BJD

T R AN SLA T IO NA L RE SE AR CH British Journal of Dermatology

Hyaluronan metabolism enhanced during epidermal

differentiation is suppressed by vitamin C*
€m a
L. Ha € l€ €rkka
ainen iD ,1 E. Ka €inen,1 P. Takabe,1 L. Rauhala,1 G. Bart,1 R. Ka
€rna
€,1 S. Pasonen-Seppa
€nen,1
1,2 1 1
S. Oikari, M.I. Tammi and R.H. Tammi
1
Institute of Biomedicine/Anatomy and 2Dentistry, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland

Linked Comment: Passi. Br J Dermatol 2018; 179:558–559.

Summary

Downloaded from https://academic.oup.com/bjd/article/179/3/651/6732402 by guest on 27 March 2023

Correspondence Background Hyaluronan is a large, linear glycosaminoglycan present throughout
Lasse H€am€al€ainen. the narrow extracellular space of the vital epidermis. Increased hyaluronan meta-
E-mail: lasse.hamalainen@uef.fi bolism takes place in epidermal hypertrophy, wound healing and cancer.
Hyaluronan is produced by hyaluronan synthases and catabolized by hyaluroni-
Accepted for publication
30 January 2018 dases, reactive oxygen species and KIAA1199.
Objectives To investigate the changes in hyaluronan metabolism during epidermal
Funding sources stratification and maturation, and the impact of vitamin C on these events.
This study was financially supported by the Fin- Methods Hyaluronan synthesis and expression of the hyaluronan-related genes
nish Dental Society Apollonia (L.H.), the Sigrid were analysed during epidermal maturation from a simple epithelium to a fully
Juselius Foundation (R.H.T., M.I.T.) and the
differentiated epidermis in organotypic cultures of rat epidermal keratinocytes
Spearhead Funds from the University of Eastern
Finland, Cancer Center of Eastern Finland
using quantitative reverse transcriptase polymerase chain reaction, immunostain-
(R.H.T., M.I.T., S.P.-S.). ing and Western blotting, in the presence and absence of vitamin C.
Results With epidermal stratification, both the production and the degradation of
Conflicts of interest hyaluronan were enhanced, resulting in an increase of hyaluronan fragments of
None to declare. various sizes. While the mRNA levels of Has3 and KIAA1199 remained stable dur-
ing the maturation, Has1, Has2 and Hyal2 showed a transient upregulation during
*Plain language summary available online
stratification, Hyal1 transcription remained permanently increased and transcrip-
DOI 10.1111/bjd.16423 tion of the hyaluronan receptor, Cd44, decreased. At maturation, vitamin C
downregulated Has2, Hyal2 and Cd44, whereas it increased high-molecular-mass
hyaluronan in the epidermis, and reduced small fragments in the medium, sug-
gesting stabilization of epidermal hyaluronan.
Conclusions Epidermal stratification and maturation is associated with enhanced
hyaluronan turnover, and release of large amounts of hyaluronan fragments. The
high turnover is suppressed by vitamin C, which is suggested to enhance normal
epidermal differentiation in part through its effect on hyaluronan.

What’s already known about this topic?

• The extracellular matrix polysaccharide hyaluronan is abundant in human epider-
mis and plays an important role in skin homeostasis and wound healing.
• Epidermis contains high levels of reactive oxygen species, which can induce
hyaluronan degradation.

What does this study add?

• Hyaluronan metabolism is activated during epidermal stratification but stabilized
again upon maturation. A large part of hyaluronan exists as fragments, which may
have signalling functions.
• Vitamin C further stabilized epidermal hyaluronan by suppressing both synthesis
and degradation, which is suggested to contribute to normal differentiation.

© 2018 British Association of Dermatologists British Journal of Dermatology (2018) 179, pp651–661 651

652 Epidermal maturation, vitamin C and hyaluronan metabolism, L. Ha € la
What is the translational message?
• Insults to epidermal homeostasis, such as trauma, inflammation and ultraviolet
radiation, activate hyaluronan turnover, which maintains an inflammatory state.
• The present data show that by slowing down hyaluronan turnover, vitamin C has
potential as a suppressor of epidermal inflammation.
Epidermis is formed of continuously proliferating and differ-
entiating keratinocytes.1 Hyaluronan is a very hydrophilic,
high-molecular-mass polysaccharide distributed in the extra-
cellular space throughout the human epidermis up to the
granular layer.2 During fetal development the loss of hyaluro-
nan staining precedes the formation of the granular and corni-
fied layers.3,4 Enzymatic removal of hyaluronan facilitates the
expression of late differentiation markers,5 suggesting a sup-
pressive effect on terminal differentiation. Accordingly,
hyaluronan accumulation is associated with delayed differenti-
ation and epidermal hyperplasia.4,6–9 Hyaluronan forms a gel,
facilitating the diffusion of nutrients, waste products and sig-
nalling molecules, which is of particular importance in tissues
lacking blood and lymphatic vessels. Hyaluronan contributes
to physiological processes, including cell proliferation, migra-
tion and differentiation.10
Hyaluronan is synthesized at the plasma membrane by
hyaluronan synthases 1–3 (HAS1–3). All three are expressed
in keratinocytes, but reports of their relative contributions to
epidermal hyaluronan production and roles in epidermal dif-
ferentiation have varied considerably.11–15
Epidermal hyaluronan turnover is rapid [half-life < 24 h in
human skin and organotypic rat epidermal keratinocyte (REK)
cultures],16,17 whereas in monolayer REKs the catabolic activ-
ity is limited (R. Tammi, unpublished data). The epidermal
staining pattern of the hyaluronan receptor CD44 correlates
with that of hyaluronan, suggesting their co-regulation and a
role for CD44 in hyaluronan turnover.18,19 The hyaluronidases
(HYAL) 1 and 2 are expressed in the epidermis.9,13,20 HYAL1
probably degrades hyaluronan in the granular cells,20 whereas
the role of HYAL2 is poorly understood. There are no reports
concerning the epidermal expression of KIAA1199 (CEMIP),
which is involved in hyaluronan degradation by fibroblasts.21
Hyaluronan is also fragmented by reactive oxygen species
(ROS).22,23 ROS are abundant in the epidermis,24,25 and ROS
scavengers inhibit epidermal hyaluronan degradation.26
We explored the influence of epidermal maturation on
hyaluronan content, size and metabolism using REKs, which
form a fully differentiated epidermis without the help of der-
mal feeder cells.27 We also studied the influence of vitamin C,
reported to facilitate normal epidermal differentiation,28 on
hyaluronan metabolism. During maturation from a simple
epithelium into a multilayered, stratified epidermis, both the
production and degradation of hyaluronan increased through
upregulation of Has1, Has2, Hyal1 and Hyal2. Vitamin C tended
to restrict the expression of Has2, Hyal1, Hyal2 and Cd44, and
British Journal of Dermatology (2018) 179, pp651–661
€ma
€inen et al.
stabilize hyaluronan by suppressing both its synthesis and
degradation.
Downloaded from https://academic.oup.com/bjd/article/179/3/651/6732402 by guest on 27 March 2023
Materials and methods
Cell culture
REKs were maintained as monolayers in Minimum Essential
Medium (MEM; Gibco/Thermo Fisher Scientific, Waltham,
MA, U.S.A.),29 10% fetal bovine serum [HyClone; Thermo
Fisher Scientific (Appendix S1 and Fig. S1; see Supporting
Information)], 4 mmol L 1 L-glutamine (Euroclone, Milan,
Italy), 50 lg mL 1 streptomycin (Euroclone) and 50 U mL 1
penicillin (Euroclone). Organotypic cultures were seeded with
100 000 REKs on polycarbonate inserts [0
4-lm pores, 12-
well dishes (Thermo Fisher Scientific); modified from Tammi
et al.].17 After 2 days, the upper-well medium was removed
and lower-well medium was replaced with Dulbecco’s Modi-
fied Eagle Medium (Gibco/Thermo Fisher Scientific) contain-
ing the same supplements as above  50 lg mL 1 L-ascorbic
acid (Sigma-Aldrich, St Louis, MO, U.S.A.).
Histology
Organotypic cultures were either fixed in 2% paraformalde-
hyde (Electron Microscopy Sciences, Hatfield, PA, U.S.A.) or
Histochoiceâ MB (Amresco, Solon, OH, U.S.A.), embedded in
paraffin, sectioned and stained with haematoxylin and eosin.
For hyaluronan staining,2 overnight incubation with biotiny-
lated hyaluronan binding complex was followed by avidin–bi-
otin peroxidase (Vector Laboratories, Burlingame, CA, U.S.A.),
diaminobenzidine (Sigma-Aldrich) plus H2O2 and Mayer’s
haematoxylin.30
For CD44,9 antigen retrieval was performed (Dako,
Glostrup, Denmark) before sequential incubations with the
anti-CD44 antibody (OX-50, 1 : 200; Chemicon/Millipore,
Billerica, MA, U.S.A.) and biotinylated antimouse antibody
(1 : 200; Vector Laboratories), followed by avidin–biotin
peroxidase.
Small interfering RNA transfections
Cells grown overnight on six-well plates were transfected with
small interfering RNA (siRNA; final concentration
30 nmol L 1) against rHas3 (Eurogentech, Liege, Belgium),
rHas2 and rHyal2 (Ambion, Austin, TX, U.S.A.) or control
© 2018 British Association of Dermatologists

