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ORIGINAL PAPER
Melanoma cell-derived factors stimulate hyaluronan synthesisin dermal fibroblasts by upregulating HAS2 throughPDGFR-PI3K-AKT and p38 signaling
Sanna Pasonen-Seppanen • Piia Takabe •
Michael Edward • Leena Rauhala • Kirsi Rilla •
Markku Tammi • Raija Tammi
Accepted: 6 July 2012 / Published online: 24 July 2012
� Springer-Verlag 2012
Abstract In many cancers hyaluronan content is
increased, either by tumor cells or the surrounding stromal
cells and this increased hyaluronan content correlates with
unfavorable clinical prognosis. In the present work, we
studied the effects of melanoma cell (aggressive melanoma
cell line C8161)-derived factors on fibroblast hyaluronan
synthesis, intracellular signaling, MMP expression and
invasion. Treatment of the fibroblast cultures with mela-
noma cell conditioned medium (CM) caused accumulation
of hyaluronan in the culture medium and formation of thick
pericellular hyaluronan coat and hyaluronan cables. The
expression of Has2 was increased approximately 20-fold
by the C8161 melanoma cell CM, while Has1 and Has3
were increased twofold. Knock-down of Has2 expression
with siRNA showed that Has2 was responsible for the
increased hyaluronan synthesis induced by the melanoma
cell CM. To find out the signaling routes, which led to
Has2 upregulation, the phosphorylation profiles of 46
kinases were screened with phosphokinase array kit. Mel-
anoma cell CM treatment strongly induced a rapid phos-
phorylation of p38, JNK, AKT, CREB, HSP27, STAT3 and
cJUN. Treatment of the fibroblasts with specific inhibitors
of PI3K, AKT and p38 reduced the melanoma cell CM-
induced hyaluronan secretion, while the inhibitor of
PDGFR totally blocked it. In addition, siRNA for
PDGFRa/b inhibited Has2 upregulation in melanoma cell
CM-treated fibroblasts. In parallel with the increased hya-
luronan synthesis the melanoma cell CM-treated fibroblasts
showed spindle shape, numerous long cell protrusions,
enhanced MMP expression and increased invasion into
collagen-Cultrex matrix. siRNA blocking of Has2 or
PDGFRa/b expression reversed the stimulatory effect of
melanoma cell CM on fibroblast invasion. PDGF secreted
by melanoma cells thus mediated fibroblasts activation,
with HAS2 upregulation as a major factor in the fibroblast
response. This effect on stromal matrix is suggested to
favor tumor growth.
Keywords Hyaluronan � Melanoma � Fibroblast �Interaction � Invasion � Extracellular matrix
Abbreviations
CM Conditioned medium
ECM Extracellular matrix
bHABC Biotinylated hyaluronan binding complex
ELSA Enzyme-linked sorbent assay
HAS Hyaluronan synthase
Introduction
Tumor stroma consists of extracellular matrix (ECM) as
well as various cell types like fibroblasts, endothelial cells
and immune cells. It has been recognized that the stromal
cells have an active role in tumor progression. Cancer cells
are in constant interaction with the stromal cells; all of
these cells release different mitogenic, angiogenic and
Electronic supplementary material The online version of thisarticle (doi:10.1007/s00418-012-1000-x) contains supplementarymaterial, which is available to authorized users.
S. Pasonen-Seppanen (&) � P. Takabe � L. Rauhala � K. Rilla �M. Tammi � R. Tammi
School of Medicine, Institute of Biomedicine/Anatomy,
University of Eastern Finland, Kuopio, Finland
e-mail: [email protected]
M. Edward
Section of Dermatology, School of Medicine,
University of Glasgow, Glasgow G12 8QQ, UK
123
Histochem Cell Biol (2012) 138:895–911
DOI 10.1007/s00418-012-1000-x
lymphangiogenic growth factors, which support tumor
progression. One major contributor to cancer is the
dynamic ECM in the tumor microenvironment, which is
extensively remodeled during tumor development. The
amount of hyaluronan, a ubiquitous ECM-molecule, is
elevated in many cancers (Sironen et al. 2011; Tammi et al.
2008). Hyaluronan is a high molecular weight, linear, non-
sulfated glycosaminoglycan composed of repeating disac-
charide units of glucuronic acid and N-acetylglucosamine.
