ORIGINAL PAPER

Role of CD44 in the organization of keratinocyte pericellularhyaluronan

Sanna Pasonen-Seppanen • Juha M. T. Hyttinen •

Kirsi Rilla • Tiina Jokela • Paul W. Noble •

Markku Tammi • Raija Tammi

Accepted: 25 October 2011

� Springer-Verlag 2011

Abstract CD44 is a ubiquitous cell surface glycoprotein,

involved in important cellular functions including cell

adhesion, migration, and modulation of signals from cell

surface receptors. While most of these CD44 functions are

supposed to involve hyaluronan, relatively little is known

about the contribution of CD44 to hyaluronan maintenance

and organization on cell surface, and the role of CD44 in

hyaluronan synthesis and catabolism. Blocking hyaluronan

binding either by CD44 antibodies, CD44-siRNA or hya-

luronan decasaccharides (but not hexasaccharides) removed

most of the hyaluronan from the surfaces of both human

(HaCaT) and mouse keratinocytes, resembling results on

cells from CD44-/- animals. In vitro, compromising

CD44 function led to reduced and increased amounts,

respectively, of intracellular and culture medium hyaluro-

nan, and specific accumulation below the cells. In vivo,

CD44-deficiency caused no marked differences in hyalu-

ronan staining intensity or localization in the fetal skin or in

adult ear skin, while tail epidermis showed a slight reduc-

tion in epidermal hyaluronan staining intensity. However,

CD44-deficient tail skin challenged with retinoic acid or

tape stripping revealed diffuse accumulation of hyaluronan

in the superficial epidermal layers, normally negative for

hyaluronan. Our data indicate that CD44 retains hyaluronan

in the keratinocyte pericellular matrix, a fact that has not

been shown unambiguously before, and that hyaluronan

abundance in the absence of CD44 can result in hyaluronan

trapping in abnormal locations possibly interfering there

with normal differentiation and epidermal barrier function.

Keywords CD44 � Hyaluronan � Keratinocyte �Epidermis � Pericellular matrix

Abbreviations

bHABC Biotinylated hyaluronan binding complex

ELSA Enzyme linked sorbent assay

Introduction

CD44, a ubiquitously expressed, single-pass transmem-

brane glycoprotein, is the main cell surface receptor for

hyaluronan, binding hyaluronan on the plasma membrane

of many cell types (Knudson et al. 1996), and assisting in

hyaluronan degradation by functioning as an endocytosis

receptor (Culty et al. 1992; Hua et al. 1993; Tammi et al.

2001). In addition to these functions in hyaluronan

metabolism, it has been shown to induce intracellular sig-

nals either on its own or through modulating growth factor

receptor activities that support cell proliferation, migration,

and invasion (reviewed in Toole 2004; Thorne et al. 2004;

Heldin et al. 2008).

CD44 is expressed in several isoforms due to its dif-

ferential, cell type-specific mRNA splicing and post-

translational modifications (Knudson et al. 1999). All

members of the CD44 family contain an NH-terminal link

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00418-011-0883-2) contains supplementarymaterial, which is available to authorized users.

S. Pasonen-Seppanen (&) � J. M. T. Hyttinen � K. Rilla �T. Jokela � M. Tammi � R. Tammi

Department of Anatomy, Institute of Biomedicine,

University of Eastern Finland, P.O.B. 1627,

70211 Kuopio, Finland

e-mail: Sanna.Pasonen@uef.fi

P. W. Noble

Duke University School of Medicine, Durham, NC, USA

123

Histochem Cell Biol

DOI 10.1007/s00418-011-0883-2

module through which they can bind hyaluronan (Teriete

et al. 2004; Banerji et al. 2007). However, the actual

capacity of CD44 to bind hyaluronan varies between cell

types, depending on the splice variant, post-translational

modifications, interactions with cytoskeleton, and aggre-

gation of CD44 (Lesley et al. 2000; Teriete et al. 2004;

Thorne et al. 2004; Banerji et al. 2007). Epidermal kerat-

inocytes express mainly CD44 isoforms which contain

variant exons v3–v10 and heparan sulfate (HS) (Kugelman

et al. 1992; Tuhkanen et al. 1998). The content of CD44 in

the epidermis is high, the HS substituted CD44 forming

one of the major proteoglycans in the epidermis (Tuhkanen

et al. 1998). In human epidermis hyaluronan and CD44

show close colocalization (Wang et al. 1992), and clinical

conditions showing upregulation of epidermal hyaluronan

expression are mirrored by similar changes in CD44

expression and localization (Tammi et al. 1994; Karvinen

et al. 2003a), suggesting that CD44 in the epidermis

functions as a hyaluronan receptor. This conclusion was

further supported by our experiments on cultured rat

keratinocytes (Tammi et al. 2001; Pasonen-Seppanen et al.

2003); however, the data were somewhat contradictory as

the displacement of hyaluronan with blocking CD44 anti-

bodies was incomplete and the competition of hyaluronan

binding required hyaluronan decasaccharides instead of

hexasaccharides typical for CD44 (Tammi et al. 1998).

In the present work, we demonstrate that CD44 is indeed

involved in retaining and organizing hyaluronan on the

keratinocyte plasma membrane. Compromising CD44

either by knocking out CD44 with siRNA or using anti-

CD44 blocking antibodies or hyaluronan oligosaccharides

in HaCaT keratinocytes leads to reduced hyaluronan con-

tent on the dorsal side of cell membrane and to hyaluronan

accumulation under the cells similarly as in mouse

CD44-/- keratinocytes. In CD44-/- keratinocytes,

CD44 transfection returned hyaluronan localization to the

dorsal cell surface, indicating the importance of CD44 in

the organizing of keratinocyte pericellular hyaluronan

matrix. Moreover, hyaluronan may be bound to keratino-

cytes via CD44 independent mechanism(s), perhaps

through hyaluronan synthases.

Materials and methods

Animals

The CD44-/- mice were obtained from Dr. Paul Noble,

West Haven, CT, USA and originally developed by

Dr. Tak Mak, Toronto, Canada (Schmits et al. 1997). Wild-

type mice (C57Bl/J, Jackson laboratory) were supplied by

the National Laboratory Animal Center (University of

Kuopio, Finland). The Animal Care and Use Committee of

the University of Kuopio approved the study protocols used

in these experiments, and they followed the National

Institutes of Health guidelines for animal care.

