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Tammi and Markku I. Tammi
Makkonen, Jarmo T. Laitinen, Raija H.
Tiina A. Jokela, Riikka Kärnä, Katri M.
Synthesis of Keratinocytes
Promoter, Stimulating Hyaluronan )HAS2Binding to Hyaluronan Synthase 2 (
Phosphorylation and
705
3 (STAT3) Tyr
Transducer and Activator of Transcription
Receptor and Induces Signal
14
Extracellular UDP-Glucose Activates P2Y
Glycobiology and Extracellular Matrices:
doi: 10.1074/jbc.M114.551804 originally published online May 20, 2014
2014, 289:18569-18581.J. Biol. Chem.
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Extracellular UDP-Glucose Activates P2Y
14
Receptor and
Induces Signal Transducer and Activator of Transcription 3
(STAT3) Tyr
705
Phosphorylation and Binding to Hyaluronan
Synthase 2 (HAS2) Promoter, Stimulating Hyaluronan
Synthesis of Keratinocytes
*
Received for publication, January 20, 2014, and in revised form, May 15, 2014 Published, JBC Papers in Press, May 20, 2014, DOI 10.1074/jbc.M114.551804
Tiina A. Jokela
‡1
, Riikka Kärnä
‡
, Katri M. Makkonen
‡§
, Jarmo T. Laitinen
‡
, Raija H. Tammi
‡
, and Markku I. Tammi
‡
From the Institutes of
‡
Biomedicine and
§
Dentistry, School of Medicine, University of Eastern Finland, P. O. Box 1627,
FIN-70210 Kuopio, Finland
Background: The secretion and possible functions of extracellular UDP-sugars in epidermal keratinocytes are not known.
Results: UDP-glucose activates P2Y
14
receptor and JAK2, increases STAT3 Tyr
705
phosphorylation, and enhances transcription
of hyaluronan synthase 2 (HAS2).
Conclusion: UDP-glucose release signals for enhanced HAS2 expression by keratinocytes.
Significance: Stimulation of hyaluronan synthesis is an inherent part of epidermal keratinocyte activation and injury response.
Hyaluronan, a major matrix molecule in epidermis, is often
increased by stimuli that enhance keratinocyte proliferation
and migration. We found that small amounts of UDP-sugars
were released from keratinocytes and that UDP-glucose (UDP-
Glc) added into keratinocyte cultures induced a specific, rapid
induction of hyaluronan synthase 2 (HAS2), and an increase of
hyaluronan synthesis. The up-regulation of HAS2 was associ-
ated with JAK2 and ERK1/2 activation, and specific Tyr
705
phos-
phorylation of transcription factor STAT3. Inhibition of JAK2,
STAT3, or G
i
-coupled receptors blocked the induction of HAS2
expression by UDP-Glc, the latter inhibitor suggesting that the
signaling was triggered by the UDP-sugar receptor P2Y
14
. Chro-
matin immunoprecipitations demonstrated increased pro-
moter binding of Tyr(P)
705
-STAT3 at the time of HAS2 induc-
tion. Interestingly, at the same time Ser(P)
727
-STAT3 binding to
its response element regions in the HAS2 promoter was
unchanged or decreased. UDP-Glc also stimulated keratinocyte
migration, proliferation, and IL-8 expression, supporting a
notion that UDP-Glc signals for epidermal inflammation,
enhanced hyaluronan synthesis as an integral part of it.
Hyaluronan is a large, ubiquitous glycosaminoglycan, con-
sisting of alternating N-acetylglucosamine (GlcNAc) and glu-
curonic acid (GlcUA) repeating units. It occupies the pericellu-
lar and extracellular space of many cell types, including basal
and spinous cell layers of skin epidermis (1). It acts as a highly
hydrated space filler, but also stimulates proliferation and
migration through binding to its receptors, like CD44 and
RHAMM (2–4). In the epidermis, hyaluronan synthesis has
been shown to increase rapidly in tissue activation, for exam-
ple, due to injury like epidermal wounding (5–8), presum-
ably to help cell growth and movement to cover the wound.
Hyaluronan disappears in stratum granulosum before termi-
nal differentiation of the keratinocytes, and reduction of epi-
dermal hyaluronan promotes differentiation (9, 10). Accord-
ingly, stimulation of hyaluronan synthesis and increase of its
content associates with compromised epidermal water bar-
rier and morphologically incomplete differentiation (11).
Moreover, hyaluronan increases in epidermal hyperprolif-
eration and squamous cell cancer induced by UV irradiation
(12).
The three mammalian HAS isoforms are multispan trans-
membrane proteins. They are active when inserted in the
plasma membrane, transferring GlcNAc and GlcUA from the
corresponding cytosolic UDP-sugars to the reducing end of
the growing hyaluronan chain (13) that is extruded into extra-
cellular space through a pore formed by the enzyme itself (14,
15). Among the three HAS genes HAS2 shows the highest
expression in keratinocytes, and is up-regulated by epidermal
growth factor (11), keratinocyte growth factor (16), TNF
␣
(17),
interferon-
␥
(18), and all-trans-retinoid acid (19), whereas
transforming growth factor

(TGF

) down-regulates its
expression in keratinocytes (11). The regulation of HAS2
expression involves several transcription factors with func-
tional response elements in its promoter. These include reti-
noic acid receptor, nuclear factor
B (NF-
B), cAMP response
element-binding protein 1 (CREB1), specificity protein 1 (SP1),
yin-yang 1 (YY1), and STAT (20, 21). For example, EGF recep-
tor activation enhances tyrosine 705-phosphorylated STAT3
binding to the promoter, inducing HAS2 gene expression (21).
The expression of HAS2 is also influenced by cellular supply
of its own substrate UDP-GlcNAc, the abundance of which
triggers a suppressive feedback loop mediated by transcription
factors SP1 and YY1. Their binding to the HAS2 promoter is
*This work was supported by grants from the Academy of Finland (to M. I. T.),
The Sigrid Juselius Foundation (to M. I. T. and R. H. T.), the EVO Funds of the
Kuopio University Hospital (to M. I. T.), The Mizutani Foundation (to M. I. T.),
the Cancer Center of the University of Eastern Finland (to M. I. T. and
R. H. T.), Glycoscience Graduate School (to T. A. J. and M. I. T.), Paavo Koisti-
nen Foundation (to T. A. J.), and Kuopio University foundation (to T. A. J.).
