlation of hyaluronan in medium relative to cell density
(Fig. 2C), with sense and
antisense curves clearly displaced; again M2 at an intermediate level.
Cell size, morphology and spreading rate
Examination of the cell lines by inverted phase contrast microscopy
suggested distinct differences in cell size, with antisense cells appearing
smaller than the sense cells. However, there were no significant differences
in cell volume between Has2 sense, antisense and mock cell lines as measured
with FACS (data not shown), suggesting that the established cell lines most
probably differ in their ability to extend on the substratum. Antisense cells
produced several smaller lamellae, as compared with control cells that had a
single, wide lamellipodium (Fig.
3A). These findings prompted us to determine the spreading of the
different cell lines by measuring the areas covered by individual cells 6
hours after plating (Fig. 3B).
The area covered by the mocktransfected lines was close to that of the
wildtype. The average areas occupied by the sense cell lines were generally
similar to those of the mock-transfected and parental controls
(Fig. 3b). In contrast, four
out of the six antisense cell lines showed less spreading than any of the
controls. In three of these the difference was statistically significant
(Fig. 3B) and remained so at
least for 24 hours (Fig. 3C).
The result suggests that Has2 is involved and necessary in the spreading of
keratinocytes. However, addition of Streptomyces hyaluronidase to the
plating medium at 1 TRU/ml, a concentration that eventually removes
pericellular hyaluronan (Tammi et al.,
2001), did not reduce the spreading rate in the wild type, mock
(M1), and the sense (S27 and S29) cell lines (data not shown). This indicates
that high molecular weight hyaluronan can be removed shortly after its
synthesis without an effect on spreading, or that the cellular compartment
where hyaluronan exerts its influence is shielded from the hyaluronidase in
the growth medium.
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Confocal analysis showed that both in mocktransfected and sense cells, hyaluronan was relatively abundant at the undersurface of the spreading cells, whereas the signal for hyaluronan was low under the antisense cell lines (Fig. 4A). By contrast, the staining for vinculin, representing the adhesion plaques to the substratum, appeared more prominent in the antisense cell lines compared with those of the mock and especially of sense cells (Fig. 4A). Hyaluronan was excluded from clusters of vinculin-positive adhesion plaques and vice versa; hyaluronan-rich areas were devoid of large adhesion plaques (Fig. 4A). In general, focal adhesions were less abundant in polarized cells with wide lamellapodia and high hyaluronan expression. The total area of focal adhesions per cell area as indicated by vinculin positivity in the undersurface of the cells was significantly higher in four of the Has2 antisense cells compared with the mock-transfected cell lines, whereas none of the sense cell lines differed significantly from the mock lines (Fig. 4B). Blocking Has2 thus allows an apparently tighter cell attachment through the vinculin-containing adhesion plaques.
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Proliferation
The effect of the Has2 gene transfections on cell proliferation
was studied in three randomly selected representatives of the sense and
antisense cell lines and compared to one mock cell and the wild-type cells.
Equal numbers of the cells from each line were plated and counted after
different time periods in culture. The number of Has2 antisense cells did not
increase at all during the first 24 hours after plating
(Fig. 5A). Consequently, their
cell numbers lagged behind but did reach the same level as control and
wild-type cells at confluency (Fig.
5A).
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Two of the three Has2 sense cell lines grew at the same rate as the controls, but one (S27) appeared to remain at a lower density when reaching confluency (Fig. 5B). These observations were documented in several repeated experiments. Analysis of variance applied to the data set with all cell lines and time points indicated that the antisense lines differed significantly from all other cell types on day 1. The calculated doubling times of the antisense cell lines on day 0-1 were on average 41 hours, compared with 21-25 hours in the other cell lines. However, there were no differences between the cell lines on days 1-2 and 2-3 in this parameter, with all showing an average of 17 hours.