In wound healing experiments, tail skin was repeatedly

stripped with Scotch tape until a mild erythema was

induced (10–15 times) and skin samples were collected for

histology 2–10 days after the wounding. In retinoic acid

experiments, tail and ear skin was treated with an all-trans

retinoic acid (RA) containing cream, Avitcid 0.05%

(Tretinoin) once a day up to 14 days. Before each RA

treatment, skin was treated with 70% ethanol to weaken

epidermal permeability barrier. Control animals received

only 70% ethanol. Twenty-four hours after the last appli-

cation, the animals were killed by cervical dislocation and

the samples were collected for histology. The skin speci-

mens were fixed by overnight incubation in 2% parafor-

maldehyde in 0.1 M phosphate buffer, pH 7.2, followed by

dehydration and embedding in paraffin using standard

procedures. Sections of 3-lm thickness were cut and

stained with hematoxylin and eosin, and for hyaluronan as

described before (Tammi et al. 1994). Briefly, deparaffin-

ised sections were blocked with 1% bovine serum albumin

for 30 min, followed by an overnight incubation with

bHABC (3 lg/ml) (biotinylated hyaluronan binding com-

plex). The bound bHABC was visualized using avidin–

biotin-peroxidase complex (Vector laboratories, CA) and

diaminobenzidine (Sigma) as a chromogen as described

previously (Wang et al. 1992). The specificity of the

staining was checked with Streptomyces hyaluronidase

(Seikakagu Kogyo Co, Tokyo, Japan), in the presence of

protease inhibitors (Tammi et al. 1989).

Cell culture

HaCaT cells, developed by Boukamp et al. (1988) were

obtained from CLS (Heidelberg, Germany). They were

cultured in DMEM (high glucose, Life Technologies,

Paisley, Scotland) supplemented with 10% serum (FCS,

PAA Laboratories GMbH, Pasching, Austria), 2 mM glu-

tamine (Sigma) and 50 lg/ml streptomycin sulfate, and

50 U/ml penicillin (Sigma). For passaging, cells were

treated with EDTA (0.05% in PBS, Sigma) for 10 min, and

then with 0.05% trypsin (w/v), and 0.02% EDTA (w/v)

in phosphate-buffered saline (PBS) (Biochrom, Berlin,

Germany) for 10 min. The cells were plated at 1:10 split

ratio twice a week for maintenance.

Primary newborn mouse epidermal keratinocytes were

isolated and cultured as described by Hager et al. (1999).

Epidermis and dermis were separated by an overnight

incubation at 4�C in Dispase (type II, Boehringer-Mann-

heim, Mannheim, Germany). Keratinocytes were isolated

from epidermis with a 10-min 0.05% trypsin and 0.02%-

EDTA (Biochrom AG, Berlin, Germany)—treatment at

Histochem Cell Biol

123

37�C, suspended in N-MEM, and plated at 50,000 cells/

cm2 on dishes coated with 1 lg/cm2 of type IV collagen

(BD Biosciences, Bedford, MA). The N-MEM was based

on 50% 3T3-fibroblast conditioned medium and 50%

E-MEM (Biowhittaker, without calcium), and contained

8% Chelex-treated (BioRad, Hercules, CA, USA) fetal

bovine serum (FBS, HyClone, Logan, UT, USA), 0.06 mM

Ca2?, 0.4 lg/ml hydrocortisone (hydrocortisone hemisuc-

cinate, Sigma), 0.75 mM aminoguanidine nitrate (Aldrich,

Steinheim, Germany), 2 ng/ml EGF (Sigma), and 10-10 M

cholera toxin (Sigma). The medium was changed once a

day.

3T3-fibroblasts (a gift from Dr. Donald MacCallum,

University of Michigan, Ann Arbor, MI, USA) were cul-

tured in DMEM (high glucose, Life Technologies, Paisley,

UK) with 10% calf serum supplemented with iron (Sigma)

until subconfluent, washed twice with PBS, and supple-

mented with low Ca2? E-MEM with 8% Chelex-treated

serum. The medium was collected after 2 days and sterile

filtered.

Immunohistochemical stainings of cell cultures

Keratinocytes grown in 8-well chamber slides were fixed

with 2% paraformaldehyde for 20 min, permeabilized with

0.1% Triton X-100 in 1% bovine serum albumin-phosphate

buffer (BSA-PB) for 10 min, and stained for hyaluronan

using bHABC. The bound probe was visualized by incu-

bation with avidin–biotin-peroxidase complex (ABC,

Vector Laboratories Inc., Burlingame, CA, USA) for 1 h

and with 0.05% 3030-diaminobenzidine (DAB, Sigma) and

0.03% H2O2 for 5 min. For fluorescence microscopy,

hyaluronan was stained using streptavidin labeled either

with Texas Red or FITC (Vector, 1:1,000). To visualize the

amount of intracellular hyaluronan the cells were incubated

in the presence of Streptomyces hyaluronidase (Seikagaku

Kogyo Co., Tokyo, Japan, 10 TRU/ml, in culture medium)

for 10 min at room temperature before permeabilization

and staining for hyaluronan (Tammi et al. 2001). For dual

stainings of bHABC with CD44, the primary antibody for

CD44; Hermes 3 (1:200) for HaCaT cells, a generous gift

of professor Sirpa Jalkanen (University of Turku, Turku,

Finland), was mixed with bHABC, and the secondary

antibody FITC-anti-mouse (1:200) with TR-Streptavidin

(Vector, 1:1,000). For the dual stainings of hyaluronan and

ezrin, bHABC was mixed with anti-ezrin antibody (1:200,

LabVision, Fremont, CA, USA). Nuclei were labeled with

DAPI (1 lg/ml, Sigma-aldrich, St Louis, MO, USA). The

fluorescently labeled specimens were viewed with an

UltraView confocal scanner (PE-Wallac-LSR, Oxford,

UK), built on a Nikon TE300 microscope or with Zeiss

Axio Observer inverted microscope (40 9 NA 1,3 oil or

63 9 NA 1,4 oil-objectives) equipped with Zeiss LSM 700

confocal module (Carl Zeiss Microimaging GmbH, Jena,

Germany). The 3-dimensional rendering of images and

further modification was performed using ImageJ 1.32

software (http://www.rsb.info.nih.gov/ij/) or ZEN 2009

software (Zeiss). Further image processing was done with

Adobe Photoshop 6.0 software (Adobe, Mountain View,

CA, USA).

For measurement of the staining intensities, the HaCaT

cell cultures stained for total and intracellular hyaluronan

using FITC-streptavidin were systematically photographed

using Nikon Eclipse TE300 microscope equipped with an

Ultraview confocal scanner (Perkin Elmer Life Sciences)

taking 30 images per culture using 60 9 NA 1.4 oil

immersion objective using same microscopic settings for

all samples from the same experiment. The hyaluronan-

positive area exceeding the level of background staining

intensity was measured using the Image J-program.