1
To whom correspondence should be addressed. E-mail: tiina.jokela@
gmail.com.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 26, pp. 18569–18581, June 27, 2014
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
JUNE 27, 2014•VOLUME 289• NUMBER 26 JOURNAL OF BIOLOGICAL CHEMISTRY 18569
at UNIVERSITETSBIBLIO I BERGEN on July 2, 2014http://www.jbc.org/Downloaded from
subject to regulation by their O-GlcNAc modification that is
dependent on the cellular concentration of UDP-GlcNAc (22).
Cytosolic UDP-GlcNAc has thus a double function; it stimu-
lates hyaluronan synthesis as a crucial substrate of the HAS
enzyme, and as a stabilizer of the HAS2 enzyme (23), but inhib-
its it through transcriptional HAS2 protein suppression of the
synthesis.
It has been recently confirmed that UDP-sugars exist also in
extracellular fluids (24), released by cellular injury or, as sug-
gested recently, in a regulated fashion (24, 25). The idea of reg-
ulated secretion is in line with the finding that increasing UDP-
sugar transport into the Golgi apparatus stimulates UDP-sugar
release through vesicular transport (24, 25). Interestingly, there
isaG
i
-protein-coupled purinergic plasma membrane receptor
(P2Y
14
) specific for UDP-sugars (26), suggesting a biological
signaling function for extracellular UDP-sugars. Release of
UDP-sugars might thus serve as an autocrine or paracrine sig-
naling system. The system may serve as a warning signal after
tissue injury, because thrombin has been shown to stimulate
the release of UDP-Glc (24), and receptor binding of UDP-Glc
induces the expression of IL-8, a mediator of inflammation (27).
Most potent agonist of the P2Y
14
is UDP-Glc (26).
P2Y
14
has a relatively wide distribution in human tissues,
with highest expression levels in placenta, adipose tissue, stom-
ach, and intestine, and moderate levels in the brain, spleen,
lung, and liver (28). P2Y
14
is an important regulator of mesen-
chymal differentiation, especially adipogenesis (29). Activation
of P2Y
14
receptor by UDP-Glc promotes MAP kinase signaling
(30) and mobilizes intracellular Ca
2⫹
stores (27). Extracellular
UDP-Glc promotes IL-8 secretion (27) and stimulates mast cell
degranulation (31).
Keratinocytes express several subtypes of P2Y receptors (32),
known to regulate their proliferation and differentiation (33).
However, nothing is known about the function of the P2Y
14
receptor and extracellular UDP-sugars in keratinocytes. In this
article we show that extracellular UDP-Glc stimulates HAS2
expression, hyaluronan synthesis, proliferation, and migration
of cultured human keratinocytes. The up-regulation of HAS2 is
mediated through a G
i
-linked P2Y receptor, most likely P2Y
14
,
and phosphorylation of JAK and STAT3, the latter specifically
in tyrosine 705, which correlates with its binding to the HAS2
promoter after UDP-Glc treatment.
EXPERIMENTAL PROCEDURES
Cell Culture—The human immortalized epidermal keratino-
cyte cell line HaCaT (34) was cultured in DMEM (Sigma) sup-
plemented with 10% FBS (Hyclone, Logan, UT), 2 mML-gluta-
mine (Euroclone, Milan, Italy), 50 units/ml of penicillin, and 50
g/ml of streptomycin (Euroclone). UDP-Glc was purchased
from Sigma.
RNA Extraction and q-PCR—q-PCR
2
analyses were done
from total RNA extracted with Eurozol (Euroclone) after cDNA
synthesis with a Verso cDNA kit (Thermo Fischer, Waltham,
MA), on an MX3000P thermal cycler (Stratagene, La Jolla, CA),
using the Fast Start universal SYBR Green Master (ROX)
(Roche Applied Science). Fold-inductions were calculated
using the formula 2
⫺(⌬⌬Ct
), where ⌬⌬C
t
is ⌬C
t(sample)
⫺
⌬C
t
(non-treated replicate1)
,⌬
Ct
is C
t(gene of interest)
⫺C
t(ARPO)
and the
C
t
is the cycle at which the threshold is crossed. The gene-
specific primers for the genes analyzed are shown in Table 1.
PCR product quality was monitored using post-PCR melt curve
analysis.
Enzyme-linked Sorbent Assay for Hyaluronan—Media from
the cultures were assayed for the concentration of hyaluro-
nan using a sandwich-type enzyme-linked sorbent assay as
described previously (35). The hyaluronan released into the
media was normalized per 10,000 cells, counted by using a
hemocytometer.
Anion Exchange HPLC of UDP-Glc—Cells were cultured
until 80% confluence. After treatments, cells were counted
from one plate for normalization of the results, whereas cells
and media from parallel plates were used to measure UDP-Glc.
Cells were washed with cold PBS on ice. Cold acetonitrile was
added to precipitate proteins and extract UDP-sugars. Cells
were scraped off and the acetonitrile with cell debris was trans-
ferred into microcentrifuge tubes, the plates were washed with
1 ml of deionized water and combined with the first extract,
centrifuged at 6000 ⫻gfor 20 min, the supernatants transferred
to clean tubes, evaporated in a vacuum centrifuge, and dis-
solved in PBS for further purification by solid phase extraction
on Superclean Envi-Carb SPE cartridges (Sigma) as described
previously (36).
Culture media were rapidly frozen, lyophilized, and redis-
solved in 250
l of water. Salts and glucose were separated from
UDP-sugars on a Superdex Peptide威column eluted at 1 ml/min
with 12 mMNH
4
HCO
3
. The fractions containing UDP-sugars
(prechecked using UDP-Glc standard) were combined and
purified further with Superclean Envi-Carb SPE, as above.
Purified samples were evaporated by vacuum centrifugation
and dissolved in 300
l of water for anion-exchange HPLC on a
CarpoPac
TM
PA1 column (4 ⫻250 mm, Dionex, Sunnyvale,
CA). The column was eluted at 1 ml/min with a gradient made
of ultrapure H
2
O (A), 1.3 Msodium borate, pH 7.0 (B), 1 M
sodium acetate, pH 7.0 (C), and 1.5 Msodium borate, pH 7.5 (D).
The column was equilibrated with 45/55/0/0% (v/v/v/v) of buf-
fers A, B, C, and D, respectively. Elution was performed with the
following program: T0 ⫽45/55/0/0%, T23 ⫽40/55/0/5%,
T48 ⫽36/55/0/9%, T65 ⫽20.6/12.3/61.8/5.3%, T69 ⫽20.6/
12.3/61.8/5.3%, T70 ⫽10.4/12.3/72/5.3%, T82 ⫽45/55/0/0%,
and T88 ⫽45/55/0/0%. Integrated peak areas were calculated
and compared with those of standard nucleotide sugars.