This simple sugar molecule is synthesized at the plasma
membrane by three hyaluronan synthases (HAS1-3). In
several cancers, hyaluronan overproduction, either by
tumor cells or the surrounding stromal cells, has been
shown to enhance tumor cell invasion and metastasis and
promote drug resistance leading to a worse clinical prog-
nosis (Auvinen et al. 2000; Sironen et al. 2011; Wang and
Bourguignon 2011). Furthermore, hyaluronan-rich tumor
microenvironment may operate in the recruitment of
inflammatory cells to the tumor area (Kobayashi et al.
2010), which facilitates tumor progression. However, the
exact molecular mechanisms for these events are not clear.
Cutaneous melanoma is one of the most deadly human
cancers. It metastasizes readily and is thus difficult to cure.
Genetic mutations associated with cell cycle regulators and
signaling molecules (NRAS, BRAF, PTEN and AKT3) are
common in melanoma cells. MAPK and AKT signaling
pathways are often constitutively activated (Meier et al.
2005; Satyamoorthy et al. 2003), and their activation is
strongly involved in melanoma dissemination. The com-
bined inhibition of MAPK and AKT signaling pathways
prevents the invasive growth of melanoma cells and
induces their death (Lasithiotakis et al. 2008; Meier et al.
2007). For melanoma development and progression, the
crosstalk between melanoma cells and fibroblasts is vital.
Paracrine growth factors, like platelet-derived growth fac-
tor (PDGF), interleukin 1 alpha (IL-1a), transforming
growth factor beta (TGF-b) and basic fibroblast growth
factor (bFGF) regulate stromal fibroblasts and modulate the
tumor stroma to the benefit of melanoma growth and
invasion (Lee and Herlyn 2007; Melnikova and Bar-Eli
2009). These paracrine signals activate stromal fibroblasts
to produce mitogenic factors for melanoma cells and to
degrade the ECM by MMPs, which facilitates the invasion
and metastasis of tumor cells and the angiogenesis.
Previous data from our group (Edward et al. 2005, 2010)
and by others (Willenberg et al. 2012) have shown that
melanoma cell conditioned medium activates hyaluronan
synthesis in dermal fibroblasts. However, the signaling
mechanisms leading to this and the consequences of
increased hyaluronan production to fibroblast behavior
have remained obscure. In this study we have explored
these questions, and found that the CM collected from the
C8161 cells activated several signaling pathways in the
fibroblasts which acquired an activated phenotype with
increased proliferation, MMP1, 9 and 14 expression and
invasion into type I collagen-Cultrex gel. Experiments with
specific inhibitors and siRNA’s indicated that PDGFR
signaling was involved in Has2 upregulation and hyalu-
ronan accumulation, and that the fibroblast invasion was
dependent both on PDGFR signaling and increased Has2
expression. The results suggest that the activated PDGFR
signaling in melanoma stroma may have an important
effect on tumor progression.
Materials and methods
Cell culture
Fibroblasts were isolated from forearm skin dermis and
used between 5 and 20 passages. Normally, fibroblasts
were cultured in Dulbecco’s modified Eagle Medium
(DMEM) (HyClone, high glucose) supplemented with
10 % inactivated FBS (HyClone, Thermo Scientific,
Epsom, UK), 2 mM L-glutamine (Euroclone, Pavia, Italy)
and penicillin–streptomycin (50 lg/ml streptomycin, 50 U/ml
penicillin; Euroclone), but certain experiments were also
carried out in low glucose DMEM (HyClone). Cells were
passaged twice a week at 1:2 or 1:3. The C8161 melanoma
cell line, originally isolated from abdominal wall metas-
tasis (Welch et al. 1991) was cultured in DMEM (Gibco,
high glucose) supplemented with 10 % FBS, 2 mM
L-glutamine and penicillin–streptomycin. Melanoma cells
were subcultured three times a week at 1:6.
Melanoma conditioned medium
Melanoma cells were grown in T75-cell culture flasks in
their own medium until the flasks were 60 % confluent.