Epidermal maturation, vitamin C and hyaluronan metabolism, L. Ha €la
siRNA (Origene, Rockville, MD, U.S.A.). siRNA and RNAiMAX
reagent (Thermo Fisher Scientific) in a-MEM (Euroclone)
were added to antibiotic-free MEM. After 4–6 h, transfection
medium was replaced with normal medium. The cultures
were harvested 2 days later.
RNA isolation, cDNA synthesis and quantitative reverse
transcriptase polymerase chain reaction
Epidermis was lysed and RNA extracted with TRI Reagentâ
(Molecular Research Center, Cincinnati, OH, U.S.A.). The purity
and the quantity of total RNA were measured with a NanoDrop
ND-1000 spectrophotometer (Thermo Fisher Scientific).
One microgram of total RNA was used for cDNA synthesis
with a VersoTM cDNA Synthesis Kit (Thermo Fisher Scientific).
Quantitative reverse transcriptase polymerase chain reaction
(qRT-PCR) was performed with MX3000P (Agilent Technolo-
gies/Stratagene, Santa Clara, CA, U.S.A.) using FastStart Univer-
sal SYBR Green Master with Rox (Roche, Mannheim, Germany).
Table S1 (see Supporting Information) lists the gene-specific
primers (Rplp0 used for normalization) and the cycling condi-
tions. Fold changes were calculated as described previously.31
Quantification of hyaluronan
Epidermis was digested with 0
05% (w/v) proteinase K (Fer-
mentas/Thermo Fisher Scientific) in 0
1 mol L 1 sodium
phosphate pH 6
5, 10 mmol L 1 ethylenediaminetetraacetic
acid at 60 °C for 1 h. After boiling for 10 min, hyaluronan
was measured by a competitive enzyme-linked immunosor-
bent assay (ELISA) kit (Echelon Biosciences, Salt Lake City,
UT, U.S.A.) or by a sandwich-type ELISA-like assay.32
For normalization, DNA was measured using bis-benzimida-
zole (Hoechst 33258; Sigma-Aldrich),33 with calf thymus
DNA standard (Sigma-Aldrich) in a fluorescence reader (Tecan
Group, M€annedorf, Switzerland).
Hyaluronan molecular mass analyses
Hyaluronan molecular size distribution was determined using
a Sephacryl S-1000 1 9 30 cm column.17 Hyaluronan
[2500 kDa (Hyalose, Oklahoma City, OK, U.S.A.) and GV-
Healon (Abbott Laboratories, Chicago, IL, U.S.A.)], hyaluronan
oligosaccharides (HA14, ~2
8 kDa; Seikagaku, Tokyo, Japan)
and glucuronic acid (Sigma-Aldrich) were used for calibration.
Oligosaccharides and glucuronic acid were assayed as described
(Table S2; see Supporting Information).34 Hyaluronan in the
fractions was measured after concentration (10 : 1) by both
competitive and sandwich-type ELISA-like assays. The fractions
in the low-molecular-mass peak were pooled, divided into two
equal portions, digested with proteinase K (200 lg mL 1 at
60 °C for 2 h), boiled, glycosaminoglycans precipitated with
ethanol and dissolved in 150 mmol L 1 sodium acetate (pH
6
8). One of the portions was subjected to Streptomyces hyaluro-
nidase (5 turbidity reducing units mL 1, 2 h at 60 °C), and
boiled and analysed with competitive ELISA.
© 2018 British Association of Dermatologists
€ma
€inen et al. 653
Protein isolation and Western blotting
Epidermis extracted in RIPA buffer [150 mmol L 1 NaCl,
50 mmol L 1 Tris, 1% Nonidet, 0
5% sodium deoxycholate,
0
1% sodium dodecyl sulfate (SDS); pH 7
5] was sonicated
on ice. In total, 10–15 lg protein,35 separated with 10%
SDS polyacrylamide gel electrophoresis, was transferred by
semi-dry blotting (Biometra GmbH, G€ ottingen, Germany)
to Protranâ nitrocellulose (GE Healthcare, Little Chalfont,
U.K.). Blocking with 5% milk powder or 1% bovine serum
albumin (for hyaluronidases 1 and 2, respectively), was
followed by incubation with anti-HYAL1 (1 : 500; LifeSpan
BioSciences, Seattle, WA, U.S.A.), anti-HYAL2 (1 : 100;
Abcam, Cambridge, U.K.) or anti-b-actin (1 : 4000; Sigma-
Downloaded from https://academic.oup.com/bjd/article/179/3/651/6732402 by guest on 27 March 2023
Aldrich), and fluorescent antirabbit or antimouse secondary
antibodies (1 : 4000; Thermo Fisher Scientific). The blots
were quantified with Odysseyâ Infrared System (LI-COR
Biosciences, Lincoln, NE, U.S.A.).
Analyses of uridine diphosphate glucose sugars
Eight- and 12-day specimens were homogenized in ice-cold
PBS in Lysing Matrix D tubes (MP Biomedicals, Santa Ana, CA,
U.S.A.) using a FastPrepâ homogenizer (Savant; Thermo
Fisher Scientific), whereas 4-day cultures were suspended in
ice-cold PBS and sonicated. After centrifugation, supernatants
were purified by EnviCarb columns (Supelco; Sigma-Aldrich)
before high-performance liquid chromatography.36 Total pro-
tein was quantified for normalization (Pierce BCA kit; Thermo
Fisher Scientific).
Statistical analysis
Data were analysed with SPSS Statistics, version 19 (IBM,
Armonk, NY, U.S.A.). Log transformation or Friedman test
was used if the data were not normally distributed or the vari-
ances were unequal. Mixed-model ANOVA and pairwise compar-
isons between the treatments were performed using the
estimated marginal means (least squares difference). The con-
trols (set as 1) and treatments were compared using the
cumulative distribution function p-norm in R for Windows,
version 2.14.1,37 and corrected for multiple comparisons.
One-sample t-test was used for comparisons of a single treat-
ment normalized to control (set as 1).
Results
Hyaluronan fragments accumulate in the epidermis
during maturation
REK layers on polycarbonate inserts were 1–2 cells thick on
day 4, i.e. 1 day after lifting to the air–liquid interface
(Fig. 1a). By day 8, the epidermis consisted of 3–5 vital layers
and a thin cornified layer (Fig. 1b). By day 12, a fully
matured epidermis with 5–6 vital cell layers and a thick stra-
tum corneum was observed (Fig. 1c).
British Journal of Dermatology (2018) 179, pp651–661
654 Epidermal maturation, vitamin C and hyaluronan metabolism, L. H€am€ala
€inen et al.