2
The abbreviations used are: q-PCR, quantitative PCR; HAS, hyaluronan syn-
thase; UDP-GlcNAc, UDP-N-acetylglucosamine; UDP-GlcUA, UDP-glucu-
ronic acid; UDP-Glc, UDP-glucose; P2Y
14
,G
i
-protein-coupled purinergic
receptor specific for UDP-sugars; PTX, pertussis toxin.
TABLE 1
Primer sequences for q-PCR of the reverse transcribed human genes
Gene name Primer sequence (5ⴕto 3ⴕ)
ARPO Forward: AGATGCAGCAGATCCGCAT
Reverse: GTGGTGATACCTAAAGCCTG
HAS2 Forward: CAGAATCCAAACAGACAGTTC
Reverse: TAAGGTGTTGTGTGTGACTGA
IL-8 Forward: GAGTGGACCACACTGCGCCAA
Reverse: TCCACAACCCTCTGCACCCAGTT
P2Y
14
Forward: TCAGCAGATCATTCCTGTGC
Reverse: GGCTCATCACAAAGTCAGCA
Extracellular UDP-Glucose Regulates HAS2 Expression
18570 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 •NUMBER 26 •JUNE 27, 2014
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Proteome Profiler
TM
Array—HaCaT cells were incubated for
30 min in medium supplemented with 100
MUDP-Glc. Cells
were washed with PBS, and cell lysis and protein extraction was
performed as described in the protocol of the Proteome Profiler
Array, Human phosphokinase array kit (R&D Systems, Minne-
apolis, MN).
Western Blotting—Proteins were extracted on ice with RIPA
lysis buffer (PBS, pH 7.4, with 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 100
g/ml of phenylmethylsulfonyl
fluoride, 10
g/ml of sodium orthovanadate, and 1% phospha-
tase inhibitor mixture 2, and 0.5% protease inhibitor mixture
(Sigma)). The samples (20
g of protein) were resolved by 10%
SDS-PAGE, followed by transfer onto nitrocellulose membrane
(Whatman) by 35 mA/cm
2
constant current with a semidry
blotter (Biometra, Göttingen, Germany). The membrane was
placed in a SNAP blotting system (Millipore, Bedford, MA),
blocked with 1% BSA, washed with Tris-buffered saline (TBS),
0.1% Tween, and incubated with the primary antibodies: phos-
pho-JAK2 (Tyr
1007/1008
), phospho-STAT3 (Tyr
705
) 1:1000,
phospho-STAT3 (Ser
727
) 1:1000 (all from Cell Signaling, Dan-
vers, MA), phospho-ERK 1:500 (Santa Cruz), and

-actin,
1:2500 (Sigma). After washes, the membrane was incubated
with the fluorescent secondary antibodies anti-rabbit 680 and
anti-mouse 800, 1:5000 (Pierce). Protein bands were visualized
and quantified with Odyssey威infrared imaging system (Li-Cor
Bioscience, Lincoln, NE). The results represent the ratio of
band intensities between the protein of interest and

-actin.
Signaling Inhibitors—Cells were treated with pertussis toxin
(PTX, 100 ng/ml, Tocris Bioscience, Southampton, UK), an
inhibitor of G
i
-coupled receptors, for 17 h prior to UDP-Glc
addition. Cells were treated for 2 h with 30
MAG490 (Sigma),
an inhibitor of JAK2, 50
MSTAT3 inhibitor IX (Calbiochem),
0.5–2
MPD98059 (Calbiochem), and 0.5–2
MUO126 (Cal-
biochem), inhibitors of MEK1/2, before adding UDP-Glc. PTX
was dissolved in water, AG490 in ethanol, and STAT3 inhibitor
IX, PD98059, and UO126 in dimethyl sulfoxide. Equal amounts
of those solvents were used as controls.
Chromatin Immunoprecipitation—After 2-h incubations
with or without 100
MUDP-Glc, nuclear proteins were cross-
linked to DNA by adding formaldehyde directly to the medium
to a final concentration of 1% for 10 min at room temperature.
Cross-linking was stopped by adding glycine to a final concen-
tration of 0.15 Mand incubating for 10 min at room
temperature.
The medium was removed and the cells were washed twice
with ice-cold PBS. The cells were then collected in ice-cold PBS
supplemented with a protease inhibitor mixture (Sigma). After
centrifugation, the cell pellets were resuspended in lysis buffer
(1% SDS, 10 mMEDTA, protease inhibitors, 50 mMTris-HCl,
pH 8.1) and incubated for 10 min at room temperature. The
lysates were sonicated until DNA fragments of 300 to 1000 bp in
length were obtained (in preliminary tests).
Cellular debris was removed by centrifugation. At this step
20
l of the supernatant was taken as the input sample and
diluted 1:5 in ChIP dilution buffer (0.01% SDS, 1.1% Triton
X-100, 1.2 mMEDTA, 167 mMNaCl, protease inhibitors, 16.7
mMTris-HCl, pH 8.1). The rest of the supernatant was divided
into the aliquots that were diluted 1:10 in ChIP dilution buffer
and incubated with the indicated antibodies (1:100–1:400)
overnight at 4 °C with rotation. The antibodies against rabbit
IgG (sc-2027), pSTAT3 (Tyr
705
) (sc-7993), were obtained from
Santa Cruz Biotechnologies and pSTAT (Ser
727
) (number
9134) and total STAT3 (number 9132) were obtained from Cell
Signaling.
The immunocomplexes were collected with 20
l of protein
G-magnetic beads (Millipore) for1hat4°Cwith rotation. The
beads were separated from the supernatant using a magnetic
rack (Qiagen, Valencia, CA). The pellets were washed sequen-
tially for 3 min by rotation with 1 ml of each of the following
buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM
EDTA, 150 mMNaCl, 20 mMTris-HCl, pH 8.1), high salt wash
buffer (0.1% SDS, 1% Triton X-100, 2 mMEDTA, 500 mMNaCl,
20 mMTris-HCl, pH 8.1), and LiCl wash buffer (0.25 MLiCl, 1%
Nonidet P-40, 1% sodium deoxycholate, 1 mMEDTA, 10 mM
Tris-HCl, pH 8.1). Finally, the beads were washed twice with 1
ml of TE buffer (1 mMEDTA, 10 mMTris-HCl, pH 8.1).