Thereafter cells were washed twice with Hank’s Balanced
Salt Solution (HBSS, Lonza, Verviers, Belgium) and fresh
medium (DMEM with 1 % FBS) was added. After 48 h,
medium was harvested, sterile filtered, concentrated
40-fold using a Millipore ultrafiltration membrane with a
molecular weight cut-off of 30 kDa (Millipore, Tullagreen,
Cork, Ireland), and diluted 1:10 with fresh DMEM
(HyClone, 1 % FBS) (=30 kDa CM). Control medium
(DMEM, 1 % FBS) was treated similarly as the melanoma
cell conditioned medium (=30 kDa control).
Reagents
The PI3K inhibitor, Wortmannin (2 lM), AKT kinase
inhibitor VIII (1 lM), MEK kinase inhibitor UO126
(5 lM), PDGFR inhibitor AG1296 (5 lM) and p38 inhib-
itor SB203580 (15 lM) were purchased from Calbiochem
896 Histochem Cell Biol (2012) 138:895–911
123
(La Jolla, CA, USA). The EGF-receptor inhibitor AG1478
(0.1 lM) was obtained from LC Laboratories (Woburn,
MA, USA). The inhibitors were dissolved in sterile filtered
DMSO or sterile water, protected from light and stored
frozen.
Hyaluronan ELSA
To study the effects of melanoma cell CM on fibroblast
hyaluronan synthesis, fibroblasts were pre-treated with 1 %
FBS containing DMEM for 3 h before the cells were
treated with melanoma cell CM with or without the
inhibitors for 5, 10 or 24 h. Thereafter the cells were
counted and medium was collected to measure hyaluronan
amount using an enzyme-linked sorbent assay (ELSA) as
described in detail previously (Hiltunen et al. 2002).
Hyaluronan binding complex (HABC) prepared from car-
tilage aggrecan and link protein and biotinylated HABC
(bHABC) used in the assay were prepared in house as
described (Tammi et al. 1994). Ninety-six-well Maxisorp
Plates (Nunc, Roskilde, Denmark) were coated with HABC
(1 lg/ml) at 37 �C for 2 h, washed with 0.5 % Tween-PBS
and blocked with 1 % BSA for 1 h at 37 �C. The dilutions
of standard hyaluronan (Provisc, Algon Laboratories, Fort
Worth, TX, USA) and the samples were aliquoted to the
wells. After a 1-h incubation at 37 �C, the plates were
washed with Tween-PBS, incubated with 1 lg/ml bHABC
for 1 h and washed with Tween-PBS. The bound hyalu-
ronan was visualized using a horseradish peroxidase
streptavidin complex (1:20,000, Vector Laboratories Inc.,
Burlingame, CA, USA) and TMB, 3,30,5,50-tetramethyl-
benzidine (Sigma-Aldrich, St Louis, MO, USA) in 0.1 M
sodium acetate buffer containing 0.005 % H2O2. The
absorbances were read at 450 nm. Each sample and stan-
dard were prepared in triplicate. The amount of hyaluronan
in the 30 kDa control and melanoma cell conditioned
medium, *25 and *70 ng/ml, respectively, was sub-
tracted from the samples hyaluronan content. The results
were normalized to cell numbers.
Molecular mass of hyaluronan
To analyze the size of secreted hyaluronan in control and
melanoma cell CM-treated fibroblast cultures, medium
samples were collected after 24 h incubation. Medium
samples were chromatographed on an 1 9 30 cm column
of Sephacryl S-1000 (Amersham Biosciences), equilibrated
and eluted at 4 ml/h with 150 mM sodium acetate, 0.1 %
CHAPS (Sigma), pH 6.8. Hyaluronan content in each
fraction was analyzed with ELSA. The size distribution of
hyaluronan in the samples was estimated from the peak
fractions of known hyaluronan standards (150, 500 and
2,500 kDa), provided by Hyalose (L.C.C., Oklahoma City,
OK, USA).
RNA isolation and quantitative RT-PCR
150,000 cells/well were seeded on 6-well plates, cultured
for 48 h before a 3-h pre-incubation in DMEM with 1 %
FBS and followed by a 24-h treatment with melanoma cell
CM or control medium. Cells were detached and lysed by
adding 1 ml of the RNA extraction reagent/well (TRI
Reagent�, Molecular Research Center Inc., Cincinnati,
USA), and the samples were stored at -70 �C. The total
RNA was extracted with chloroform-isopropanol according
to the standard procedure, washed once with 75 % ethanol
and dissolved in sterile water.