Cell counts after 4 hours (plating efficiency) indicated that a similar number of transfected and parental cells adhered (data not shown). The number of apoptotic cells was also similar in all cell lines, which excludes cell death as a cause of the lag in the early growth of the antisense cells. Likewise, the numbers of floating cells were not different between the cells lines (data not shown). Flow cytometric analysis was performed on one of the antisense cell lines to determine the relative DNA content shortly after plating. As deduced from the DNA content at 16 hours, the percentage of the A22 antisense cells in the cell cycle phases G0/G1, S and G2/M were 52.8%, 34.0% and 11.0%, respectively, whearas the corresponding figures in the wild-type cells were 44.4% 35.7% and 18.9%. The difference persisted but was reduced at 24 hours. These results indicated that after plating, the reduced Has2 expression in antisense cells caused a transient delay in entering the S-phase of the cell cycle.
Migration
Wounding induced the wild-type REKs to migrate from the edge of the cleared
area at a rate of 7 µm/hour on average. The migration of the
mock-transfected cells (M1, M2 and M2) did not markedly differ from that of
the wild-type cells (Fig. 6). Three of the Has2 overexpressing cell lines migrated significantly faster
compared with mock-transfected cells, whereas migration of four cell lines
with the Has2 antisense gene was significantly reduced
(Fig. 6). These differences in
motility were also seen in non-wounded, non-confluent monolayer cultures where
Has2 overexpressing cells appeared to have wider lamellipodia and fill the
empty areas more efficiently than Has2 antisense cells.
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The role of hyaluronan synthesis in the present migration model was further examined by localization of newly synthesized hyaluronan in the wounded cultures. Confluent cultures were digested with Streptomyces hyaluronidase to remove existing hyaluronan, washed, wounded and stained 8 hours later for the new hyaluronan chains emerging on the cells. In mock-transfected cultures, numerous hyaluronan-positive spots were found in cells close to the wound edge, whereas cells in non-wounded areas showed less newly synthesized hyaluronan (Fig. 7). By contrast, antisense cells rarely showed this wound-edge induction of hyaluronan expression whereas the cell lines carrying the Has2 gene in sense orientation not only showed intensely stained cells in the wound edge but also an elevated hyaluronan staining in non-wounded areas (Fig. 7). This suggests that hyaluronan synthesis was specifically induced in keratinocytes lining the cleared area and that this hyaluronan synthesis contributed to the migratory activity of the cells.
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To exclude the chance that the observed differences in migration were related to different proliferation rates of the cells, cultures were labeled with bromodeoxyuridine 14-16 hours after wounding. The number of labeled nuclei representing cells in the cell cycle S phase were counted from a 250-300 µm wide field close to the wound edge. In wild-type cultures, 32% of the cells in this field were labeled, whereas the transfected cell lines showed slightly higher labeling indices (38.4±2.6% in three mock lines, 39.5±2.5% in five sense lines, and 40.0±2.8% in six antisense lines, means±s.e.). This indicates that cell proliferation differences in the wound edge did not contribute to the differences of migration between the sense and antisense cell lines.
The addition of highly purified hyaluronan of the same size as that newly
synthesized by REKs did not significantly increase the migration rate in mock,
sense and antisense cell lines (Fig.
8A). Importantly, the reduced migration of antisense cells could
not be restored by this exogenous hyaluronan. Moreover, when cell surface
hyaluronan was first cleared with Streptomyces hyaluronidase
treatment, and 1 U/ml of the enzyme was added to the medium for the following
24 hour migration experiments, there was no marked change in the migration of
mock-transfected, Has2 antisense and Has2 sense cells
(Fig. 8B). Experiments were
also done with wild-type cells in the presence of 450 µg/ml of purified
hyaluronan decasaccharides. The decasaccharide-treated cultures showed
97±13% (mean±s.d., six separate experiments) of the migration in
control cultures (Fig. 8C),
even though this oligosaccharide size and concentration are known to displace
receptor-bound hyaluronan and about half of the total cell surface hyaluronan
in confluent REK cultures (Tammi et al.,
1998). Taken together, these experiments suggest that exogenous
hyaluronan has a limited effect on the migration of the REK cells and that
continuous, gradual cleavage of endogenous, newly synthesized hyaluronan or
blocking its binding to cell surface receptors has little influence on the
Has2-modulated migratory response.