The immunocomplexes were eluted by adding 350
l of elu-
tion buffer (1% SDS, 100 mMNaHCO
3
), incubating at 65 °C for
30 min, and separating the beads from the supernatant on a
magnetic rack. This 350-
l sample was the enriched output
sample. The cross-linking was reversed and the remaining pro-
teins were digested by adding 1.8
l of proteinase K (final con-
centration 110
g/ml, Fermentas) to both the input and output
samples, and incubating overnight at 65 °C. The DNA was
recovered by using the QIAquick威PCR purification kit
(Qiagen).
The DNA was used as a template for PCR with the following
profile: 10 min preincubation at 95 °C, and 45 cycles of 30 s
denaturation at 95 °C, 30 s annealing at 60 °C, and 30 s elonga-
tion at 72 °C, with one final incubation for 10 min at 72 °C. The
primers for the amplification are presented in Table 2. The
resulting PCR products were quantified and expressed as per-
cent of the input control. Fold-changes were calculated using
the formula 2
(⌬Ct)
, where ⌬C
t
is C
t
output ⫺C
t
input and C
t
is
the cycle at which the threshold is crossed. The results are pre-
sented as fold-changes over the value of samples precipitated
with normal rabbit IgG. Input samples were diluted 13.3 times
compared with outputs.
Proliferation and Migration—For the proliferation assay
20,000 HaCaT cells were seeded on a 24-well plate and after 4 h
UDP-Glc was added to a final concentration of 100
M. The
media containing UDP-Glc was replaced each day. Cells were
detached with trypsin-EDTA at 1–5 days following plating and
counted with hemocytometer.
TABLE 2
Sequence and location of the PCR primers used in the ChIP assays of
human HAS2 gene promoter
Region Location Primer sequence (5ⴕto 3ⴕ)
1 (TSS) ⫺32 to ⫹57 Forward: GGAGGCAGAAGGGCAACAAC
Reverse: GTTCAATGGGCTGCTCGAAGC
2⫺481 to ⫺244 Forward: GTTACTTAGCTGAAGGGCACC
Reverse: GGCCGGTTCTAAACTCCAATG
3⫺1048 to ⫺655 Forward: CAGTCATCAGCAGGCTTGTTG
Reverse: CTGACGTCAAGTGTCAAAGCC
4⫺1896 to ⫺1554 Forward: GGTATTCCCGCATTACGTGTC
Reverse: CACTGATTTCCCCCAGCAAC
Extracellular UDP-Glucose Regulates HAS2 Expression
JUNE 27, 2014•VOLUME 289• NUMBER 26 JOURNAL OF BIOLOGICAL CHEMISTRY 18571
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Cell proliferation was also studied by BrdU staining. HaCaT
cells were seeded on 8-well chamber slides (Nalge Nunc,
Naperville, IL), cultured to 80% confluence, and treated with
100
MUDP-Glc overnight. The next day the cultures were
incubated with 5-bromo-2⬘deoxyuridine (1:1000, BrdU, Roche
Applied Science) in DMEM for 1 h, washed with PBS, and fixed
with 4% paraformaldehyde on ice. The slides were washed with
100 mMsodium phosphate, pH 7.4 (PB), incubated with 70%
EtOH for 10 min, and washed with PB again. The slides were
treated with 2 MHCl for 30 min at 37 °C, washed with PB, and
blocked with 1% BSA/PB for 10 min at room temperature, then
immunostained with the anti-BrdU antibody (1:500, Sigma) in
1% BSA/PB for2hat37°C,washed with PB, incubated with
biotinylated anti-mouse antibody (1:200, Vector Laboratories,
Burlingame, CA) in 1% BSA/PB for 30 min at 37 °C, and washed
with PB. The bound antibodies were visualized with the avidin-
biotin peroxidase (1:200, ABC-standard kit, Vector) in PB for
1 h at room temperature, washed with PB, and incubated with
0.05% 3,3⬘-diaminobenzidine and 0.03% hydrogen peroxide for
5 min. The nuclei were stained with hematoxylin. The cells
were mounted using Supermount (BioGenex, San Ramon, CA).
The number of nuclei showing positive staining and the num-
ber of all nuclei were counted from 10 fields, randomly photo-
graphed using a ⫻10 objective.
For studies on apoptosis HaCaT cells were seeded on 96-well
plates (Cell Star威, Greiner Bio-One, Kremsmunster, Austria).
The next day, fresh medium containing 100
MUDP-Glc was
changed. After a 24-h incubation, living and dead cells were
determined with a kit according to the instructions of the man-
ufacturer (CytoTox-Glo
TM
Cytotoxicity Assay, Promega).
For the migration assay HaCaT cultures were grown to conflu-
ence and an artificial wound was introduced to the cell layer with a
pipette tip. Fresh medium with 100
MUDP-Glc were changed.
The cell-free area was measured immediately after scraping and
24 h later using an Olympus CK2 phase-contrast microscope
(Olympus Optical Co. Ltd., Tokyo, Japan) and NIH Image soft-
ware. The newly covered wound area was calculated and con-
verted to an average migration distance from the wound edge.
Statistical Analysis—Normally distributed data were analyzed
by paired samples ttests when comparing control and UDP-Glc-
treated cultures, and by one-way analysis of variance with Dun-
nett’s post hoc test, when many treatments were tested.
RESULTS
Extracellular UDP-Glc Stimulates HAS2 Expression and
Induces Hyaluronan Synthesis—A signaling function has been
established for extracellular UDP-sugars, activating several
intracellular pathways and modulating cell functions. Given the
FIGURE 1. Extracellular UDP-sugars regulate HAS2 expression and hyaluronan synthesis. A, HaCaT cultures were treated for 2 h with 100
Mof the
indicated UDP-sugars and analyzed for HAS2 expression. B, UDP-Glc as the most powerful stimulator was used to study hyaluronan release into growth
medium, analyzed 4 and 6 h after adding UDP-Glc. C, the time course of HAS2 induction using 100
Mconcentration of UDP-Glc. D, effect of UDP-Glc
concentration on HAS2 expression during a 2-h incubation. In panels A and Cthe data represent mean ⫾S.E. of three independent experiments, each done as
duplicate. The statistical significance of the differerences between control and UDP-sugars were tested using Dunnett’s test. In panels B and Dthe data
represent mean ⫾S.E. of 5 and 4 independent experiments, respectively. Statistical significance was estimated using paired sample ttest. *, p⬍0.05; **, p⬍
0.01; and ***, p⬍0.001.