Hyaluronan visualization for TEM was done as descri-

bed previously with minor modifications (Karvinen et al.

2003b). Briefly, HaCaT cultures were fixed with 2%

paraformaldehyde and 0.5% glutaraldehyde and blocked

with 3% BSA in 0.05% Saponin for 10 min. Thereafter the

cultures were incubated with bHABC (10 lg/ml) over-

night, washed, and incubated with streptavidin-HRP

(Vector, 1:500), followed by treatment with DAB. After

post-fixation with reduced osmium the cells were dehy-

drated and embedded in Spurrs resin. Thin sections were

stained with uranyl acetate and viewed using a JEOL 1200

EX microscope.

Manipulation of CD44-expression and hyaluronan

binding

To compete the hyaluronan binding to cell surface recep-

tors, we treated nearly confluent HaCaT cultures with

hyaluronan oligosaccharides consisting of six or ten

monosaccharide units in length (HA6, HA10) (from Sei-

kagaku Kogyo Co., Tokyo, Japan) at final concentrations of

0.2 mg/ml for 2 h. To compete the hyaluronan binding to

CD44, HaCaT cultures were incubated with the anti-CD44

Hermes 1 (Developmental Studies Hybridoma, 5 lg/ml,

developed under the auspices of the NICHD, and main-

tained by The University of Iowa, Department of Biolog-

ical Sciences, Iowa City, USA), or with non-immune rat

IgG (Sigma, 5 lg/ml) for 0.5, 2, 4, 6 and 24 h prior fixa-

tion, and the mouse cells with anti-mouse CD44 antibody

KM201 (10 lg/ml, Southern Biotechnology, Birmingham,

Alabama, USA) for 2 h.

To inhibit CD44 expression, HaCaT keratinocytes were

transfected with siRNAs specific for human CD44 (Am-

bion, Austin, TX, USA). Scrambled siRNA (Silencer�

Negative control #2 siRNA, Ambion) was used as a neg-

ative control. Subconfluent cultures were transfected with

Histochem Cell Biol

123

50 nM siRNAs using LipofectamineTM 2000 (InVitrogen,

Carlsbad, CA, USA) according to manufacturer’s instruc-

tions. Transfection medium was removed after 6 h of

incubation and replaced with ordinary culture medium. The

efficacy of knock-down was confirmed by Q-PCR, and

western blotting and immunostaining using anti-CD44

antibody Hermes 3. CD44 silencing of HaCaT keratino-

cytes caused an 80% reduction in CD44 mRNA expression

and a 50% reduction in protein level (supplementary

Fig. 1a–c). The cultures were fixed and stained for CD44

and hyaluronan 2 days after the transfection. Collection of

the media for hyaluronan synthesis measurements was

started 48 after the transfection and stopped at 72 h.

The plasmid containing human CD44 standard form

(a generous gift from Dr. Clare Isacke, Imperial College,

G) was transfected to mouse keratinocytes using ExGen

500 transfection reagent (Fermentas, Life Sciences, EU)

according to manufacturer’s instructions. 0.5 lg of plas-

mid DNA was mixed with 100 ll of 0.15 M NaCl and

3 ll of ExGen, and 20 ll of this solution was pipetted to

200 ll of medium per a well in 8-well chamber slide.

The cultures were fixed and stained for hyaluronan

and human CD44 as described above 2 days after the

transfection.

RNA isolation and quantitative RT-PCR

Forty-eight hours after CD44 siRNA transfection, HaCaT

cells were detached and lysed by adding 1 ml of the RNA

extraction reagent/well (EuroGOLD RNAPure, Euro-

clone), and the samples were stored at -70�C. The total

RNA was extracted with chloroform–isopropanol accord-

ing to the standard procedure, washed once with 75%

ethanol, and dissolved in sterile water.

The transcript levels of CD44 in the HaCaT cultures

were measured using quantitative real-time PCR (QRT-

PCR). Eight hundred nanograms of total RNA was reverse

transcribed and real-time PCR was performed with a

MX3000P thermal cycler (Stratagene, La Jolla, CA, USA)

using Brilliant SYBR Green q-PCR master mix (Strata-

gene). At the end of each run a melt curve was obtained to

monitor the quality of the amplicon. Fold inductions were

calculated using the formula 2�DDCt , where DDCt is the DCt

(treatment)-DCt (control). DCt is Ct Target gene-Ct Arpo

(acidic ribosomal phophoprotein, used to normalize tran-

script levels between samples), and Ct is where the detec-

tion threshold is crossed.

Western blotting

Fifteen micrograms of protein was resolved in a 10%

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) and transferred onto ImmobilonTM-NC

membranes (Millipore, Bedford, MA, USA) by 35 mA per

cm2 constant current with a SammyTM semidry blotter

(Schleicher and Schuell, Dassel, Germany). The blots were

blocked for 30 min at room temperature in 10 mM Tris,

150 mM NaCl, pH 7.4 (Tris-saline blocking buffer) con-

taining 1% fat-free milk powder and 0.1% Tween-20.

Thereafter the blots were incubated with primary antibody,

anti-CD44 (Hermes 3, 1:100) overnight at ?4�C. After

washing with 0.1% Tween-20 in Tris-saline buffer, the

blots were incubated with the horse-radish peroxidase-

conjugated secondary antibody, anti-mouse, for 1 h

(1:5,000, Santa Cruz). The protein bands were visualized

using the NEN chemiluminescent detection system (Life

Science Products, Boston, MA, USA) and Kodak Biomax

Light Film (Eastman Kodak Company, NY).

After CD44 visualization, the bound antibodies were

removed by NaOH (0.2 M) treatment for 5 min at room

temperature followed by incubation with an antibody

against actin (diluted 1:1,000, Sigma) in TBS containing

1% milk overnight at 4�C. After washes, the blots were

incubated with goat anti-rabbit IgG, (1:5,000 dilution in

TBS containing 1% milk, Santa Cruz) for 1 h at room

temperature.

Hyaluronan assay

HaCaT keratinocytes transfected with control and CD44

siRNAs were trypsinized and seeded into six-well plates

at 300,000 cells/well, and grown for 1 day. The medium

was changed and the incubation continued for 24 h. The

media were collected and the cell layers washed once

with EDTA, which were combined to the media. After

releasing the cells with trypsin, the cells were pelleted

and counted for normalization, while the supernatants

were boiled for 10 min to inactivate the trypsin. Hyalu-

ronan contents in the media and trypsinates were mea-

sured using enzyme-linked sorbent assay (ELSA) of

hyaluronan, performed as described earlier (Hiltunen et al.