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Downloaded from https://academic.oup.com/bjd/article/179/3/651/6732402 by guest on 27 March 2023

(j) (k)

Fig 1. Hyaluronan production and distribution in organotypic keratinocyte cultures during differentiation. Organotypic rat epidermal keratinocyte
cultures were harvested after 4, 8 and 12 days of growth, fixed and processed for histology. Samples were stained with (a–c) haematoxylin and
eosin for morphology and with (d–f) biotinylated hyaluronan binding complex for hyaluronan, using diaminobenzidine as a chromogen. White
arrowheads indicate stratum corneum and black arrowhead loss of hyaluronan staining in granular layer. Scale bars = 50 lm. (g) Epidermal, (h)
medium and (i) total hyaluronan was quantified from organotypic keratinocyte cultures collected on the indicated days using a commercial,
competitive enzyme-linked immunosorbent assay (ELISA) kit. The concentration of hyaluronan in the unused medium (157 ng mL 1) was
subtracted from the hyaluronan detected in the used culture media. The data represent mean  SEM from five individual experiments with two
replicates in each. Statistical significance for log-transformed values was tested using a mixed-model analysis comparing the values to day 4
(**P < 0
01; ***P < 0
001). The molecular mass of hyaluronan was determined using S1000 size-exclusion chromatography, and competitive
hyaluronan (HA) ELISA from (j) epidermis and (k) culture media of 4- and 12-day-old organotypic keratinocyte cultures (means  SEM from
three independent experiments). The positions of hyaluronan standards (Hyalose) and hyaluronan oligosaccharides (HA14) are indicated by
arrows, and the three different size pools by bars.

Initially, all cell layers exhibited equal hyaluronan staining
(Fig. 1d), whereas the signal was depleted in the uppermost
vital layer during stratum corneum formation (Fig. 1f).
Hyaluronan staining resided on cell surfaces with some intra-
cellular signal detectable (Fig. 1e, f).
The concentration of hyaluronan in the unused medium
(derived from serum) was 157 ng mL 1 (mean of three sepa-
rate assays). This value, comprising approximately 25% and
7% of the hyaluronan in the culture media collected on days
4 and 12, respectively, has been subtracted from the values
presented in Figure 1h, i.
Culture media were changed 24 h before collecting the
samples for the hyaluronan assays. The content of hyaluronan
that diffused into medium during the 24-h incubation
exceeded that present in the epidermis (Fig. 1g–i). Hyaluro-
nan contents in the epidermis and medium were stable until
day 6, then doubled by day 8–10, with little further change
on day 12 (Fig. 1g–i).
The size distribution of hyaluronan in the culture medium
and epidermis could be divided into three major pools: one
corresponding to high-molecular-mass hyaluronan, one repre-
senting small fragments close to the inclusion volume of the
column (indicated by HA14; Fig. 1j) and one between them.
In the 4-day-old epidermis they comprised 38%, 41% and
21% of total hyaluronan, respectively (Fig. 1j), and 17%, 44%
and 39% in the mature (12-day-old) epidermis, indicating a

British Journal of Dermatology (2018) 179, pp651–661 © 2018 British Association of Dermatologists
 Epidermal maturation, vitamin C and hyaluronan metabolism, L. H€am€ala
€inen et al. 655

shift towards low-molecular-mass species. In the medium,
there was no obvious difference in the hyaluronan size distri-
bution between the immature and mature cultures (Fig. 1k).
The high-molecular-mass hyaluronan comprised the minority
of total hyaluronan in the medium (range 8–22%) (Fig. 1k).
The medium-sized fragments were the dominant species in
one of the three experiments, whereas the small fragments
dominated in two experiments. On average, the small species
constituted 54% of the hyaluronan in medium (Fig. 1k).
Expression of Has1 and Has2 transiently increase during
epidermal maturation
Using 3-day-old cultures as a reference point, Has3 and Has2
culture medium (Fig. S2a; see Supporting Information), as
previously reported.9 Cell-associated hyaluronan at cell–cell
contacts and apical cell surfaces was also depleted, especially
when combining Has2 and Has3 siRNAs (Fig. S2c–e).
The supply of precursor sugars also strongly influences
hyaluronan synthesis.38–40 We therefore analysed the concentra-
tions of UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-
glucuronic acid (UDP-GlcUA) and UDP-glucose (UDP-Glc),
which showed a progressive decline with maturation (Fig. 2d–f),
thus excluding elevated substrate levels as the cause for the
increased hyaluronan content in mature epidermis.
The expressions of Hyal1 and Hyal2 increase during