|
CD44 expression
Immunocytochemical stainings for CD44 of all the cell lines showed a strong
signal on plasma membranes and no apparent changes in the localization of the
signal (data not shown). Flow cytometric analysis of CD44-associated
fluorescence intensities in mock transfected (75±3, mean±s.e.,
three cell lines), antisense (56±5, six cells lines), and sense
(53±4, five cell lines) cells indicated no correlation between Has2
expression level and cell surface CD44, suggesting that Has2-associated
phenotypes were not caused by changes in the quantity of cell surface
CD44.
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Discussion |
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---|
Hyaluronan and keratinocyte migration
The strong control that Has2 has on keratinocyte motility in an in vitro
wound healing assay shows that hyaluronan synthesis is not just coincident
with the migratory activity but is required for efficient motility of these
cells. It was recently shown that transfection of the Has genes into
fibroblasts enhances cell motility (Itano
et al., 2002). Furthermore, hyaluronan synthesis was specifically
induced at the wound edge, suggesting that Has upregulation, quite probably of
Has2, is an inherent feature of the proper wound healing response. Wounding of
mesothelial cell monolayers in a similar manner to the present cultures
induces Has2 expression and hyaluronan synthesis in the cells that begin
migration from the wound edges (Yung et
al., 2000
). Human keratinocytes migrating to cover a gingival
wound show elevated hyaluronan staining
(Oksala et al., 1995
), and
hyaluronan accumulates in wounded mouse epidermis (R.T., unpublished),
indicating that this hyaluronan synthesis response is also a part of wound
healing in vivo.
Hyaluronan is thought to act as a signaling ligand that influences the activity of the intracellular locomotory system through cell surface receptors. However, the present experiments show that adding soluble, purified hyaluronan of the same high molecular weight as that synthesized by the cells themselves did not markedly increase the reduced migration rate caused by the Has2 antisense gene. This indicates that exogenous hyaluronan either does not have access to the receptors used by endogenous hyaluronan or that the mechanism of Has2 action is not simple hyaluronan receptor activation by ligand binding.
Another way of probing the role of receptor-mediated signaling in migration was to add hyaluronan decasaccharides to displace endogenous high molecular weight hyaluronan bound to cell surface receptors. The oligosaccharide treatment had no influence on migration, suggesting that continuous occupation of cell surface receptors by intact high molecular weight hyaluronan or the formation of a hyaluronan coat is not necessary for keratinocyte migration in the present assay. This conclusion was supported by the fact that continuous fragmentation of the cellular hyaluronan coat by Streptomyces hyaluronidase did not inhibit the migration of keratinocytes.
Hyaluronan synthesis and cell adhesion
The adhesive properties of the keratinocytes were clearly changed in Has2
antisense cells as indicated by the area of adhesion plaques. While just a
small proportion of hyaluronan lies under the cell, this may still be
important in the dynamic turnover of the focal adhesions that occurs in
migration. If hyaluronan synthase is inserted or activated in the plasma
membrane domain under a cell, the rapidly extruded chain can create pressure
that may make existing adhesions more labile or reduce the number or size of
new contacts. Hyaluronidase added in the culture medium may not have free
access to this domain. Electron microscope analysis shows plasma membrane
distension away from the substratum at hyaluronan deposits under the cell
(Pienimäki et al., 2001),
and the present data indicated that adhesion plaques are excluded in the cell
underside domains occupied by hyaluronan. The view that Has2 controls cell
adhesion is supported by the finding that its overexpression reduces contact
inhibition of cell growth and the organization of cadherin and filamentous
actin (Itano et al.,
2002
).