2002). Briefly, Maxisorp Plates (Nunc, Roskilde, Den-

mark) coated with 1 lg/ml of the non-biotinylated HABR,

were blocked with 1% BSA, and incubated with standard

hyaluronan (ProVisc�, Alcon, Fort Worth, TX, USA) and

samples for 1 h at 37�C. The bound hyaluronan was

detected using bHABR (1 lg/ml), visualized with horse-

radish peroxidase streptavidin (Vector), and O-phenyl-

enediamine dihydrochloride (Sigma) in 0.03% H2O2. The

reaction was stopped with 4 M H2SO4 and the absor-

bances were read at 490 nm. Each sample and standard

was done in triplicate. The hyaluronan contents of the

media and trypsinates were combined to represent total

amount of hyaluronan in the culture.

Histochem Cell Biol

123

Metabolic labeling assay

HaCaT keratinocytes were seeded into 12-well plates at

100,000 cells/well, and grown until subconfluent (2 days).

Fresh medium was added to the cells, and [3H]-glucosa-

mine (final concentration 20 lCi/ml) (Perkin Elmer, Life

Sciences Inc.) and 35SO4 (final concentration 100 lCi/ml)

(Amersham, Little Chalfont, UK) were added at 0-, 3-, 6-

or 9-h time points followed by either 3 or 12 or 24 h

incubations. The medium and one 0.15 ml HBSS (Euro-

clone, Milano, Italy) wash of the cell layer were combined

and designated ‘medium’. Cell surface-associated hyalu-

ronan was detached with 0.25 ml 0.05% trypsin (w/v)/

0.02% EDTA (w/v) for 10 min at 37�C, the cells pelleted,

and washed with 150 ll of HBSS. The trypsin solution and

the HBSS wash were combined and designated ‘pericel-

lular’ while the cell pellet was designated as the ‘cells’

representing the trypsin-resistant hyaluronan pool con-

taining the intracellular hyaluronan and the possible con-

tamination from the pericellular pool which could not be

removed by the trypsin (Tammi et al. 2001). Hyaluronan

and other glycosaminoglycans were purified and quanti-

tated from the different cellular compartments after deter-

mination of the specific activity of the hexosamines as

described in detail earlier (Tammi et al. 2000). Briefly,

carrier hyaluronan (4 lg in 40 ll, Healon; Pharmacia,

Uppsala, Sweden) was added to each medium, trypsinate,

and intracellular sample to evaluate the recovery of the

samples. Papain (Sigma) digestion was performed at 60�C

overnight in 150 mM sodium acetate, pH 5.8 containing

5 mM cysteine-HCl, and 5 mM sodium-EDTA. The sam-

ples were heated at 100�C for 10 min, centrifuged, and

supernatants containing hyaluronan and other glycosami-

noglycans recovered. 1% cetylpyridinium chloride (CPC)

in 20 mM NaCl was added to each supernatant, followed

by incubation for 1 h at room temperature. The samples

were centrifuged at 13,000g for 15 min at room tempera-

ture and each supernatant was carefully removed by aspi-

ration. The CPC-precipitates were washed with H2O and

dissolved in 50 ll 4 M guanidine-HCl, and reprecipitated

with 900 ll of ethanol at -20�C for 1 h. The samples were

centrifuged and dissolved in 50 ll 0.5 M ammonium ace-

tate, pH 7.0, and digested for 3 h at 37�C with 25 mU

chondroitinase ABC and 1 mU of Streptococcal hyal-

uronidase (both from Seikagaku Kogyo, Tokyo, Japan),

and 39 ll injected onto a 1 9 30 cm Superdex Peptide

column (Pharmasia), eluted at 0.5 ml/min with 12 mM

NH4HCO3. The eluent was monitored at 232 nm, and ali-

quots of the 350-ll fractions were counted for [3H] and

[35S]. The carrier hyaluronan produced a disaccharide peak

at 232 nm, which was used to correct for any losses in the

purification (recovery 60–70%). The chemical quantitation

of hyaluronan and other glycosaminoglycans was done

from different compartments as described earlier (Tammi

et al. 2000).

Molecular mass of hyaluronan

Subconfluent keratinocytes were incubated in a medium

with 20 lCi/ml [3H]-glucosamine for 18 h. Aliquots of the

culture media and trypsinates, collected as described

above, were subjected to gel filtration on a Sephacryl

S-1000 column (Pharmacia, Sweden) as described previ-

ously (Karvinen et al. 2003b). The amount of labeled

hyaluronan was measured by incubating parallel aliquots

from each fraction in the presence and absence of Strep-

tomyces hyaluronidase (Seikagaku Co, 12.5 mU), both

precipitated with 1% cetylpyridinium chloride (Sigma) in

the presence of 5 lg of carrier hyaluronan. The increase of

[3H]-glucosamine in the supernatant of the hyaluronidase-

treated aliquot was a specific measure of hyaluronan.

Statistical analysis

Student’s t test for paired and unpaired samples was used to

test the significance of differences.

Results

CD44 retains hyaluronan on the apical cell surface

in cultured human keratinocytes

The HaCaT human keratinocyte cell line synthesized

0.74–1.48 ng of hyaluronan per 10,000 cells in an hour

when measured using metabolic labeling with [3H]-gluco-

samine and [35S]-sulphate (Table 1), while the total

amount of hyaluronan accumulated during 12 h of con-

tinuous labeling was 5 ng/10,000 cells (Table 1). After 3 h

of labelling, during 12 h about half of the newly synthe-

sized hyaluronan was found in the pericellular matrix

Table 1 HaCaT cells were metabolically labelled with [3H]-gluco-

samine and [35S]-sulphate and analyzed for hyaluronan as described

in ‘‘Materials and methods’’

Time

(h)

Total

(ng/10,000 cells)

Medium

(%)

Trypsinate

(%)

Cells

(%)

0–3 0.97 ± 0.03 36 ± 7 52 ± 6 12 ± 0 .5

3–6 0.80 ± 0.14 27 ± 0.5 64 ± 1.8 9 ± 1.1

6–9 1.48 ± 0.36 28 ± 1.8 51 ± 0.2 21 ± 1.5

9–12 0.74 ± 0.02 22 ± 0.7 53 ± 1.3 24 ± 0.6

0–12 4.88 ± 0.66 69 ± 0.8 21 ± 0.1 10 ± 0.1

0–24 6.27 ± 0.45 79 ± 0.3 13 ± 0.1 7.6 ± 0.3

The means and ranges of duplicate measurements from one experi-

ment of two with similar results are shown

Histochem Cell Biol

123

(released by trypsin), 9–24% resistant to trypsin and

22–36% in the medium, indicating that part of the newly

synthesized hyaluronan is rapidly either released to the

medium or internalized. Twelve and twenty-four hour

continuous labelling shows that hyaluronan tends to

cumulate in the medium (Table 1), while less changes

occur in the quantities on cell surface and that endocytosed.