Downloaded from https://academic.oup.com/bjd/article/179/3/651/6732402 by guest on 27 March 2023

expression levels were higher in the organotypic cultures than
in the monolayers (qRT-PCR DCt-values for Has3 were 12 vs.
16 and for Has2 were 9 vs. 11), whereas Has1 expression was
approximately the same (DCt value: 13 vs. 14). The level of
Has3 mRNA did not significantly change during epidermal
maturation, whereas Has1 and Has2 increased approximately
twofold on days 6 and 4, respectively, and returned to the ref-
erence level in fully differentiated cultures (Fig. 2a–c).
The contributions of the different Has isoenzymes to
hyaluronan accumulation were also probed with siRNAs. Con-
sistently, attempts to silence Has1 failed, but ~60% knockdown
of Has2 and Has3 each reduced hyaluronan by 44% in the
epidermal maturation
Among the proteins involved in hyaluronan metabolism, the
expression of (based on day 3 qRT-PCR DCt values), Hyal2
and Cd44 mRNA levels were relatively high (DCt values: 9 and
8, respectively), whereas Hyal1 and KIAA1199 (CEMIP) were
lower (DCt values: 14 and 12, respectively). Hyal1 mRNA
increased from day 3 to day 6 (2
5-fold), remaining elevated
after that, whereas Hyal2 peaked on day 4 and returned close
to the initial level by day 6 (Fig. 3a,b). Cd44 mRNA was sig-
nificantly decreased on day 10 (0
7-fold; Fig. 3c), whereas
KIAA1199 mRNA did not significantly change during matura-
tion (Fig. 3d). With the anti-HYAL1 antibody, two faint

(a) (b) (c)

(d) (e) (f)

Fig 2. Has1, Has2 and Has3 exhibit distinct mRNA expression patterns during epidermal maturation. Relative mRNA expression was determined
using quantitative reverse transcriptase polymerase chain reaction from organotypic keratinocyte cultures on cultivation days 3–12. All results were
normalized to Rplp0, with day 3 as a calibrator (= 1). Data represent means  SEM from four individual experiments with two replicates in each.
Statistical significance was analysed using a mixed-model ANOVA, comparing the values from days 4–12 to day 3 (set as 1) using the pnorm-
function and correcting for multiple comparisons. For Has2 the nonparametric Friedman test was used owing to unequal variances. Epidermal
sheets from rat epidermal keratinocyte organotypic cultures on days 4, 8 and 12 were harvested and (d) UDP-N-acetylglucosamine (UDP-GlcNAc),
(e) UDP-glucuronic acid (UDP-GlcUA) and (f) UDP-glucose (UDP-Glc) analysed as described in the ‘Materials and methods’. The data represent
means  SEM from five independent experiments (four experiments for day 12) with 1–2 replicates in each. Statistical significance was analysed
using a mixed-model ANOVA, comparing the values from days 8 and 12 to day 4. *P < 0
05; **P < 0
01; ***P < 0
001.

© 2018 British Association of Dermatologists British Journal of Dermatology (2018) 179, pp651–661
 €ma
656 Epidermal maturation, vitamin C and hyaluronan metabolism, L. Ha €l€ainen et al.

(a) (b) (c)

(d)
(e)

Downloaded from https://academic.oup.com/bjd/article/179/3/651/6732402 by guest on 27 March 2023

(f)
(i)

(j)

(g) (h)

(k)

Fig 3. Hyaluronidase expression is altered during epidermal maturation. Relative mRNA expression of (a) Hyal1, (b) Hyal2, (c) Cd44 and (d)
KIAA1199 were determined using quantitative real-time polymerase chain reaction. mRNA levels were normalized to Rplp0 and day 3 was used as a
calibrator (= 1). The data represent means  SEM of four (Hyal1 and Hyal2) and three (Cd44 and KIAA1199) individual experiments with two
biological replicates in each. Statically significant comparing to day 3 were calculated as described in Figure 2. (e) HYAL1 and (f) HYAL2 protein
expression was determined from 4-, 8- and 12-day-old organotypic cultures using Western blot analysis. (g, h) HYAL2 was quantified after
normalization to b-actin. The data represent means  SEM from four independent experiments. Statistically significant differences at the 8- and
12-day time points compared with day 4 and between control and vitamin C-treated (12+C) samples at day 12 were calculated using a mixed-
model ANOVA correcting for multiple comparisons. (i–k) The distribution of CD44 was analysed by immunostaining with the OX50 antibody, as
described in ‘Materials and methods’. Arrowheads indicate stratum corneum. Scale bar = 50 lm. **P < 0
01; ***P < 0
001.

bands (~55 and 48 kDa, respectively) emerged in the mature

cultures (Fig. 3e), as reported previously,41 whereas samples
from early cultures were blank (Fig. 3e). This is in line with
the initially low Hyal1 mRNA, and its increase with epidermal
differentiation (Fig. 3a, e).
The anti-HYAL2 antibody also detected two bands (~56 and
52 kDa) in Western blots. Both were quenched by the Hyal2
siRNA, suggesting that they represent HYAL2 (Fig. 3f). Band
intensities were ~10-fold higher in the fully differentiated cul-
tures (Fig. 3f, g).
CD44 in the single-layered epidermis was mainly found on
apical and lateral cell surfaces (Fig. 3i). Its staining intensity
increased in the basal and spinous layers between culture days
8 (Fig. 3j) and 12 (Fig. 3k), but was always absent in the
uppermost vital cells in fully differentiated cultures (Fig. 3k).
Vitamin C slows down epidermal hyaluronan metabolism
As vitamin C exerts a powerful effect on epidermal differentia-
tion, we probed its influence on hyaluronan metabolism. In
day 8 samples, only the expression of Has1 was consistently
influenced by vitamin C, showing a significant upregulation
(Fig. 4). Has3 expression was strongly downregulated in two
of the three experiments, but it did not reach statistical signifi-
cance (Fig. 4c). In the fully differentiated cultures a marked
decrease in the expressions of Has2, Cd44 and Hyal2 was
observed, with a similar trend in Hyal1, but no changes in
Has1, Has3 and KIAA1199 (Fig. 4). The decrease of HYAL2 was
also detected by Western blotting (Fig. 3f, g). These changes
did not influence the spatial distribution of hyaluronan
(Fig. 5a, b) or CD44 (Fig. 5c, d). However, they were

British Journal of Dermatology (2018) 179, pp651–661 © 2018 British Association of Dermatologists