Further, the strong influence of hyaluronan synthesized endogenously as
compared with that added in the medium is probably due to the correct
positioning of the endogenous molecule, whether it is involved in signaling
and/or associated with cell adhesion or plasma membrane dynamics
(Oliferenko et al., 2000).
Impairment of proliferation of the antisense cells
The delay in the initiation of the proliferation cycle in antisense cells
was not completely unexpected, since active hyaluronan synthesis is often
correlated with rapid proliferation. However, there was no evidence in the
antisense cells for a block in the G2 phase of the cell cycle, as when the
receptor RHAMM is inhibited (Mohapatra et
al., 1996), or during cytokinesis, as occurs when hyaluronan
synthesis is inhibited by periodate oxidized UDP-glucuronic acid
(Brecht et al., 1986
). On the
contrary, the cellular DNA content indicated a reduced proportion of cells
with duplicated DNA, suggesting that there is a regulatory point in the G1
stage of the cell cycle that is influenced by hyaluronan synthesis. The
present finding of delayed entry into the S-phase of the cell cycle in the
Has2 antisense cells fits well with a recent report of increased proportion of
cells in the S and G2/M phases associated with Has2 overexpression
(Itano et al., 2002
). The
delay in the initiation of proliferation was correlated with reduced
spreading, but further experiments are needed to reveal more of the molecular
mechanisms that may involve signaling through the PI-kinase
(Itano et al., 2002
).
Lamellipodia extension and hyaluronan synthesis
Extension of plasma membrane into lamellipodia is the prerequisite for cell
spreading and migration and involves vectorial growth bursts of actin filament
bundles and arrays (Svitkina and Borisy,
1999). The formation of lamellipodia is triggered, for instance,
by the GTPase Rac, which can be activated by the interaction of the hyaluronan
receptor CD44 and the guanidine nucleotide exchange factor Tiam1
(Bourguignon et al., 2000
). The
expression level of CD44 correlates with migratory activity
(Peck and Isacke, 1996
;
Thomas et al., 1992
), and
blocking of CD44 reduces cell spreading and migration in some cell types
(Ichikawa et al., 1999
;
Ladeda et al., 1998
;
Oliferenko et al., 2000
). We
found no significant difference in CD44 levels between Has2 sense and
antisense cells, suggesting that the reduced spreading in antisense cells is
not due to alterations in CD44 expression. Thus, although CD44 may be
essential, it apparently cannot support lamellipodia formation in
keratinocytes without sufficient Has2 expression and hyaluronan synthesis.
Rapidly fluctuating hyaluronan synthesis, controlled, for example, by growth
factors (Pienimäki et al.,
2001
), seems a good regulator of the dynamic changes of
keratinocyte migration.
Expression of the transfected genes in keratinocytes
Although the sets of mock, sense and antisense clones significantly
differed from each other in their behaviour, in the individual clones the
magnitude of these functional properties was not correlated with the level of
Has2 expression. The mechanisms of this clonal variation are not known, but
may involve concurrent changes in gene regulation, in some cases preventing
the penetration of the phenotype. This is likely with genes vital for
survival, such as Has2 (Camenisch et al.,
2000). In the future, experiments with conditional expression of
the sense and antisense regulators of Has2 might be used to avoid these
problems.
The present findings suggest that keratinocytes represent a cell type in
which hyaluronan synthesis is strictly regulated and that this regulated
synthesis level influences major cell functions. We collected a number of
individual transfected clones of keratinocytes, some with multiple sense or
antisense gene copies, and all displayed a moderate change in the level of
hyaluronan secretion consistent with their level of Has2 expression. This is
unlike the marked increase previously noted in some of the clones from other
Has2-transfected cells types (Brinck and
Heldin, 1999; Itano et al.,
2002
; Kosaki et al.,
1999
; Watanabe and Yamaguchi,
1996
). Although the upper limit of hyaluronan synthesis is
probably specific for keratinocytes, we believe that the lower limit was
determined by cell viability. The cells carrying the antisense gene(s) grew
very slowly in the clonal densities following transfection (data not shown),
and it is conceivable that transfectants with more efficient antisense Has2
expression might have been excluded owing to a growth disadvantage. No
previous studies on stable antisense Has transfections are currently available
for comparison.