Extracellular UDP-Glucose Regulates HAS2 Expression
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importance of the intracellular UDP-sugars for hyaluronan
synthesis (23, 35, 37) and HAS expression (22, 38) we examined
whether UDP-sugars are also active when added in the extra-
cellular compartment. Introduction of UDP-Glc induced a
clear increase of HAS2 mRNA although UDP-GlcUA caused a
modest increase, and UDP-Gal and UDP-GlcNAc did not pres-
ent significant effects (Fig. 1A). The activation of HAS2 expres-
sion was rapid, with a significant increase already 90 min after
the addition of UDP-Glc, whereas the expression level had
returned back to control level after 6 h (Fig. 1C). The expression
of HAS2 showed a steep increase between 10 and 50
M, but
without a significant further increase above 100
M, as if cross-
ing a threshold concentration would trigger a rapidly saturable
response (Fig. 1D). UDP-Glc also slightly increased the expres-
sion of HAS3, which was not further analyzed, whereas HAS1
expression was too low for reliable analysis. To see if the rise in
HAS2 mRNA expression was reflected in hyaluronan secretion,
its content in the culture medium was assayed. A significant
175% rise in the amount of hyaluronan was found in samples
collected 6 h after the addition UDP-Glc (Fig. 1B). This change
was not observed in samples collected at the 4-h time point. The
delay in the increased hyaluronan production was in line with
an idea that transcriptional activation preceded the increased
hyaluronan production.
Extracellular Decay of UDP-Glc—Because the effect of UDP-
Glc on HAS2 transcription was relatively short-lived, we mea-
sured the decay rate of the UDP-Glc in the medium. As shown
in Fig. 2A, UDP-Glc content was decreased after 30 min incu-
bation to 80% and after6hto25%oftheoriginal level. The
endogenously produced UDP-Glc in the medium varied
between 1 and 2 pmol/10,000 cells.
We also studied how the addition of UDP-Glc influenced its
intracellular content. 30 min after addition of 100
MUDP-Glc
to the culture medium there was already a 1.8-fold increase, and
at the 2-h time point the intracellular level of UDP-Glc was
increased by 2.7-fold (Fig. 2B), suggesting that part of UDP-Glc
was endocytosed.
The Effect of UDP-Glc on HAS2 Transcription Is Mediated by
G
i
-coupled P2Y Receptors—UDP-Glc has been shown to act as
an agonist of the G
i
-coupled receptor P2Y
14
(25). Three G
i
-cou-
pled P2Y receptor subtypes have been identified, namely P2Y
12
,
P2Y
13
, and P2Y
14
. ADP activates the former two, and P2Y
14
is
the only subtype known to be activated by UDP-Glc (39). P2Y
14
is expressed in HaCaT cells (32, 40). To study the involvement
of P2Y
14
, experiments were done in cells pretreated with PTX,
FIGURE 2. Added UDP-Glc decreases in the HaCaT culture medium and increases in the cells. HaCaT cells were incubated for 1, 30, 120, and 360 min with
100
MUDP-Glc. Extracellular (A) and intracellular (B) UDP-Glc was analyzed with anion exchange HPLC, with mean ⫾S.E. of 3 and 4 independent experiments,
respectively. In A, the time point at 1 min was taken as the control (100%), indicating the starting level of the UDP-Glc in the medium. In B, the time point 0
(100%) indicates the average intracellular UDP-Glc level in non-treated cultures. The statistical significance between non-treated and UDP-Glc-treated cultures
at each time point indicate the results of Dunnett’s test: *, p⬍0.05; **, p⬍0.01; and ***, p⬍0.001.
FIGURE 3. Pertussis toxin inhibits UDP-Glc-induced expression of HAS2
and IL-8.HaCaT cells were preincubated overnight with or without 100 ng/ml
of PTX before UDP-Glc was added to 100
Mfinal concentration and incuba-
tion continued for 2 h. HAS2 (A) and IL-8 (Band C) mRNA were measured by RT
q-PCR. Data represent mean ⫾S.E. of four experiments, ***, p⬍0.001, Dun-
nett’s test (A), of five experiments, *, p⫽0.017, paired sample ttest (B) and in
chart (C) mean ⫾range of one experiment with replicate samples.
Extracellular UDP-Glucose Regulates HAS2 Expression
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which specifically inhibits signal transduction through G
i
-cou-
pled receptors by catalyzing ADP-ribosylation of the
␣
subunit
of G
i
protein, thereby uncoupling the G protein from receptor
(41). We used 100 ng/ml of PTX, previously reported to effec-
tively block G
i
-coupled receptors (31). PTX did not show toxic
effects on the HaCat culture, as indicated by cell morphology
and the absence of increased apoptosis in the cytotoxicity assay,
but it significantly inhibited the stimulation of the HAS2
expression evoked by UDP-Glc (Fig. 3A), indicating that UDP-
Glc signaling involves G
i
-coupled receptors, very likely P2Y
14
.
For background, we checked whether UDP-Glc also induces
signals other than those leading to HAS2 expression. As
enhanced secretion of IL-8 was previously reported in airway
epithelial cells treated with UDP-Glc (27), we measured its
expression in HaCaT cells. Indeed, IL-8 mRNA was signifi-
cantly up-regulated by UDP-Glc simultaneously with HAS2
(Fig. 3B), indicating a wider range of signals activated by UDP-
Glc in HaCaT cells. The induction of IL-8 expression was also
blocked by PTX (Fig. 3C). The findings support the idea that the
HAS2 response is part of an inflammatory reaction.
UDP-Glc Stimulates ERK and STAT3 Phosphorylation—To
study which signaling pathways are involved in the up-regula-
tion of HAS2 expression, we used a phosphokinase array (Fig.
4A). After a 30-min incubation of HaCaT cells with UDP-Glc,
the most interesting finding was the Tyr
705
phosphorylation of
STAT3, an important transcriptional regulator of HAS2 (11,
21) and its upstream activator ERK (42, 43). The increase of
ERK and STAT3-Tyr
705
phosphorylations by UDP-Glc was
confirmed by Western blotting (Fig. 4, Band C).