Confocal microscopy showed that hyaluronan is local-

ized on the apical cell surfaces and at cell–cell contacts

(Fig. 1a–d), showing a partial colocalization with ezrin

(Fig. 1a, b) and CD44 (Fig. 1c, d). The patchy layer of

hyaluronan (Fig. 1b, d) was probably due to association

with small, ezrin-positive microspikes on the plasma

membrane (Fig. 1a, b, e). Hyaluronan was also found

Fig. 1 Pericellular hyaluronan staining is perturbed by CD44-

antibody blocking and hyaluronan decasaccharides. HaCaT cells

were dual stained for hyaluronan and ezrin (a, b; HA, green; ezrin,

red) and hyaluronan and CD44 (c, d, k, l; HA, red; CD44, green), and

for hyaluronan alone (e). In f–h, red colour indicates hyaluronan and

nuclei are blue. In f insert, cells were stained for hyaluronan (red),

CD44 (green) and nuclei (blue). In i and j, hyaluronan was visualized

using DAB as a chromogen. The specimen in (e) was stained for

hyaluronan, and processed in Epon and viewed in TEM. The cultures

in (g) and (h) were treated with hyaluronan hexasaccharides

(g) and decasaccharides (h) (0.2 mg/ml) for 2 h prior to the fixation.

In j, k and l the cultures were treated with blocking CD44 antibody

(Hermes 1, 5 lg/ml) for 24 (j), and 6 h (k, l), prior to the fixation and

staining for hyaluronan. Culture in (i) was treated with control IgG

(5 lg/ml) for 24 h. Arrows in (b) and (l) indicate hyaluronan below

the cells, asterisks in (d) intracellular hyaluronan, arrowheads in

(e) indicate hyaluronan on cell surface. c is a compressed image stack

and b, d and f insert Z-sections created from stacks. Other confocal

images represent single optical sections, l being focused on the

bottom of the cell layer; others are focused to intersect the nuclei.

Magnification bars represent 10 lm in (a), (c), (k) and l, 20 lm in

(f) insert and f–h, 1 lm in e, and 50 lm in i and j

Histochem Cell Biol

123

within cells, localized in vesicles close to the nucleus

(Fig. 1a, d, f insert). When HaCaT cells were treated with

short hyaluronan oligosaccharides consisting of ten or more

monosaccharide units, the staining for hyaluronan was

reduced compared with untreated cultures (Fig. 1h, f,

respectively), while hyaluronan hexasaccharides had no

effect on staining intensity (Fig. 1g). Treatment of HaCaT

cultures with Hermes 1 antibody, which partially blocks

binding of hyaluronan to human CD44 (Jalkanen et al.

1987), clearly reduced the intensity of pericellular hyalu-

ronan (Fig. 1i, j), although some staining was left on cell

surface, inside the cells (Fig. 1k), and below the cells

(Fig. 1l). In conclusion, as indicated by decasaccharide

competition, a large part of hyaluronan on human kerati-

nocyte surface is receptor bound, most likely to CD44 since

hyaluronan staining was reduced by an antibody specific to

CD44 (Hermes 1) (Fig. 1i, j).

To reduce CD44 expression, HaCaT cells were treated

with CD44-targeted siRNAs. CD44 silencing with siRNA

caused an 80% reduction in mRNA expression level and a

50% reduction in protein level as studied with qRT-PCR

and western blotting (supplementary Fig. 1a–c). The

staining of CD44 was markedly reduced as compared with

control siRNA (Fig. 2a, b), although some intensely posi-

tive cells remained (Fig. 2b), suggesting that some cells

were probably not transfected (supplementary Fig. 2) or

these cells may originally highly express CD44 and thus

contain some even after transfection. In line with the

antibody blocking experiments, the CD44 siRNAs

decreased hyaluronan staining intensity (Fig. 2c, d, sup-

plementary Fig. 2f). Image analysis of the stainings indi-

cated that the CD44 siRNA transfection reduced both total

cell-associated hyaluronan (Fig. 2e), as well as the intra-

cellular hyaluronan (Fig. 2f).

Quantification of hyaluronan with ELSA in the medium

and pericellular compartments showed that CD44 siRNA

treatment caused approximately 20% increase in the total

amount of hyaluronan in HaCaT cultures during a 24-h

study period (Fig. 2g). The increased amount, together

with the reduced intracellular hyaluronan staining, sug-

gested that CD44 knock-down slowed hyaluronan uptake

and catabolism in HaCaT cells. However, as the increase in

the total hyaluronan amount was moderate (*20%), the

proportion of catabolized hyaluronan seems to be relatively

low corresponding to the proportion internalized in the

metabolic labelling experiment (Table 1).

CD44-deficient mouse keratinocytes show low

hyaluronan staining intensity

The residual hyaluronan on HaCaT cell plasma membrane

following treatments with CD44 siRNA and CD44 anti-

body could either be due to another binding partner or

incomplete elimination of CD44 (Fig. 2b). To study the

latter possibility we took advantage of mice missing a

functional CD44 gene. Primary keratinocytes isolated

from the CD44-/- mice showed very low hyaluronan

staining intensity (Fig. 3a), compared with wild-type cells

(Fig. 3c). Wild-type mouse keratinocytes presented a

strong hyaluronan signal on the apical cell surface,

colocalizing with CD44 (Fig. 3f). Approximately 90% of

CD44-/- keratinocytes appeared either completely neg-

ative (Fig. 3a, asterisk), or showed a few faintly stained

hyaluronan spots on their apical surface (Fig. 3a, b insert,

arrows). In addition, about 10% of the CD44-/- cells

contained intensely stained, large hyaluronan deposits

below the cells (Fig. 3b, e). Interestingly, treatment of the

wild-type keratinocytes with anti-CD44 antibody, which

blocks hyaluronan binding, created similar hyaluronan

deposits below the cells, in addition to reducing the

hyaluronan staining on the apical cell surfaces (Fig. 3k, l),

while the control antibody did not alter hyaluronan

staining pattern (Fig. 3m).