Finally, the continuous rat keratinocyte parental cell line used in this
study is unique among available keratinocyte models. The cells can be cloned
while maintaining their ability to undergo epidermal differentiation in the
absence of a feeder layer (Tammi et al.,
2000). This property allowed selection of the stably transfected
clones used in this study and demonstrates the potential of these cells for
transfection with other genes that may influence keratinocyte biology. The
ability of the sense and antisense Has2 clones to undergo epidermal
differentiation is currently under investigation.
![]() |
Acknowledgments |
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akiyama, Y., Jung, S., Salhia, B., Lee, S., Hubbard, S., Taylor, M., Mainprize, T., Akaishi, K., van Furth, W. and Rutka, J. T. (2001). Hyaluronate receptors mediating glioma cell migration and proliferation. J. Neurooncol. 53,115 -127.[CrossRef][Medline]
Andreutti, D., Geinoz, A. and Gabbiani, G. (1999). Effect of hyaluronic acid on migration, proliferation and alpha-smooth muscle actin expression by cultured rat and human fibroblasts. J. Submicrosc. Cytol. Pathol. 31,173 -177.[Medline]
Anttila, M. A., Tammi, R. H., Tammi, M. I., Syrjänen, K.
J., Saarikoski, S. V. and Kosma, V. M. (2000). High levels of
stromal hyaluronan predict poor disease outcome in epithelial ovarian cancer.
Cancer Res. 60,150
-155.
Assmann, V., Jenkinson, D., Marshall, J. F. and Hart, I. R.
(1999). The intracellular hyaluronan receptor RHAMM/IHABP
interacts with microtubules and actin filaments. J. Cell
Sci. 112,3943
-3954.
Auvinen, P., Tammi, R., Parkkinen, J., Tammi, M., Ågren,
U., Johansson, R., Hirvikoski, P., Eskelinen, M. and Kosma, V. M.
(2000). Hyaluronan in peritumoral stroma and malignant cells
associates with breast cancer spreading and predicts survival. Am.
J. Pathol. 156,529
-536.
Baden, H. P. and Kubilus, J. (1983). The growth and differentiation of cultured newborn rat keratinocytes. J. Invest. Dermatol. 80,124 -130.[Abstract]
Bourguignon, L. Y., Zhu, H., Shao, L. and Chen, Y. W.
(2000). CD44 interaction with tiam1 promotes Rac1 signaling and
hyaluronic acidmediated breast tumor cell migration. J. Biol.
Chem. 275,1829
-1838.
Bourguignon, L. Y., Zhu, H., Shao, L. and Chen, Y. W.
(2001). CD44 interaction with c-Src kinase promotes
cortactin-mediated cytoskeleton function and hyaluronic acid-dependent ovarian
tumor cell migration. J. Biol. Chem.
276,7327
-7336.
Brecht, M., Mayer, U., Schlosser, E. and Prehm, P. (1986). Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem. J. 239,445 -450.[Medline]
Brinck, J. and Heldin, P. (1999). Expression of recombinant hyaluronan synthase (HAS) isoforms in CHO cells reduces cell migration and cell surface CD44. Exp. Cell Res. 252,342 -351.[CrossRef][Medline]
Calabro, A., Benavides, M., Tammi, M., Hascall, V. C. and
Midura, R. J. (2000). Microanalysis of enzyme digests of
hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted
carbohydrate electrophoresis (FACE). Glycobiology
10,273
-281.