Epidermal maturation, vitamin C and hyaluronan metabolism, L. Ha €l€ainen et al. 657
(a) (b) (c)
(d) (e) (f)
(g)
Fig 4. Influence of vitamin C on hyaluronan-related genes in
organotypic cultures. Vitamin C (50 lg mL 1) was added every other
day to rat epidermal keratinocyte organotypic culture medium from
day 4 onwards. (a–g) mRNA expression of hyaluronan-related genes
was quantified by quantitative reverse transcriptase polymerase chain
reaction and normalized to Rplp0. Controls at each day were set as 1.
Data represent means  SEM from three individual experiments.
Statistically significant difference at each time point was calculated
using a mixed-model ANOVA correcting for multiple comparisons. The
treatments were compared with the controls (Ctrl; set as 1) using the
pnorm-function [*P < 0
05; ***P < 0
001 (compared to day 3)].
accompanied by a reduced leakage of hyaluronan into the
medium on day 8 (Fig. 5f), and followed by increased reten-
tion in the epidermis on day 12 (Fig. 5e). Hyaluronan size
distribution was shifted slightly towards larger species in both
the epidermis (from medium-sized to high-molecular-mass
hyaluronan; Fig. 5h) and in the medium (from small frag-
ments to medium-sized hyaluronan; Fig. 5i).
Discussion
This work showed that during the active tissue reorganization
from a single layer of keratinocytes into a multilayered epider-
mis, the metabolism of hyaluronan was stimulated, eventually
leading to an elevated hyaluronan content. Interestingly, most
of the hyaluronan comprised of medium-sized and small frag-
ments. At maturation, the hyaluronan content remained high,
© 2018 British Association of Dermatologists
€ma
even though the Has enzymes declined back to their prestratifi-
cation level, and their UDP sugar substrates were depleted.
Importantly, vitamin C further enhanced the differentiated
state by suppressing both synthesis and catabolism, facilitating
epidermal retention of high-molecular-mass hyaluronan.
The increased hyaluronan content during maturation of the
organotypic epidermal cultures resembles the findings in
human reconstituted skin.42 HAS2 and HAS3 were the main
hyaluronan producers in REK, and their expression levels were
higher in the three-dimensional model than in conventional
monolayers. The high but relatively stable Has3 expression
during stratification suggests that it produced hyaluronan at an
unchanged rate, not related to the exact maturation status, in
line with the results of Sayo et al.11 In contrast, the expression
Downloaded from https://academic.oup.com/bjd/article/179/3/651/6732402 by guest on 27 March 2023
of Has1 and Has2 increased transiently during stratification. Of
these, HAS2 was probably the main isoenzyme responsible for
hyaluronan production, as suggested by the Has2 and Has3
siRNA silencing experiments, and the relatively low expression
and low enzymatic activity of HAS1.43
The increased production of hyaluronan and Has2 expres-
sion during the stratification phase in our model resembles
those observed in epidermal hyperplasia during wound heal-
ing and growth factor treatment.4,7,12 Considerable amounts
of hyaluronan are detected in the hyperplastic epidermis of
wounded Has1/3 knockout mice, confirming the importance
of Has2 in activated epidermis.44 While the epidermal hyaluro-
nan synthesis does not directly correlate with cell prolifera-
tion,45 terminal differentiation is delayed by hyaluronan
synthesis,5 thereby contributing to epidermal hypertrophy by
increasing the number of vital cell layers.
Previous contradictory findings concerning Has2 expression
in keratinocytes may reflect specific factors in the experimental
conditions:13,14,46 growth factors present in serum are known
to upregulate Has2,7,12,47 whereas hydrocortisone and vitamin
C (present findings) considerably decrease it.48–50 Serum-free
media for keratinocytes often include both hydrocortisone and
vitamin C, resulting in suppressed Has2 expression.
Hyaluronan turnover is considerably higher in organotypic
REK cultures compared with the slow degradation in mono-
layers (~20% per day, R. Tammi, unpublished).17 This is
reflected by the large amounts of short hyaluronan fragments
found in the organotypic culture medium, previously underes-
timated because of the types of hyaluronan assays used.9,16,19
For example, the sandwich-type hyaluronan ELISA virtually
ignored fragments smaller than ~50 kDa. The presence of the
small fragments indicates that the final steps in the degrada-
tion process lag behind the initial extracellular fragmentation.
How abundant these fragments are in vivo, and whether they
are produced for a specific biological purpose, is unknown.
Interestingly, ~50-kDa hyaluronan fragments appear to pro-
mote the expression of genes related to epidermal differentia-
tion and tight junction complexes.51 The small fragments may
also have signalling functions for dermal cells, or they could
accumulate in the stratum corneum, affecting barrier charac-
teristics,52 and enhance bacterial defence mechanisms in skin
by inducing the secretion of b-defensins via activation of Toll-
British Journal of Dermatology (2018) 179, pp651–661
 €ma
658 Epidermal maturation, vitamin C and hyaluronan metabolism, L. Ha €l€ainen et al.

(a) (b)

(c) (d)

(e) (f) (g)

Downloaded from https://academic.oup.com/bjd/article/179/3/651/6732402 by guest on 27 March 2023

(h) (i)

Fig 5. Influence of vitamin C on CD44 and hyaluronan (HA) content and hyaluronan molecular mass distribution in organotypic cultures. Twelve-
day-old rat epidermal keratinocyte organotypic cultures (b, d) with or (a, c) without vitamin C treatment were stained for (a, b) hyaluronan and (c,
d) CD44, as described in ‘Materials and methods’. Arrowheads indicate stratum corneum. Scale bar = 50 lm. (e–g) Hyaluronan in the epithelium
and in the medium in control (Ctrl) and vitamin C-treated cultures was quantified with competitive hyaluronic acid enzyme-linked immunosorbent
assay. Data represent means  SEM from four individual experiments, with two replicates in each. The statistical difference at each time point
compared with the corresponding control was calculated using a mixed-model ANOVA (*P < 0
05; **P < 0
01; ***P < 0
001; ns, nonsignificant).
The molecular mass distribution of hyaluronan was determined from (h) epidermis and (i) culture media from 12-day-old organotypic cultures,
with or without vitamin C treatment, using S1000 gel filtration analysis. Means  SEM from three independent experiments are shown. The arrows
indicate the elution positions of hyaluronan standards and hyaluronan oligosaccharides (HA14), and the bars the different size pools.

like receptors (TLRs).53 Hyaluronan fragments may also act as
a self-sustaining regulatory loop to propagate further hyaluro-
nan production. They were also reported to increase hyaluro-
nan and CD44 staining in the mouse epidermis,54 together
with increased expression of Has2, Has3 and Hyal2.54,55 The
signalling route involved was independent of CD44, but
remained otherwise unrevealed. Engagement of TLRs by
hyaluronan fragments activates nuclear factor jB (NF-jB),56,57
which, in turn, can control Has2 expression.58,59
During epidermal stratification, hyaluronan fragments
increased concomitantly with the upregulation of Hyal2 and
Hyal1. The increase in medium-sized and short hyaluronan
fragments matches the properties of HYAL2, with a reported
maximal degradation down to ~20-kDa fragments.56
KIAA1199 (CEMIP) is another protein that could account for
the hyaluronan fragments,21 but its mRNA expression was
unaffected by epidermal differentiation and was actually high-
est in the monolayers (data not shown). While both HYAL1
and HYAL2 are most active at lysosomal pH, the presence of
HYAL2 on the plasma membrane suggests a contribution to
the extracellular degradation of hyaluronan.60 Thus, HYAL2
and ROS are good candidates for producing the hyaluronan
fragments observed.
The low levels of Hyal1 mRNA and protein in the organ-
otypic REK cultures, and appearance of the HYAL1 protein late
in maturation, are in line with its strict location in the