To confirm that the lack of hyaluronan in the apical cell

surface of KO cells was actually due to deficiency of

CD44, CD44-/- keratinocytes were transfected with a

plasmid construct containing human CD44. Transfection

resulted in the reappearance of hyaluronan on the apical

cell surfaces, colocalizing there with the transfected CD44

(Fig. 3d, g, h), confirming the ability of CD44 to retain

hyaluronan on the apical cell surfaces.

To test if the remaining hyaluronan on CD44-negative

keratinocytes was bound to receptors, the cells were

incubated with hyaluronan oligosaccharides. The CD44-

deficient keratinocytes treated with HA-decasaccharides

(HA10) showed similar low-level staining on the apical

cell surfaces (Fig. 3i), as the untreated CD44-/- cells,

with occasional cells showing intensely stained patches on

the basal cell surfaces (Fig. 3j). Similar results were seen

when HA6 was used. The data suggest that the pericellular

hyaluronan in CD44-negative keratinocytes was bound

either to an unknown receptor type resistant to HA10

competition, or, more likely, to hyaluronan synthase(s).

We hypothesized that CD44 might stabilize the growing

hyaluronan chain, preventing its premature detachment,

thus promoting the synthesis of longer hyaluronan chains.

To check this, we analyzed the molecular mass distribution

of hyaluronan secreted by CD44-/- keratinocytes. Sev-

enty-six percentage of the newly synthesized hyaluronan

was released into the culture medium, while 20% was

found in the trypsinate and 4% was resistant to the trypsin

(Fig. 4a, b). Hyaluronan present in the pericellular matrix

(trypsin-releasable) and in the culture medium (Fig. 4a)

was mainly of high molecular mass ([2 million Da). This

indicates that CD44 is not necessary for hyaluronan chain

elongation.

Histochem Cell Biol

123

The hyaluronan resistant to trypsin (cells) contained

both high- and low-molecular-weight molecules, similar to

the equivalent fraction in rat (Tammi et al. 1998), and

human keratinocytes (Tammi et al. unpublished). The

presence of lower molecular weight species in this pool

suggests that some degradation has taken place. As

pericellular hyaluronan may not have been completely

removed by the trypsin digestion, we confirmed the pres-

ence of intracellular hyaluronan in these cells by histo-

chemical staining of hyaluronan by removing the

pericellular hyaluronan with Streptomyces hyaluronidase

prior to the permeabilization of the cell membranes

0

10

20

30

40

50

60

70

2

4

6

8

10

12

14

CD44 immunostainingControl siRNA

CD44 immunostainingCD44 siRNA

Hyaluronan stainingControl siRNA

Hyaluronan staining CD44 siRNA

5

10

15

20

25

0 0ControlsiRNA

ControlsiRNA

ControlsiRNA

CD44siRNA

CD44siRNA

CD44siRNA

Cell associated Intracellular

*

*

Hya

luro

nan

posi

tive

area

(%

)

Hya

luro

nan

posi

tive

area

(%

)

Hya

luro

nan

(ng/

10 0

00 c

ells

/24h

)**

a b

c d

e f g80 30

Fig. 2 siRNA block of CD44 expression reduces pericellular and

intracellular hyaluronan and retards hyaluronan turnover in HaCaT

cell cultures. HaCaT cells were transfected either with control

siRNAs (a, c) or with CD44-targeted siRNAs (b, d), cultured for

2 days after transfection and stained for CD44 (a, b) and hyaluronan

(c, d). Magnifying bar 50 lm. Hyaluronan-positive area was

measured from specimens where hyaluronan was visualized using

fluorescently labelled streptavidin using image analyses (e, f). The

data represent means and SEM from five (e) and eight (f) experiments.

The total amount of hyaluronan in the HaCaT cultures (g) was

measured using an ELSA (means and SEM of eight experiments). The

differences between groups treated with control siRNA and CD44

siRNA were tested using Student’s t test for paired samples,

*p \ 0.05, **p \ 0.01

Histochem Cell Biol

123

(Fig. 4c). The number of cells showing a clear intracellular

hyaluronan signal varied between experiments being

always less than 20% of all cells.

CD44 deficiency causes minor changes in mouse

epidermal hyaluronan

Little hyaluronan was present in the epidermis collected

from the wild-type (C57Bl/J, Jackson) mouse under normal

tissue homeostasis (Fig. 5a, d), a finding similar to other

mice lines [C57Bl/J subline, Harlan laboratory (Tammi

et al. 2005), K-mice (Siiskonen et al. in press) and hairless

SKH-1 mice (Maytin et al. 2004)]. The epidermis in ear

skin showed occasional low-intensity staining around a few

basal cells (Fig. 5a). In the tail skin most of the hyaluronan

was associated with hair follicles (data not shown) as

described previously (Tammi et al. 2005), while the

interfollicular areas were either negative or faintly positive

in the basal and lower spinous cells (Fig. 5d). In CD44-

deficient mice both ear and tail epidermis showed either

no hyaluronan staining or very low-intensity staining

(Fig. 5b, e).

During the embryonic period when hyaluronan expres-

sion in the epidermis is high (Tammi et al. 2005), CD44-

deficient mice (Fig. 5k) showed intense plasma membrane-

associated hyaluronan staining similar to the wild-type

mice (Fig. 5j). At E17 stage intense hyaluronan staining

was found in the basal and intermediate cell layers of the

Fig. 3 CD44-deficient mouse keratinocytes show reduced capacity to

retain hyaluronan in the pericellular matrix. Keratinocytes isolated

from CD44-negative (CD44-/-) and wild-type (WT) newborn mice

were cultured for 3–5 days as described in ‘‘Materials and methods’’.

Cells were stained for hyaluronan (red) and ezrin (green) or CD44

(green, f, h). Panels (d, g, h) represent CD44-/- keratinocytes

transfected with a human CD44 construct. Panels (i, j) represent

CD44-/- keratinocytes treated with hyaluronan decasaccharides

(HA10) for 5 (i) and 2 (j) hours prior to fixation and staining for

hyaluronan. WT cells in (k, l) were treated with a blocking anti-CD44

antibody KM201 (10 lg/ml) for 2 h prior to fixation and staining,

while the cell in (m) received non-immune IgG. Compressed stacks of

horizontal optical sections are shown in (c, d, k). Images in (b, j) were

focused on the basal surface of the cells, and those in (a, i, b insert) on

the upper cell surface. Z-sections created from the image stacks are

shown in (e, f, g, h, l, m). Most of the CD44-/- cells were either

hyaluronan negative (stars in a, i) or showed very low hyaluronan

signal on the apical cell surface (arrows in a, i), while those

expressing the transfected hCD44 showed a stronger pericellular

hyaluronan staining, mainly localized on the apical cell surface

(arrow in g). In some of the CD44-deficient cells hyaluronan

accumulated in the basal side of the cells (b, e). WT control

keratinocytes showed a strong hyaluronan staining on the apical cell

surface (f). Horizontal magnification bar represents 40 lm for the

horizontal sections, and the vertical bar represents 5 lm for the

Z-sections

Histochem Cell Biol

123

epidermis, while the more differentiated superficial layers

contained less hyaluronan (Fig. 5j, k). In general, there

were no obvious differences between wild-type and CD44-

deficient mice in hyaluronan localization or content in

epidermis or adjacent dermis in contrast to mice with

keratinocyte-targeted deletion of CD44 (Kaya et al. 1997).