Camenisch, T. D., Spicer, A. P., Brehm-Gibson, T., Biesterfeldt,
J., Augustine, M. L., Calabro, A., Jr, Kubalak, S., Klewer, S. E. and
McDonald, J. A. (2000). Disruption of hyaluronan synthase-2
abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation
of epithelium to mesenchyme. J. Clin. Invest.
106,349
-360.
Darbre, P. and King, R. J. (1984). Progression to steroid autonomy in S115 mouse mammary tumor cells: role of DNA methylation. J. Cell Biol. 99,1410 -1415.[Abstract]
Dorsch, M. and Goff, S. P. (1996). Increased
sensitivity to apoptotic stimuli in c-abl-deficient progenitor B-cell lines.
Proc. Natl. Acad. Sci. USA
93,13131
-13136.
Hall, C. L., Lange, L. A., Prober, D. A., Zhang, S. and Turley, E. A. (1996). pp60(c-src) is required for cell locomotion regulated by the hyaluronan receptor RHAMM. Oncogene 13,2213 -2224.[Medline]
Hofmann, M., Assmann, V., Fieber, C., Sleeman, J. P., Moll, J., Ponta, H., Hart, I. R. and Herrlich, P. (1998). Problems with RHAMM: a new link between surface adhesion and oncogenesis? Cell 95,591 -592.[Medline]
Ichikawa, T., Itano, N., Sawai, T., Kimata, K., Koganehira, Y.,
Saida, T. and Taniguchi, S. (1999). Increased synthesis of
hyaluronate enhances motility of human melanoma cells. J. Invest.
Dermatol. 113,935
-939.
Itano, N., Sawai, T., Yoshida, M., Lenas, P., Yamada, Y.,
Imagawa, M., Shinomura, T., Hamaguchi, M., Yoshida, Y., Ohnuki, Y. et al.
(1999). Three isoforms of mammalian hyaluronan synthases have
distinct enzymatic properties. J. Biol. Chem.
274,25085
-25092.
Itano, N., Atsumi, F., Sawai, T., Yamada, Y., Miyaishi, O.,
Senga, T., Hamaguchi, M. and Kimata, K. (2002). Abnormal
accumulation of hyaluronan matrix diminishes contact inhibition of cell growth
and promotes cell migration. Proc. Natl. Acad. Sci.
USA 99,3609
-3614.
Kaya, G., Rodriguez, I., Jorcano, J. L., Vassalli, P. and Stamenkovic, I. (1997). Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation. Genes Dev. 11,996 -1007.[Abstract]
Kosaki, R., Watanabe, K. and Yamaguchi, Y.
(1999). Overproduction of hyaluronan by expression of the
hyaluronan synthase Has2 enhances anchorage-independent growth and
tumorigenicity. Cancer Res.
59,1141
-1145.
Ladeda, V., Aguirre Ghiso, J. A. and Bal de Kier Joffe, E. (1998). Function and expression of CD44 during spreading, migration, and invasion of murine carcinoma cells. Exp. Cell Res. 242,515 -527.[CrossRef][Medline]
Lesley, J., Hascall, V. C., Tammi, M. and Hyman, R.
(2000). Hyaluronan binding by cell surface CD44. J.
Biol. Chem. 275,26967
-26975.
Lewis, C. A., Townsend, P. A. and Isacke, C. M. (2001). Ca(2+)/calmodulin-dependent protein kinase mediates the phosphorylation of CD44 required for cell migration on hyaluronan. Biochem. J. 357,843 -850.[CrossRef][Medline]
Li, R., Wong, N., Jabali, M. D. and Johnson, P.
(2001). CD44-initiated cell spreading induces Pyk2
phosphorylation, is mediated by Src family kinases, and is negatively
regulated by CD45. J. Biol. Chem.
276,28767
-28773.