British Journal of Dermatology (2018) 179, pp651–661 © 2018 British Association of Dermatologists

Epidermal maturation, vitamin C and hyaluronan metabolism, L. Ha
granular layer,20 where it has been suggested to act as a gate-
keeper, restricting high-molecular-mass hyaluronan entering
the stratum corneum.20
CD44 is an abundant cell-surface protein in keratinocytes,
important for binding and organizing pericellular hyaluronan,
and contributing to its endocytosis.18,61,62 The Cd44 mRNA
level was reduced during epidermal maturation. However, no
such trend was found in the CD44 content during immuno-
histochemical examination of the basal and spinous layers. As
in fetal epidermal maturation,4,63 CD44 disappeared from the
superficial layers upon formation of the granular layer and the
diffusion barrier. The disappearance of CD44 and increased
HYAL1 activity both probably contribute to the lack of
hyaluronan in the granular layer.
Vitamin C has a dual-sided effect on free radicals. At low
doses it acts as an antioxidant, protecting cells from the harmful
effects of ROS,64 but at high (mmol L 1) doses it can be a pro-
oxidant, leading to apoptosis.65 The topical formulations pro-
tecting against ultraviolet (UV) radiation contain relatively high
concentrations of vitamin C (15–20%),64,66 but the biologically
effective dose may be considerably lower owing to the poor
penetration of vitamin C through stratum corneum.67
In the present study, the relatively low, close to the physio-
logical concentration of vitamin C appeared to slow down
hyaluronan metabolism in the reconstituted epidermis.68 At
present, the mechanism by which this happens remains open
to discussion. Although ROS can directly fragment hyaluronan
chains non-enzymatically,69,70 and scavenging of free radicals
by the use of catalase or superoxide dismutase delayed degra-
dation of epidermal hyaluronan in human skin organ cul-
tures,26 the fact that vitamin C in REK cultures strongly
influenced the expression levels of the enzymes involved in
hyaluronan degradation, as well as synthesis, suggests that
other mechanisms were involved. Vitamin C is known to reg-
ulate intracellular signalling pathways like NF-jB via both
ROS-dependent and independent mechanisms.71
As hyaluronan metabolism is stimulated in activated ker-
atinocytes, for example owing to wounding, irritation, UV
light, growth factors, malignant transformation or inflamma-
tion,72 the finding that vitamin C can normalize hyaluronan
turnover fits well with previous reports of the beneficial effects
of vitamin C on ceramide synthesis, formation of cornified
envelopes and barrier properties of the stratum corneum.28,73,74
The contribution of hyaluronan metabolism to these effects
appears to be an interesting topic for further research.
Acknowledgments
We are very grateful to Ms Eija Rahunen, Ms Eija Kettunen,
Ms Arja Ven€al€ainen, Ms Silja Pyysalo and Mr Kari Kotikumpu
for their excellent technical assistance.
References
1 Matsui T, Amagai M. Dissecting the formation, structure and barrier
function of the stratum corneum. Int Immunol 2015; 27:269–80.
© 2018 British Association of Dermatologists
€m€ala
€inen et al. 659
2 Wang C, Tammi M, Tammi R. Distribution of hyaluronan and its
CD44 receptor in the epithelia of human skin appendages. Histo-
chemistry 1992; 98:105–12.
3 
Agren UM, Tammi M, Ryyn€anen M, Tammi R. Developmentally
programmed expression of hyaluronan in human skin and its
appendages. J Invest Dermatol 1997; 109:219–24.
4 Tammi R, Pasonen-Sepp€anen S, Kolehmainen E, Tammi M. Hyaluro-
nan synthase induction and hyaluronan accumulation in mouse epi-
dermis following skin injury. J Invest Dermatol 2005; 124:898–905.
5 Passi A, Sadeghi P, Kawamura H et al. Hyaluronan suppresses epi-
dermal differentiation in organotypic cultures of rat keratinocytes.
Exp Cell Res 2004; 296:123–34.
6 Maytin EV, Chung HH, Seetharaman VM. Hyaluronan participates
in the epidermal response to disruption of the permeability barrier
in vivo. Am J Pathol 2004; 165:1331–41.
Downloaded from https://academic.oup.com/bjd/article/179/3/651/6732402 by guest on 27 March 2023
7 Pasonen-Sepp€anen SM, Maytin EV, T€ orr€onen KJ et al. All-trans
retinoic acid-induced hyaluronan production and hyperplasia are
partly mediated by EGFR signaling in epidermal keratinocytes.
J Invest Dermatol 2008; 128:797–807.
8 Siiskonen H, T€orr€onen K, Kumlin T et al. Chronic UVR causes
increased immunostaining of CD44 and accumulation of hyaluro-
nan in mouse epidermis. J Histochem Cytochem 2011; 59:908–17.
9 Rauhala L, H€am€al€ainen L, Salonen P et al. Low dose ultraviolet B
irradiation increases hyaluronan synthesis in epidermal ker-
atinocytes via sequential induction of hyaluronan synthases Has1-3
mediated by p38 and Ca2+/calmodulin-dependent protein kinase
II (CaMKII) signaling. J Biol Chem 2013; 288:17999–8012.
10 Toole BP. Hyaluronan: from extracellular glue to pericellular cue.
Nat Rev Cancer 2004; 4:528–39.
11 Sayo T, Sugiyama Y, Takahashi Y et al. Hyaluronan synthase 3 reg-
ulates hyaluronan synthesis in cultured human keratinocytes. J
Invest Dermatol 2002; 118:43–8.
12 Pienim€aki JP, Rilla K, F€ op C et al. Epidermal growth factor acti-
ul€
vates hyaluronan synthase 2 in epidermal keratinocytes and
increases pericellular and intracellular hyaluronan. J Biol Chem
2001; 276:20428–35.
13 Averbeck M, Gebhardt CA, Voigt S et al. Differential regulation of
hyaluronan metabolism in the epidermal and dermal compartments
of human skin by UVB irradiation. J Invest Dermatol 2007; 127:687–97.
14 Kakizaki I, Itano N, Kimata K et al. Up-regulation of hyaluronan
synthase genes in cultured human epidermal keratinocytes by UVB
irradiation. Arch Biochem Biophys 2008; 471:85–93.
15 Makkonen KM, Pasonen-Sepp€anen S, T€ orr€onen K et al. Regulation
of the hyaluronan synthase 2 gene by convergence in cyclic AMP
response element-binding protein and retinoid acid receptor sig-
naling. J Biol Chem 2009; 284:18270–81.