Because p-ERK can activate STAT3, we checked whether
inhibition of p-ERK blocks the up-regulation of HAS2 by UDP-
Glc. It turned out that UO126, which totally abolished ERK
phosphorylation (Fig. 5A), efficiently cut the basal expression of
HAS2 down to ⬃30% of control, whereas the relative stimula-
tion by UDP-Glc was minimally affected (Fig. 5, Aand B). This
finding was reproduced by PD98059, another inhibitor of ERK
phosphorylation (Fig. 5, Aand C). The higher efficiency of
UO126 over PD98059 has been noted before (44) and may be
due to the fact that UO126 targets both MEK1 and MEK2,
whereas the main target of PD98059 is MEK1. Data on both
inhibitors and concentrations were subjected to statistical
analysis, which indicated that UDP-Glc activation of HAS2
remained significant in the presence of ERK inhibition. This
suggests that STAT3 was mainly activated by another signal-
ing pathway.
FIGURE 4. UDP-Glc activates ERK and STAT3. A, dot blot images from a phosphokinase array on protein samples extracted from cells incubated for 30 min
with 100
MUDP-Glc. The phosphorylated proteins STAT-Tyr
705
and ERK are encircled. Other spots in array: A3A4, p38a; A7A8, JNK1/2/3; A9A10, GSK-3a/b; B5B6,
MSK1/2; B9B10, Akt1/2/3; C1C2, TOR; C3C4, CREB; C7C8, AMPKa2; C9C10,

-catenin; D1D2, Src; D7D8, STAT2; D9D10, STAT5a; E1E2, Fyn; E3E4, Yes; E9E10,
STAT5b; F1F2, Hck; F3F4, Chk-2; F5F6, FAK; F7F8, PDGF Rb; F9F10, STAT5a/b (Band C). Western blots of cells incubated for 30 –60 min with 100
MUDP-Glc with
antibodies against p-ERK and Tyr(P)
705
-STAT3. Mean ⫾S.D. of 4 independent experiments are shown. Statistical significance of the difference versus control, **,
p⫽0.002 (Dunnett’s test).
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JAK2 Phosphorylation Associates to UDP-Glc-stimulated
HAS2 Expression—Besides ERK, Janus kinase 2 (JAK2) has been
found to activate STAT3 (45). We therefore examined the level
of p-1007/1008JAK2 following introduction of UDP-Glc. There
was a transient increase of JAK2 at 10–20 min, followed by
return to the control level at 30–60 min (Fig. 6, Aand B). The
activation suggested that JAK2 could also be involved in the
up-regulation of HAS2. The contribution of JAK2 was sup-
ported by the decrease of HAS2 mRNA with AG490, an inhib-
itor of JAK2 (Fig. 6C).
UDP-Glc Causes Sequential Activation of STAT3 at Tyr
705
and Ser
727
—STAT3 has two phosphorylation sites (Tyr
705
and
Ser
727
) commonly associated with its activation (45). Time-de-
pendent changes of these two phosphorylations were analyzed
with Western blotting to correlate their levels with the tran-
sient increase in HAS2 mRNA. Tyr(P)
705
-STAT3 increased at
30 min, reached its maximum at 60 min, and declined close to
the control level at 120 min (Fig. 7, Aand B), thus preceding the
peak of HAS2 at 120 min (Fig. 1C). In contrast, there was no
significant change in the level of Ser(P)
727
at 30 and 60 min,
whereas a significant increase was observed at 120 min (Fig.
7, Aand C). This suggests that the increase of HAS2 mRNA
fits better with STAT3 phosphorylation of Tyr(P)
705
than
Ser(P)
727
.
The Tyr
705
phosphorylation of STAT3 was very efficiently
blocked by STAT3 inhibitor IX (Fig. 7D). The JAK2 inhibitor
AG490 also blocked the UDP-Glc-induced increase of
Tyr(P)
705
-STAT3 (Fig. 7D), suggesting that JAK2 was involved
in the Tyr(P)
705
-STAT3 activation. The increase of UDP-Glc-
induced HAS2 expression was completely neutralized with
STAT3 inhibitor IX, although the inhibitor itself increased the
basal level of HAS mRNA (Fig. 7E). Together, these results sug-
gest that the early JAK2 activation triggers Tyr
705
phosphory-
lation of STAT3, which is followed by an increase of HAS2
mRNA.
Tyr
705
-phosphorylated STAT3 Binds to HAS2 Promoter after
UDP-Glc Treatment—Because the data suggested involvement
of STAT3 in the UDP-Glc-induced HAS2 response, and this
transcription factor is known to have functional binding sites
on HAS2 promoter, we studied if UDP-Glc-stimulated signal-
ing increases STAT3 binding to the promoter regions with
reported STAT response elements (21) and/or to the transcrip-
tion start site. After 2 h incubation with UDP-Glc, STAT3 bind-
ing to the transcription start site containing the region was
significantly increased (Fig. 8A). Separate ChIP analyses with
Tyr(P)
705
- and Ser(P)
727
-STAT3 antibodies revealed that the
increase of STAT3 binding to the transcription start site con-
taining the region was solely due to the Tyr(P)
705
-STAT3,
whereas there was no change in the binding of Ser(P)
727
-
STAT3 (Fig. 8A). Similar findings were made in the three other
regions of the HAS2 promoter with STAT response elements,
showing increased Tyr(P)
705
-STAT3 binding and no change or
reduced binding of Ser(P)
727
-STAT3 (Fig. 8, B–D). Taken
together, the data indicate that UDP-Glc, through a G
i
-coupled
FIGURE 5. ERK contributes to the basal HAS2 mRNA level but not UDP-Glc-mediated up-regulation. A, phosphorylated ERK (pERK) levels after 120 min
preincubation with MEK inhibitors: UO126 and PD98059 followed 30 min incubation with or without 100
MUDP-Glc. Band C, cells preincubated for 120 min
in the presence of the UO126 and PD98059 were incubated for 60 min with each inhibitor with or without 100
MUDP-Glc. HAS2 expression was assayed and
compared between UDP-Glc-treated and untreated cultures, the relative increase is indicated by the percentages above the columns. Mean ⫾S.D. of 2–3
independent experiments are shown. Analysis of variance indicated that the inhibitors did not influence the increase caused by UDP-Glc. In addition, UDP-Glc
versus control in the presence of the inhibitors, p⬍0.001 (ttest).
Extracellular UDP-Glucose Regulates HAS2 Expression
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P2Y
14
receptorandJAK2activation stimulates Tyr
705
phosphor-
ylation of STAT3, leading to its binding to the HAS2 promoter
and stimulation of its transcription, resulting in enhanced hyal-
uronan synthesis.