Li, Y. and Heldin, P. (2001). Hyaluronan production increases the malignant properties of mesothelioma cells. Br. J. Cancer 85,600 -607.[CrossRef][Medline]
Mohapatra, S., Yang, X., Wright, J. A., Turley, E. A. and Greenberg, A. H. (1996). Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin B1 expression. J. Exp. Med. 183,1663 -1668.[Abstract]
Nishida, Y., Knudson, C. B., Nietfeld, J. J., Margulis, A. and
Knudson, W. (1999). Antisense inhibition of hyaluronan
synthase-2 in human articular chondrocytes inhibits proteoglycan retention and
matrix assembly. J. Biol. Chem.
274,21893
-21899.
Ohta, S., Yoshida, J., Iwata, H. and Hamaguchi, M. (1997). Hyaluronate activates tyrosine phosphorylation of cellular proteins including focal adhesion kinase via CD44 in human glioma cells. Int. J. Oncol. 10,561 -564.
Okamoto, I., Kawano, Y., Murakami, D., Sasayama, T., Araki, N.,
Miki, T., Wong, A. J. and Saya, H. (2001). Proteolytic
release of CD44 intracellular domain and its role in the CD44 signaling
pathway. J. Cell Biol.
155,755
-762.
Oksala, O., Salo, T., Tammi, R., Häkkinen, L., Jalkanen,
M., Inki, P. and Larjava, H. (1995). Expression of
proteoglycans and hyaluronan during wound healing. J. Histochem.
Cytochem. 43,125
-135.
Oliferenko, S., Kaverina, I., Small, J. V. and Huber, L. A.
(2000). Hyaluronic acid (HA) binding to CD44 activates Rac1 and
induces lamellipodia outgrowth. J. Cell Biol.
148,1159
-1164.
Peck, D. and Isacke, C. M. (1996). CD44 phosphorylation regulates melanoma cell and fibroblast migration on, but not attachment to, a hyaluronan substratum. Curr. Biol. 6, 884-890.[Medline]
Pienimäki, J. P., Rilla, K., Fülöp, C., Sironen,
R. K., Karvinen, S., Pasonen, S., Lammi, M. J., Tammi, R., Hascall, V. C. and
Tammi, M. I. (2001). Epidermal growth factor activates
hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular
and intracellular hyaluronan. J. Biol. Chem.
276,20428
-20435.
Ropponen, K., Tammi, M., Parkkinen, J., Eskelinen, M., Tammi, R., Lipponen, P., Ågren, U., Alhava, E. and Kosma, V. M. (1998). Tumor cell-associated hyaluronan as an unfavorable prognostic factor in colorectal cancer. Cancer Res. 58,342 -347.[Abstract]
Salustri, A., Camaioni, A., di Giacomo, M., Fülop, C. and
Hascall, V. C. (1999). Hyaluronan and proteoglycans in
ovarian follicles. Hum. Reprod. Update
5, 293-301.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (eds) (1989). Molecular cloning; A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Savani, R. C., Cao, G., Pooler, P. M., Zaman, A., Zhou, Z. and
DeLisser, H. M. (2001). Differential involvement of the
hyaluronan (HA) receptors CD44 and receptor for HA-mediated motility in
endothelial cell function and angiogenesis. J. Biol.
Chem. 276,36770
-36778.
Schor, S. L., Schor, A. M., Grey, A. M., Chen, J., Rushton, G., Grant, M. E. and Ellis, I. (1989). Mechanism of action of the migration stimulating factor produced by fetal and cancer patient fibroblasts: effect on hyaluronic acid synthesis. In Vitro Cell Dev. Biol. 25,737 -746.
Sugiyama, Y., Shimada, A., Sayo, T., Sakai, S. and Inoue, S.
(1998). Putative hyaluronan synthase mRNA are expressed in mouse
skin and TGF-beta upregulates their expression in cultured human skin cells.
J. Invest. Dermatol.
110,116
-121.