16 Tammi R, S€a€am€anen AM, Maibach HI, Tammi M. Degradation of
newly synthesized high molecular mass hyaluronan in the epider-
mal and dermal compartments of human skin in organ culture. J
Invest Dermatol 1991; 97:126–30.
17 Tammi RH, Tammi MI, Hascall VC et al. A preformed basal lamina
alters the metabolism and distribution of hyaluronan in epidermal
keratinocyte “organotypic” cultures grown on collagen matrices.
Histochem Cell Biol 2000; 113:265–77.
18 Tammi R, MacCallum D, Hascall VC et al. Hyaluronan bound to
CD44 on keratinocytes is displaced by hyaluronan decasaccharides
and not hexasaccharides. J Biol Chem 1998; 273:28878–88.
19 Tammi R, Rilla K, Pienim€aki JP et al. Hyaluronan enters ker-
atinocytes by a novel endocytic route for catabolism. J Biol Chem
2001; 276:35111–22.
20 Malaisse J, Evrard C, Feret D et al. Hyaluronidase-1 is mainly func-
tional in the upper granular layer, close to the epidermal barrier. J
Invest Dermatol 2015; 135:3189–92.
British Journal of Dermatology (2018) 179, pp651–661
 €ma
660 Epidermal maturation, vitamin C and hyaluronan metabolism, L. Ha €l€ainen et al.
21 Yoshida H, Nagaoka A, Nakamura S et al. Murine homologue of
the human KIAA1199 is implicated in hyaluronan binding and
depolymerization. FEBS Open Bio 2013; 3:352–6.
22 Soltes L, Mendichi R, Kogan G et al. Degradative action of reac-
tive oxygen species on hyaluronan. Biomacromolecules 2006;
7:659–68.
23 Duan J, Kasper DL. Oxidative depolymerization of polysaccharides
by reactive oxygen/nitrogen species. Glycobiology 2011; 21:401–9.
24 Carr WJ, Oberley-Deegan RE, Zhang Y et al. Antioxidant proteins
and reactive oxygen species are decreased in a murine epidermal
side population with stem cell-like characteristics. Histochem Cell Biol
2011; 135:293–304.
25 Hamanaka RB, Glasauer A, Hoover P et al. Mitochondrial reactive
oxygen species promote epidermal differentiation and hair follicle
development. Sci Signal 2013; 6:ra8.
26 
Agren UM, Tammi RH, Tammi MI. Reactive oxygen species con-
tribute to epidermal hyaluronan catabolism in human skin organ
culture. Free Radic Biol Med 1997; 23:996–1001.
27 Bart G, H€am€al€ainen L, Rauhala L et al. rClca2 is associated with
epidermal differentiation and is strongly downregulated by ultravi-
olet radiation. Br J Dermatol 2014; 171:376–87.
28 Pasonen-Sepp€anen S, Suhonen TM, Kirjavainen M et al. Vitamin C
enhances differentiation of a continuous keratinocyte cell line
(REK) into epidermis with normal stratum corneum ultrastructure
and functional permeability barrier. Histochem Cell Biol 2001;
116:287–97.
29 Baden HP, Kubilus J. The growth and differentiation of cultured
newborn rat keratinocytes. J Invest Dermatol 1983; 80:124–30.
30 Tammi R,  Agren UM, Tuhkanen AL, Tammi M. Hyaluronan meta-
bolism in skin. Prog Histochem Cytochem 1994; 29:1–81.
31 Livak KJ, Schmittgen TD. Analysis of relative gene expression data
using real-time quantitative PCR and the 2(-Delta Delta C(T))
Method. Methods 2001; 25:402–8.
32 Hiltunen EL, Anttila M, Kultti A et al. Elevated hyaluronan concen-
tration without hyaluronidase activation in malignant epithelial
ovarian tumors. Cancer Res 2002; 62:6410–13.
33 Jalkanen M, Larjava H, Turakainen H, Tammi M. Improved appli-
cation of the diphenylamine reaction for the determination of
DNA. J Biochem Biophys Methods 1980; 3:195–9.
34 Blumenkrantz N, Asboe-Hansen G. New method for quantitative
determination of uronic acids. Anal Biochem 1973; 54:484–9.
35 Bradford MM. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of pro-
tein-dye binding. Anal Biochem 1976; 72:248–54.
36 Oikari S, Ven€al€ainen T, Tammi M. Borate-aided anion exchange
high-performance liquid chromatography of uridine diphosphate-
sugars in brain, heart, adipose and liver tissues. J Chromatogr A
2014; 1323:82–6.
37 R Development Core Team. R: A Language and Environment for Statistical
Computing. Vienna: Foundation for Statistical Computing, 2011.
38 Jokela TA, Jauhiainen M, Auriola S et al. Mannose inhibits hyaluro-
nan synthesis by down-regulation of the cellular pool of UDP-N-
acetylhexosamines. J Biol Chem 2008; 283:7666–73.
39 Rilla K, Oikari S, Jokela TA et al. Hyaluronan synthase 1 (HAS1)
requires higher cellular UDP-GlcNAc concentration than HAS2 and
HAS3. J Biol Chem 2013; 288:5973–83.
40 Deen AJ, Arasu UT, Pasonen-Sepp€anen S et al. UDP-sugar substrates
of HAS3 regulate its O-GlcNAcylation, intracellular traffic, extracel-
lular shedding and correlate with melanoma progression. Cell Mol
Life Sci 2016; 73:3183–204.
41 Puissant E, Gilis F, Dogne S et al. Subcellular trafficking and activity
of Hyal-1 and its processed forms in murine macrophages. Traffic
2014; 15:500–15.
British Journal of Dermatology (2018) 179, pp651–661
42 Malaisse J, Pendaries V, Hontoir F et al. Hyaluronan does not regu-
late human epidermal keratinocyte proliferation and differentia-
tion. J Biol Chem 2016; 291:6347–58.
43 Itano N, Sawai T, Yoshida M et al. Three isoforms of mammalian
hyaluronan synthases have distinct enzymatic properties. J Biol Chem
1999; 274:25085–92.
44 Mack JA, Feldman RJ, Itano N et al. Enhanced inflammation and
accelerated wound closure following tetraphorbol ester application
or full-thickness wounding in mice lacking hyaluronan synthases
Has1 and Has3. J Invest Dermatol 2012; 132:198–207.
45 Tammi R, Tammi M. Correlations between hyaluronan and epider-
mal proliferation as studied by [3H]glucosamine and [3H]thymi-
dine incorporations and staining of hyaluronan on mitotic
keratinocytes. Exp Cell Res 1991; 195:524–7.
46 Malaisse J, Bourguignon V, De Vuyst E et al. Hyaluronan metabo-
Downloaded from https://academic.oup.com/bjd/article/179/3/651/6732402 by guest on 27 March 2023
lism in human keratinocytes and atopic dermatitis skin is driven
by a balance of hyaluronan synthases 1 and 3. J Invest Dermatol
2014; 134:2174–82.
47 Pasonen-Sepp€anen S, Karvinen S, T€ onen K et al. EGF upregu-
orr€
lates, whereas TGF-beta downregulates, the hyaluronan synthases
Has2 and Has3 in organotypic keratinocyte cultures: correlations