The staining patterns were similar also in the newborn

mouse tail skin. However, the staining intensity tended to

be somewhat lower in the CD44-deficient specimens

(Fig. 5m) as compared with wild-type ones (Fig. 5l).

To better reveal the effects of missing CD44-receptor

for hyaluronan localization in the adult epidermis, HAS2/3

expression and hyaluronan production were stimulated by

topical retinoic acid application (Pasonen-Seppanen et al.

2008). A clear increase in hyaluronan staining was seen in

the retinoid-treated ears concomitantly with epidermal

thickening (Fig. 5c). Hyaluronan decorated the keratino-

cyte surfaces in the basal and spinous layers without any

relocalization to superficial epidermal layers or to the

dermal side (Fig. 5c). Similarily, treatment of the tail skin

with retinoic acid (Fig. 5f), or by tape stripping caused a

marked upregulation of hyaluronan staining in the epider-

mis of CD44-deficient animals (Fig. 5g, h, i). The intensity

of the staining in the lower epidermal layers varied

between different specimens from very intense to faintly

positive (Fig. 5g vs. h and i). In addition to the location in

the basal epidermal strata, hyaluronan was also found in

the more superficial cell layers. Furthermore, while in the

basal cell layers hyaluronan was localized on the kerati-

nocyte plasma membranes, in the superficial layers it

formed diffuse, high-intensity deposits (Fig. 5f, h, i,

arrows). Due to the intensity of the staining, it is impossible

to know if hyaluronan was localized between the cells or

inside them. In between the two HA-positive strata, basal

and superficial, there was often a hyaluronan-negative layer

(Fig. 5g).

Epidermal thickness is not influenced by CD44

deficiency

Given the major changes that take place in hyaluronan

metabolism during epidermal differentiation, growth, and

Fraction numberFraction number17 21 25 29 33 37 17 21 25 29 33 37

Medium

Trypsinate

Intracellular

Hya

luro

nan

(DP

Mx1

0 )3

0

2

4

6

8

10

12

14

16

0

0.1

0.2

0.3

0.4

0.5

Vo = 19Vt = 48

Vo = 19Vt = 48

a

c

b

Intracellular HA

Fig. 4 Size distribution of hyaluronan in the culture medium,

trypsinate and cell fractions in CD44-/- keratinocytes. Cultured

mouse keratinocytes were metabolically labelled with 3H-glucosa-

mine for 20 h. Proportional aliquots from different compartments

(a medium and trypsinate; b intracellular) were analyzed using S1000

gel filtration columns as described in the ‘‘Materials and methods’’.

Each fraction was analyzed for hyaluronan by its susceptibility to

Streptomyces hyaluronidase. Void volume was at fraction 19 and total

volume at fraction 48. The figure represents data from one experiment

of two with similar results. In c, Streptomyces hyaluronidase

treatment after fixation was used to remove pericellular hyaluronan

before permeabilization to specifically visualize intracellular hyalu-

ronan. Approximately 10% of CD44-/- keratinocytes contained

intracellular hyaluronan. Magnification bar represents 20 lm

Histochem Cell Biol

123

inflammation, CD44 should also contribute to these pro-

cesses. In particular, CD44 has been shown to modulate

cell proliferation in many cell types including keratino-

cytes, via influencing growth factor signaling (Kaya et al.

1997; Barnes et al. 2010; Meran et al. 2011; Wang and

Bourguignon 2006). However, despite the prominent

expression of CD44 in the epidermis during the fetal period

(Underhill 1993; Tammi et al. 2005), CD44-KO mice

showed normal development of the epidermis. We could

not detect any tendency to epidermal atrophy in adult

animals either when studying the ear or tail epidermis

(Fig. 5), in contrast to a previous study (Bourguignon et al.

2006). To confirm this we measured the epidermal thick-

ness from tail skin specimens from age-matched animals

Fig. 5 Hyaluronan accumulation in the superficial epidermal layers

in CD44-deficient mouse tail epidermis by tape stripping and retinoic

acid. Skin specimens from wild-type (a, d, j, l) and CD44-/- (b, c, e,

f, g, h, i, k, m) animals were stained for hyaluronan. Panels (a–c) are

from ear skin and (d–i) from tail skin of adult animals, whereas

(j, k) are from D17 embryonic head skin and (l, m) from tail skin of

newborn animals. Specimens in g–i were wounded with tape stripping

2 days (g) or 3 days (h, i) before sample collection; specimens in

(c, f) were treated with retinoic acid for 4 days. Untreated adult

epidermis from both WT and CD44-/- animals shows just

low-intensity hyaluronan staining or is totally negative, while

embryonic epidermis shows a strong hyaluronan signal, and in the

newborn animals the spinous cells remain positive. Strong hyaluronan

staining is seen in tape-stripped (wounded) and retinoic acid-treated

epidermis. In tail skin (f–i) the distribution varies between the

specimens, but shows typically accumulation of hyaluronan in

superficial epidermal layers (arrows). Asterisk indicates a layer with

lower staining intensity. The dash lines denote the epidermis and

dermis junction. Magnification bars represent 25 lm

Histochem Cell Biol

123

(age 7–12 months). The epidermal thickness was slightly

lower in wild-type animals than in CD44 KO animals

(25.2 ± 0.6 lm; n 7 and 27.1 ± 0.9 lm; n 17, respec-

tively), but the difference was not significant (unpaired

t test, p = 0.221).

Discussion

The present data show that CD44 is involved in binding

and organizing pericellular hyaluronan in keratinocytes,

but at the same time suggested that there are also other

mechanisms involved in this process. CD44 is necessary

for an even distribution of hyaluronan in proper plasma

membrane domains on the apical keratinocyte surfaces in

vitro. Thus, perturbation of CD44 either by knocking it

down by gene deletion or by siRNA, or by blocking its

function with anti-CD44 antibodies or hyaluronan deca-

saccharides, leads to hyaluronan release from the cell

surface to the culture medium, where it forms loose

accumulations below the cells. Our data indicate that this

abnormal hyaluronan localization in CD44-deficient

keratinocytes is returned with CD44 transfection to

CD44-/- keratinocytes. The absence of CD44 may also in

vivo allow abnormal hyaluronan localization in epidermis

challenged with factors that increase hyaluronan synthesis.