UDP-Glc Stimulates Cell Proliferation and Migration—Be-
cause enhanced migratory and proliferative activity of epider-
mal keratinocytes is a common feature in inflammation (46)
and often associated with elevated hyaluronan synthesis (2, 47),
we studied whether UDP-Glc stimulates these cellular func-
tions. In time series experiments daily UDP-Glc treatment did
not influence cell numbers until day 5, when the cells
approached confluence (Fig. 9C). At that time a small (15%) but
statistically significant increase in cell counts was observed. To
check if the late UDP-Glc response was due to slow or indirect
mechanisms, or was related to confluence, we treated near con-
fluent cultures with UDP-Glc for 24 h and analyzed the number
of proliferating cells with BrDU labeling. A small (12%) but
consistent increase in the number BrDU-positive cells was
observed in UDP-Glc-treated cultures (Fig. 9A), suggesting that
the proliferative response may relate to the confluence or dif-
ferentiation status of the cells. UDP-Glc treatment did not
influence the number of dead cells (Fig. 9B).
In a scratch wound assay, HaCaT cells treated with UDP-Glc
for 24 h migrated significantly more than the untreated cells
(Fig. 9D). Although the difference between the means was
rather modest (⬃13%), the result was reproduced in all exper-
iments performed. To summarize these results, UDP-Glc does
not induce apoptosis, and it modestly increases cell prolifera-
tion and migration.
DISCUSSION
The present study established that HAS2 gene expression
in keratinocytes is subject to regulation by extracellular
UDP-sugars, complementing the recently described control
by intracellular UDP-sugars (22). However, the extracellular
and intracellular signaling mechanisms appear to be com-
pletely independent. The intracellular regulation comes
through the content of UDP-GlcNAc, controlling O-GlcNAc
modifications in transcription factors SP1 and YY1, and the
HAS2 enzyme itself (23). In contrast, among the different
UDP-sugars UDP-Glc is the dominant extracellular effector
on HAS2 expression, mediated by G
i
-coupled P2Y receptors,
most likely P2Y
14
, leading to JAK2 activation and Tyr
705
phosphorylation of the transcription factor STAT3, and its
binding to HAS2 promoter. The resulting increase of hyalu-
ronan synthesis occurred together with enhanced migration
and proliferation, phenotypic changes previously found to
accompany the accumulation of hyaluronan by other kerati-
nocyte activators like EGF (2), keratinocyte growth factor
(16), and retinoic acid (19).
The UDP-Glc stimulation on HAS2 expression comes up
very rapidly. Because the HA response requires more time, it is
not likely to result from direct regulation of HAS activity, but
rather be a result of increased mRNA level. JAK2-STAT3 sig-
naling appeared to be involved in HAS2 response suggesting
transcriptional regulation, although changes in mRNA stability
are also possible. HAS2 response also recedes fast, probably due
to the fast decay of UDP-Glc in the growth medium. Ectonucle-
otidases are known to catalyze hydrolysis of nucleotides on the
outer surface of plasma membrane, and in the extracellular
milieu (48), which can explain the observed decay of the
UDP-Glc.
The increase of intracellular UDP-Glc during the incubation
suggests that a part of UDP-Glc is taken up into the cells, where
it remained at an elevated level for at least 6 h. This finding is in
line with the reported recycling of nucleotides between extra-
cellular and intracellular compartments (48) but no data exist
on the mechanisms of UDP-Glc uptake. Being membrane
impermeable, and lacking known plasma membrane transport
channels, UDP-Glc can be taken up by fluid phase endocytosis
(49), with or without help from receptors. The increased cellu-
lar UDP-Glc is perhaps located in endosomes, ER, or Golgi
apparatus. However, we believe that the cellular uptake is not a
major mediator of signals induced by extracellular UDP-Glc,
considering the fact that inhibition of the UDP-Glc receptor
with pertussis toxin abolished its stimulatory effect on HAS2
expression.
UDP-Glc is an activating ligand of the G
i
-coupled receptor
P2Y
14
. The other G
i
-coupled P2Y receptor subtypes are acti-
vated by ADP (39). P2Y
14
involvement was strongly supported
by the notion that PTX, a potent inhibitor of G
i
-coupled recep-
tors, prevented the UDP-Glc-induced up-regulation of HAS2
expression. RT-PCR also confirmed that P2Y
14
was expressed
in HaCaT cells.
FIGURE 6. JAK2 phosphorylation associates with UDP-Glc-stimulated
HAS2 expression. A, Western blots of pJAK2 at the indicated times following
introduction of 100
MUDP-Glc. B, quantitation of the Western blots from
4 –6 separate experiments at each time point, normalized to

-actin. *, p⬍
0.05, UDP-Glc versus control, by paired sample ttest. C, effect of the JAK2
inhibitor AG490 (30
M)onHAS2 expression in cultures treated with or with-
out 100
MUDP-Glc. The cultures were preincubated for 120 min with the
inhibitor, followed by 120 min with or without 100
MUDP-Glc. The data
represent mean ⫾S.E. of 5 independent experiments. Statistical significance,
control versus UDP-Glc: ***, p⬍0.001 (Dunnett’s test). D, verification of AG490
effect on JAK2 phosphorylation. The cultures were preincubated for 120 min
with or without the inhibitor, followed by 120 min with or without 100
M
UDP-Glc.
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It has been reported that activation of G
i
-coupled receptors
by extracellular UDP-Glc leads to activation of ERK (30), JNK
and p38 MAP kinases (31), and mobilization of intracellular
Ca
2⫹
stores (27). In agreement with the reports (30), HaCaT
cultures showed elevated ERK phosphorylation after UDP-Glc
treatment. However, inhibition of the ERK signaling pathway
(by UO126 and PD98059) did not block the UDP-Glc-induced
stimulation of HAS2 expression (Fig. 4, Cand D), indicating
that other pathways were involved in the HAS2 response.
Indeed, phosphorylation of JAK2, often activated by inflamma-
tory cytokines, was increased by UDP-Glc, and an inhibitor of
JAK2 reduced HAS2 up-regulation, suggesting that JAK2 was a
more important contributor to the response than ERK1/2.