with epidermal proliferation and differentiation. J Invest Dermatol
2003; 120:1038–44.
48 Zhang W, Watson CE, Liu C et al. Glucocorticoids induce a near-
total suppression of hyaluronan synthase mRNA in dermal fibro-
blasts and in osteoblasts: a molecular mechanism contributing to
organ atrophy. Biochem J 2000; 349:91–7.
49 Jacobson A, Brinck J, Briskin M. J ø. Expression of human hyaluro-
nan synthases in response to external stimuli. Biochem J 2000;
348:29–35.
50 Gebhardt C, Averbeck M, Diedenhofen N et al. Dermal hyaluronan
is rapidly reduced by topical treatment with glucocorticoids. J Invest
Dermatol 2010; 130:141–9.
51 Farwick M, Gauglitz G, Pavicic T et al. Fifty-kDa hyaluronic acid
upregulates some epidermal genes without changing TNF-alpha
expression in reconstituted epidermis. Skin Pharmacol Physiol 2011;
24:210–17.
52 Sakai S, Yasuda R, Sayo T et al. Hyaluronan exists in the normal
stratum corneum. J Invest Dermatol 2000; 114:1184–7.
53 Gariboldi S, Palazzo M, Zanobbio L et al. Low molecular weight
hyaluronic acid increases the self-defense of skin epithelium by
induction of beta-defensin 2 via TLR2 and TLR4. J Immunol 2008;
181:2103–10.
54 Kaya G, Tran C, Sorg O et al. Hyaluronate fragments reverse skin
atrophy by a CD44-dependent mechanism. PLOS Med 2006; 3:e493.
55 Nikolic DS, Ziori C, Kostaki M et al. Hyalurosome gene regulation
and dose-dependent restoration of skin atrophy by retinaldehyde
and defined-size hyaluronate fragments in dermatoporosis. Derma-
tology 2014; 229:110–15.
56 Stern R. Hyaluronan catabolism: a new metabolic pathway. Eur J
Cell Biol 2004; 83:317–25.
57 Jokela TA, Makkonen KM, Oikari S et al. Cellular content of UDP-
N-acetylhexosamines controls hyaluronan synthase 2 expression
and correlates with O-linked N-acetylglucosamine modification of
transcription factors YY1 and SP1. J Biol Chem 2011; 286:33632–
40.
58 Vigetti D, Genasetti A, Karousou E et al. Proinflammatory cytokines
induce hyaluronan synthesis and monocyte adhesion in human
endothelial cells through hyaluronan synthase 2 (HAS2) and the
nuclear factor-kappaB (NF-kappaB) pathway. J Biol Chem 2010;
285:24639–45.
59 Saavalainen K, Tammi MI, Bowen T et al. Integration of the activa-
tion of the human hyaluronan synthase 2 gene promoter by
© 2018 British Association of Dermatologists
 Epidermal maturation, vitamin C and hyaluronan metabolism, L. H€am€ala
common cofactors of the transcription factors retinoic acid recep-
tor and nuclear factor kappaB. J Biol Chem 2007; 282:11530–9.
60 Andre B, Duterme C, Van Moer K et al. Hyal2 is a glycosylphos-
phatidylinositol-anchored, lipid raft-associated hyaluronidase. Bio-
chem Biophys Res Commun 2011; 411:175–9.
61 Pasonen-Sepp€anen S, Hyttinen JM, Rilla K et al. Role of CD44 in
the organization of keratinocyte pericellular hyaluronan. Histochem
Cell Biol 2012; 137:107–20.
62 Jokela T, Oikari S, Takabe P et al. Interleukin-1beta-induced reduc-
tion of CD44 Ser-325 phosphorylation in human epidermal ker-
atinocytes promotes CD44 homomeric complexes, binding to
ezrin, and extended, monocyte-adhesive hyaluronan coats. J Biol
Chem 2015; 290:12379–93.
63 Tuhkanen AL, Tammi M, Pelttari A et al. Ultrastructural analysis of
human epidermal CD44 reveals preferential distribution on plasma
membrane domains facing the hyaluronan-rich matrix pouches. J
Histochem Cytochem 1998; 46:241–8.
64 Telang PS. Vitamin C in dermatology. Indian Dermatol Online J 2013;
4:143–6.
65 Venturelli S, Sinnberg TW, Niessner H, Busch C. Molecular mecha-
nisms of pharmacological doses of ascorbate on cancer cells. Wien
Med Wochenschr 2015; 165:251–7.
66 Pinnell SR, Yang H, Omar M et al. Topical L-ascorbic acid: percuta-
neous absorption studies. Dermatol Surg 2001; 27:137–42.
67 Jung EC, Zhu H, Zou Y et al. Effect of ultrasound and heat on per-
cutaneous absorption of L-ascorbic acid: human in vitro studies on
Franz cell and Petri dish systems. Int J Cosmet Sci 2016; 38:646–50.
68 Rhie G, Shin MH, Seo JY et al. Aging- and photoaging-dependent
changes of enzymic and nonenzymic antioxidants in the epidermis
and dermis of human skin in vivo. J Invest Dermatol 2001; 117:1212–17.
69 Monzon ME, Fregien N, Schmid N et al. Reactive oxygen species
and hyaluronidase 2 regulate airway epithelial hyaluronan frag-
mentation. J Biol Chem 2010; 285:26126–34.
© 2018 British Association of Dermatologists
€inen et al. 661
70 Esser PR, Wolfle U, Durr C et al. Contact sensitizers induce skin
inflammation via ROS production and hyaluronic acid degradation.
PLOS One 2012; 7:e41340.
71 Carcamo JM, Pedraza A, B orquez-Ojeda O et al. Vitamin C is a
kinase inhibitor: dehydroascorbic acid inhibits IkappaBalpha kinase
beta. Mol Cell Biol 2004; 24:6645–52.
72 Tammi RH, Tammi MI. Hyaluronan accumulation in wounded
epidermis: a mediator of keratinocyte activation. J Invest Dermatol
2009; 129:1858–60.
73 Kim KP, Shin KO, Park K et al. Vitamin C stimulates epidermal cer-
amide production by regulating its metabolic enzymes. Biomol Ther
(Seoul) 2015; 23:525–30.
74 Savini I, Catani MV, Rossi A et al. Characterization of keratinocyte
differentiation induced by ascorbic acid: protein kinase C involve-
ment and vitamin C homeostasis. J Invest Dermatol 2002; 118:372–9.
Downloaded from https://academic.oup.com/bjd/article/179/3/651/6732402 by guest on 27 March 2023
Supporting Information
Additional Supporting Information may be found in the online
version of this article at the publisher’s website:
Appendix S1 Further information on the fetal bovine
serum used in the experiments.
Fig S1. Molecular mass of fetal bovine serum and molecular
mass dependence of the different types of enzyme-linked
immunosorbent-like hyaluronan assays.
Fig S2. Effect of Has2 and Has3 siRNA suppression on
hyaluronan synthesis.
Table S1 Cycling conditions and primer sequences used in
qRT-PCR.
Table S2 Sequences of the siRNA-oligos used.
Powerpoint S1 Journal Club Slide Set.
British Journal of Dermatology (2018) 179, pp651–661