The data suggest that the influence of CD44 is insignificant

on mouse epidermal development during fetal period and

on normal differentiation in adults, which may be due to its

compensation by another hyaluronan-binding receptor like

RHAMM (Nedvetzki et al. 2004). However, the role of

CD44 in holding hyaluronan may become important upon

skin injury.

In human and mouse keratinocytes, hyaluronan dis-

placement from CD44 required decasaccharide size hya-

luronan fragments (HA10), as found previously in rat

keratinocytes (Tammi et al. 1998). The reason for the

requirement for a longer stretch of hyaluronan for efficient

competition, untypical for CD44, remains open for further

experimentation. However, there are earlier studies, which

support the present results. Teriete et al. (2004) and Banerji

et al. (2007) have indicated that decasaccharides bind

better to CD44 than shorter oligomers, which may explain

why HA10-mers give more efficient competition than

HA6-mers. The fact that the affinity of hyaluronan to CD44

is low but is increased by CD44 clustering (Lesley et al.

2000) raises the idea that in certain conditions CD44 alone,

or together with other proteins, forms complexes that

provide extended hyaluronan binding sites.

The residual cell surface hyaluronan seen in CD44-

deficient keratinocytes may be bound to HAS enzymes as

with transfected Has genes (Rilla et al. 2005; Kultti et al.

2006). Therefore, cells in an active phase of hyaluronan

synthesis may form considerable hyaluronan pericellular

coats without CD44, a notion supported by the consider-

able HA-positive staining around keratinocytes in the fetal

epidermis of CD44-deficient mice and in the ear epidermis

stimulated by retinoic acid. Hyaluronan may also be bound

to another hyaluronan receptor like RHAMM, also

expressed by keratinocytes (Lovvorn et al. 1998; Yamano

et al. 2008). However, all known hyaluronan receptors and

hyaluronan-binding matrix proteins are displaced by

hyaluronan decasaccharides, suggesting that the residual

pericellular hyaluronan in CD44-deficient keratinocytes is

bound to HAS enzymes.

CD44 has been shown to mediate hyaluronan endocy-

tosis (Culty et al. 1992; Hua et al. 1993). Our previous data

on rat keratinocytes also suggested that CD44 may inter-

nalize hyaluronan (Tammi et al. 2001). The decreased

amount of intracellular hyaluronan in HaCaT cells treated

with CD44-specific siRNA is in line with the previous

findings. As blocking CD44 function in vitro caused accu-

mulation of hyaluronan below the cells, this domain may be

a site of active hyaluronan clearance by CD44. An alter-

native explanation, that HAS in the absence of CD44 would

specifically relocalize to the basal cell surface, seems

improbable as transfected GFP-HAS3/2 showed similar

plasma membrane distributions in CD44-deficient and

CD44-positive cells (Kultti et al. 2006). The lack of obvious

differences in hyaluronan distribution or content in

embryonic epidermis of CD44-deficient mice also suggests

that there are redundant clearance mechanisms, like frag-

mentation by free radicals or Hyal2, followed by diffusion

to dermis. Endocytosis of HAS (Rilla et al. 2005) could also

bring in the associated hyaluronan for degradation in vivo.

Our experiments with mouse tail skin in vivo suggest

that lack of CD44 may under certain circumstances abolish

the localization and clearance of hyaluronan. The diffuse

accumulation of hyaluronan in the upper epidermal layers

in the tail skin suggests that the inability to retain hyalu-

ronan on cell surface leads to its movement away from the

site of synthesis, in line with our in vitro data showing that

the importance of CD44 in immobilizing hyaluronan in the

apical pericellular matrix of keratinocytes. When the per-

meability barrier is compromised by tape stripping or ret-

inoic acid treatment, the hyaluronan which is not

immobilized by CD44 may be drawn toward the surface,

aided by the increased flux of water through the skin, and

end up in a compartment lacking a clearance mechanism

for hyaluronan. In the thin ear skin the compensatory

mechanisms for hyaluronan clearance seem to be more

effective than in the tail epidermis, providing effective

enough clearance for epidermal hyaluronan even when the

synthesis is activated by retinoic acid.

It was recently reported that CD44-/- mice show

delayed recovery after barrier disruption, with increased

Histochem Cell Biol

123

transepidermal water loss, and delayed development of

tight junctions during the fetal development (Kirschner

et al. 2011). Accumulation of the highly hydrophilic hya-

luronan in the uppermost vital cell layers could contribute

to the water loss in CD44-deficient mice. One could

hypothesize that by keeping open the intercellular spaces

hyaluronan could compromise the assembly of tight junc-

tions and thereby delay the healing process. However,

whether macromolecular hyaluronan enhances or delays

reformation of the epidermal diffusion barrier remains

controversial at the moment (Bourguignon et al. 2006;

Kirschner et al. 2011).

In conclusion, the present data show that although the

lack of CD44 deficiency has just minor consequences on

epidermal hyaluronan localization and content in mouse

skin in vivo under normal tissue homeostasis, CD44 is

involved in the binding and organizing pericellular hyalu-

ronan both in mouse and human keratinocytes. Its absence

leads to hyaluronan release from cell surface with sub-

sequent accumulation to abnormal localization below the

cells in vitro and diffuse deposits close to permeability

barrier in vivo. As a consequence, it may disturb cell to

substratum and cell to cell attachments and interfere with

differentiation-specific proteins (Passi et al. 2004) or for-

mation of proper diffusion barrier, a hypothesis which

warrants further research.

Acknowledgments We are grateful to Ms. Eija Rahunen and

Mr. Kari Kotikumpu for preparing the histological specimens, and

Ms. Riikka Karna, Arja Venalainen and Eija Kettunen for taking care

of the cell cultures and performing the hyaluronan measurements. We

thank the personnel of the Laboratory Animal Center for taking care

of the animals, and Biomater Center for providing us the confocal and

transmission electron microscopes. The work was supported by grants

from Finnish Cancer Foundation (RT), Juselius Foundation (RT, MT),

EVO Funds of the University Hospital of Kuopio (MT), funds

from BioCenter and Cancer Center of University of Eastern Finland

(RT, MT), and Academy of Finland (MT).

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