Svitkina, T. M. and Borisy, G. G. (1999). Progress in protrusion: the tell-tale scar. Trends Biochem. Sci. 24,432 -436.[CrossRef][Medline]
Takigawa, M., Tajima, K., Pan, H. O., Enomoto, M., Kinoshita, A., Suzuki, F., Takano, Y. and Mori, Y. (1989). Establishment of a clonal human chondrosarcoma cell line with cartilage phenotypes. Cancer Res. 49,3996 -4002.[Abstract]
Tammi, R., Ripellino, J. A., Margolis, R. U. and Tammi, M. (1988). Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe. J. Invest. Dermatol. 90,412 -414.[Abstract]
Tammi, R., Säämänen, A.-M., Maibach, H. I. and Tammi, M. (1991). Degradation of newly synthesized high molecular mass hyaluronan in the epidermal and dermal compartments of human skin in organ culture. J. Invest. Dermatol. 97,126 -130.[Abstract]
Tammi, R., Paukkonen, K., Wang, C., Horsmanheimo, M. and Tammi, M. (1994). Hyaluronan and CD44 in psoriatic skin. Intense staining for hyaluronan on dermal capillary loops and reduced expression of CD44 and hyaluronan in keratinocyte-leukocyte interfaces. Arch. Dermatol. Res. 286,21 -29.[Medline]
Tammi, R., MacCallum, D., Hascall, V. C., Pienimäki, J.-P.,
Hyttinen, M. and Tammi, M. (1998). Hyaluronan bound to CD44
on keratinocytes is displaced by hyaluronan decasaccharides and not
hexasaccharides. J. Biol. Chem.
273,28878
-28888.
Tammi, R., Rilla, K., Pienimäki, J. P., MacCallum, D. K.,
Hogg, M., Luukkonen, M., Hascall, V. C. and Tammi, M. (2001).
Hyaluronan enters keratinocytes by a novel endocytic route for catabolism.
J. Biol. Chem. 276,35111
-35122.
Tammi, M., Day, A. and Turley, E. A. (2002).
Hyaluronan, a balancing act. J. Biol. Chem.
277,4581
-4584.
Tammi, R. H., Tammi, M. I., Hascall, V. C., Hogg, M., Pasonen, S. and MacCallum, D. K. (2000). A preformed basal lamina alters the metabolism and distribution of hyaluronan in epidermal keratinocyte "organotypic" cultures grown on collagen matrices. Histochem. Cell Biol. 113,265 -277.[Medline]
Thomas, L., Byers, H. R., Vink, J. and Stamenkovic, I. (1992). CD44H regulates tumor cell migration on hyaluronate-coated substrate. J. Cell Biol. 118,971 -977.[Abstract]
Toole, B. P., Wight, T. N. and Tammi, M. I.
(2002). Hyaluronan-cell interactions in cancer and vascular
disease. J. Biol. Chem.
277,4593
-4596.
Turley, E. A., Austen, L., Vandeligt, K. and Clary, C. (1991). Hyaluronan and a cell-associated hyaluronan binding protein regulate the locomotion of ras-transformed cells. J. Cell Biol. 112,1041 -1047.[Abstract]
Watanabe, K. and Yamaguchi, Y. (1996).
Molecular identification of a putative human hyaluronan synthase.
J. Biol. Chem. 271,22945
-22948.
Weigel, P. H., Hascall, V. C. and Tammi, M.
(1997). Hyaluronan synthases. J. Biol.
Chem. 272,13997
-14000.
Yung, S., Thomas, G. J. and Davies, M. (2000). Induction of hyaluronan metabolism after mechanical injury of human peritoneal mesothelial cells in vitro. Kidney Int. 58,1953 -1962.[CrossRef][Medline]
Zhang, S., Chang, M. C., Zylka, D., Turley, S., Harrison, R. and
Turley, E. A. (1998). The hyaluronan receptor RHAMM regulates
extracellular-regulated kinase. J. Biol. Chem.
273,11342
-11348.