800 ng of total RNA was reverse transcribed using a
VersoTM cDNA Kit according to the manufacturer’s pro-
tocol (Thermo Fisher Scientific, Surrey, UK) and tran-
scription was performed with MJ Research PTC-200
Peltier Thermo Cycler (MJ Research Inc., Watertown,
USA). The transcript levels of Has1-3, CD44, Hyal2,
MMP1, MMP2, MMP9, MT1-MMP (MMP14) and
PDGFRa/b (primer sequences in Table 1) in the fibroblast
cultures were measured using quantitative real-time PCR
(qRT-PCR) on a MX3000P thermal cycler (Stratagene, La
Jolla, CA, USA) using a FastStart Universal SYBR Green
Master (ROX) (Roche, Indianapolis, USA). At the end of
each run a melt curve was obtained to monitor the quality
of the amplicon. Fold inductions were calculated using
the formula 2�ðDDCtÞ , where DDCt is the DCt (treatment)
- DCt (control). DCt is Ct target gene - Ct Arpo (acidic
ribosomal phosphoprotein, used to normalize transcript
levels between samples), and Ct means the threshold cycle
(the PCR cycle where the detection threshold is crossed).
Has2, Has3 and PDGFRa/b siRNA transfection
Has2/Has3 pre-designed siRNAs were purchased from
Ambion (Austin, TX, USA). Transfection was carried out
using a combination of three different Has2 and Has3
siRNAs. Scrambled siRNA (Ambion) was used as a neg-
ative control. One day before transfection, fibroblasts were
plated on 12-well plates (90,000 cells/well) or 6-well plates
(180,000 cells/well). The cells were transiently transfected
with Has2 or Has3, or control siRNA (0.1 lM) using
LipofectamineTM 2000 reagent or Lipofectamine RNAi-
MAX reagent according to the manufacturer’s instructions
(Invitrogen, Carlsbad, CA, USA). 6 h after transfection,
fresh medium (control or melanoma cell CM) was changed.
The following day (24 h), the cells were counted and
medium was analyzed for hyaluronan. The Has2/3 siRNA
experiments were repeated three times. For invasion assay
Histochem Cell Biol (2012) 138:895–911 897
123
Has2 siRNA-transfected cells were plated 24 h after
transfection. The efficacy of knock-down was confirmed by
qPCR. Has2 silencing of fibroblasts caused an 80 %
reduction in Has2 mRNA expression (data not shown).
To inhibit PDGFRa/b expression, subconfluent fibro-
blast cultures were transfected with 0.1 lM siRNAs spe-
cific for human PDGFRa and b (Eurogentec Inc., San
Diego, CA, USA) using Lipofectamine RNAiMAX reagent
according to the manufacturer’s instructions (Invitrogen,
Carlsbad, CA, USA). PDGFRa/b siRNA transfection
caused an 82 % reduction in PDGFRa expression and an
85 % reduction in PDGFRb expression (data not shown).
In protein level, PDGFRb silencing caused approximately
90 % reduction (Supplementary Fig. 1). The sequences for
the used siRNAs are shown in Table 2.
Hyaluronan and CD44 staining in fibroblast cultures
Fibroblasts grown in 8-well chamber slides were fixed and
stained for hyaluronan as described before (Karvinen et al.
2003; Tammi et al. 1998). Shortly, after fixation in 4 %
paraformaldehyde for 20 min, the cells were permeabilized
with 0.1 % Triton X-100 in 1 % BSA-PB for 10 min,
followed by overnight treatment with bHABC (Tammi
et al. 1994). The bound probe was visualized with avidin–
biotin peroxidase complex (ABC, Vector Laboratories Inc,
Burlingame, CA, USA) and with 0.05 % 3,30-diam-
inobenzidine (DAB, Sigma) and 0.03 % H2O2. For fluo-
rescence microscopy, streptavidin labeled either with
Texas Red or FITC (1:1,000, Vector Laboratories), was
used as a secondary step instead of ABC and DAB.
For dual stainings for hyaluronan and CD44, the primary
antibody for CD44 (Hermes 3 (1:200), a generous gift of
professor Sirpa Jalkanen (University of Turku, Turku,
Finland), was mixed with bHABC, and the secondary
antibody FITC-anti-mouse (1:200) with TR-streptavidin
(Vector Laboratories). To visualize plasma membrane
protrusions, fibroblasts were stained for actin and CD44.