The STAT transcription factors are the established down-
stream mediators of JAK2 signaling, governing the expression
of a number of genes, especially those involved in cell prolifer-
ation (50). Given the fact that the P2Y-group receptors have
been shown to activate STAT3 (51), and keratinocytes present
functional STAT3 response elements on their HAS2 promoter
(21), it was not unexpected that UDP-Glc activated STAT3, and
a STAT3 inhibitor blocked the UDP-Glc-induced HAS2
expression. It is thus very likely that G
i
-coupled receptor acti-
FIGURE 7. UDP-Glc-induces both Tyr(P)
705
and Ser(P)
727
phosphorylation of STAT3. A, Western blots of Tyr(P)
705
-STAT3 and Ser(P)
727
-STAT3 30 –120 min
after introduction of 100
MUDP-Glc. B, quantitation of the changes in phosphorylation of Tyr(P)
705
and C, Ser(P)
727
of STAT3. Mean ⫾S.E. from 3 to 6 separate
experiments are shown. **, p⬍0.01, UDP-Glc versus control, by paired sample ttest. D, verification of the effect of STAT3 inhibitor IX and JAK2 inhibitor AG490
on STAT3 Tyr
705
phosphorylation after 120 min preincubation with the inhibitors followed by 60 min incubation with UDP-Glc. E, inhibition of the UDP-Glc-
induced HAS2 up-regulation by STAT3 inhibitor IX. The cultures were preincubated for 120 min with 50
MSTAT3 inhibitor IX, followed by 120 min in the
presence and absence of 100
MUPD-Glc. Mean ⫾S.E. of five separate experiments, *, p⬍0.05, Dunnett’s test.
Extracellular UDP-Glucose Regulates HAS2 Expression
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vation signaled the up-regulation of HAS2 expression through
the JAK2-STAT3 pathway.
On the other hand, strong down-regulation of the basal
HAS2 expression by the inhibitors of MAPK pathway is in line
with previous findings that growth factors and cytokines in the
serum activate its transcription (38) and the inhibitors block
this part of the HAS2 expression. Although we cannot com-
pletely exclude unspecific effects of the inhibitors, it was clear
that during the 4-h incubation no obvious signs of toxicity or
increased apoptosis was detected with any of them.
The STAT response elements on HAS2 promoter bind
Tyr(P)
705
-STAT3 and activate the gene (21). Our data show
that 2 h after introduction of UDP-Glc, when HAS2 mRNA
reached its peak level, HAS2 promoter binding of both total
STAT3 and Tyr(P)
705
-STAT3 are increased, whereas at the
same time Ser(P)
727
-STAT3 tended to vanish from the chroma-
tin immunoprecipitates. It has been reported that Tyr
705
phos-
phorylation of STAT3 is dependent on JAK activity, whereas
ERK and other MAP kinases have been suggested to promote
phosphorylation of Ser
727
(42, 43). Phosphorylation of Tyr
705
causes dimerization of STAT3, which leads to its nuclear local-
ization (52).
The role of Ser
727
phosphorylation of STAT3 is controversial
at the moment. It has been reported to promote the transcrip-
tional activity of STAT3 by recruiting coactivators (53–55). On
the other hand, it may enhance Tyr
705
dephosphorylation, and
thereby inhibit transcription (56). The reciprocal changes we
found in Tyr
705
and Ser
727
phosphorylations of STAT3 are in
line with the latter report, supporting the role of Tyr
705
as an
activating modification (56). Of course, the function of Ser
727
phosphorylation may depend on the specific gene and cell type.
How UDP-sugars get into the extracellular space has
remained obscure. Plasma membrane defect following cellular
injury is one potential source. On the other hand, the positive
correlation between UDP-sugar transporter activity in Golgi
membrane, and the UDP-Glc appearance in culture medium
has been suggested to indicate its controlled secretion, likely by
vesicular transport to plasma membrane (25). The concentra-
tion of UDP-Glc in astrocytoma culture medium is quite small
but is increased by thrombin treatment (24), also suggesting
regulated secretion. The concentration of UDP-Glc was quite
small in the growth medium of keratinocytes. However, it is
very difficult to estimate the effective concentration in vivo,
considering the minor intercellular space between epidermal
keratinocytes (57), the labile nature of the substance, and the
unknown mechanism of its export.
Extracellular UDP-Glc has been related to many cellular
functions. Involvement in inflammation is suggested by its
ability to induce the secretion of IL-8 in airway epithelial
cells (27) and degranulation of mast cells via activation of the
FIGURE 8. UDP-Glc treatment enhances Tyr(P)
705
-STAT3 binding to the HAS2 gene promoter. HaCaT cells were incubated for 2 h with 100
MUDP-Glc and
STAT3 binding into the HAS2 promoter was studied by ChIP. Total STAT3, Tyr(P)
705
-STAT3 and Ser(P)
727
-STAT3 binding to the transcription start site region is
shown in A, to region ⫺481 to ⫺244 in B, to region ⫺1048 to ⫺655 in C, and to region ⫺1896 to ⫺1554 in D. The data represent mean ⫾S.E. of five independent
experiments. Statistical significance control versus UDP-Glc: *, p⬍0.05 (paired sample ttest).
Extracellular UDP-Glucose Regulates HAS2 Expression
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P2Y
14
receptor (31). In rat brain expression of the P2Y
14
receptor level is up-regulated by a challenge with lipopoly-
saccharide (58, 59), suggesting a role in reactive astrogliosis.
In addition to inflammation, extracellular UDP-Glc and
P2Y
14
receptor have been associated to the commitment of
mesenchymal stem cells to adipogenic and osteogenic differ-
entiation (29), and to the chemotaxis of hematopoietic stem
cells (60). Our results suggest that extracellular UDP-Glc has
a functional role also in skin epidermis. The induction of
HAS2 and IL-8 gene expressions, and the stimulation of
migration and proliferation, suggest that its biological func-
tion may be to contribute to epidermal activation as a
response to trauma or inflammation.
Acknowledgment—Expert technical help from Tuula Venäläinen is
gratefully acknowledged.
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FIGURE 9. UDP-Glc stimulates HaCaT cell proliferation and migration.
A, cell proliferation was studied by incubating HaCaT cells for 24 h with
100
MUDP-Glc and then 1 h with BrdU. The specimens were processed
for histology, immunostained for BrdU, and the percentage of BrdU-pos-
itive cells per all cells was calculated. B, apoptosis after 24 h incubation
with 100
MUDP-Glc was studied by a cytotoxicity assay kit. C, cell num-
bers were counted in a hemocytometer following incubations in 100
M
UDP-Glc for 1–5 days. A new culture medium, including UDP-Glc, was
changed every day. D, cell migration was studied in a scratch wound
model for 24 h in presence of 100
MUDP-Glc. Data in Arepresent mean ⫾
S.E. of 4; B, mean ⫾range of 2; and Cand D, mean ⫾S.E. of 5 independent
experiments. Statistical significance, control versus UDP-Glc. *, p⬍0.05;
**, p⬍0.01 (paired samples ttest).
Extracellular UDP-Glucose Regulates HAS2 Expression
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