After incubation with FITC-anti-mouse antibody, chamber
slides were incubated with 5 lg/ml Alexa Fluor
594-Phalloidin (4 U/ml, Molecular Probes) for 1 h at room
temperature to stain actin.
To visualize the amount of intracellular hyaluronan, the
fibroblasts were incubated in the presence of Streptomyces
hyaluronidase (Seikagaku Kogyo Co., Tokyo, Japan, 10
TRU/ml in culture medium) for 10 min at room tempera-
ture before permeabilization and staining for hyaluronan.
Nuclei were labeled with DAPI (1 lg/ml, Sigma-Aldrich,
St Louis, MO, USA). The fluorescently labeled specimens
were viewed with a Zeiss Axio Observer inverted
Table 1 qRT-PCR primersGene Seguence (50–30) Product size (bp)
Arpo For-AGATGCAGCAGATCCGCAT 318
Rev-GTGGTGATGCCCAAAGCTTG
HAS1 For-CAAGATTCTTCAGTCTGGAC 124
Rev-TAAGAACGAGGAGAAAGCAG
HAS2 For-CAGAATCCAAACAGACAGTTC 186
Rev-TAAGGTGTTGTGTGTGACTG
HAS3 For-CTTAAGGGTTGCTTGCTTGC 194
Rev-GTTCGTGGGAGATGAAGGAA
CD44 For-CATCTACCCCAGCAACCCTA 153
Rev-CTGTCTGTGCTGTCGGTGAT
Hyal2 For-CCTCTGGGGCTTCTACCTCT 217
Rev-CTGAACACGGAAGCTCACAA
MMP1 For-GAAAAGCGGAGAAATAGTGG 381
Rev-TCCAGGTCCATCAAAAGG
MMP2 For-TGCTGGAGACAAATTCTGGA 200
Rev-ACTTCACGCTCTTCAGACTTTGG
MMP9 For-TGCCCGGACCAAGGATACAG 182
Rev-TCAGGGCGAGGACCATAGAG
MMP14 For-GCCTTCTGTTCCTGATAAACC 345
Rev-GCATCAATCTTGTCGGTAGG
PDGFRa For-TGGCTGCTCGCAACGTCCTC 234
Rev-GGTAAGGGGTGCCACCAAGGG
PDGFRb For-TCACCGTGGTTGAGAGCGGC 129
Rev-ACCACAGGACAGTGGGCGGT
898 Histochem Cell Biol (2012) 138:895–911
123
microscope (409 NA 1.3 oil or 639 NA 1.4 oil objectives)
equipped with Zeiss LSM 700 confocal module (Carl Zeiss
Microimaging GmbH, Jena, Germany).
Characterization of fibroblast shape
The effect of melanoma cell CM on fibroblast shape was
examined from confocal images using the ImageJ64 soft-
ware. Cell shape was quantified by the axial ratio (AR)
from 50 cells. The axial ratio is one for a circle and become
larger in an elongated cell.
Axial ratio ¼ ðmajor axis length)=ðminor axis length):
Live cell imaging
Fibroblasts were plated (10,000 cells/well) on Ibidi chamber
slides (Ibidi GmbH, Munich, Germany) and 24 h after
plating, the cells were exposed to the pre-incubation med-
ium for 3 h and thereafter the medium was changed (30 kDa
control medium or 30 kDa CM). On the following day, live
cells were stained for hyaluronan with HABC labeled in
house with Alexa fluor 568 (Molecular Probes, Eugene, OR,
USA). For the live cell imaging, a Zeiss XL-LSM S1 incu-
bator with temperature and CO2 control was utilized. ZEN
2009 software (Carl Zeiss Microimaging GmbH) was used
for image processing, measurements and 3D rendering.
Scanning electron microscopy (SEM)
For SEM, fibroblasts were grown on 13-mm coverslips
until subconfluent. After that cells were washed and
pre-treated with 1 % FBS-DMEM medium for 3 h and
thereafter 30 kDa CM or 30 kDa control medium was
added. After 24 h treatment, the cultures were washed and
fixed with 4 % paraformaldehyde for 2 h at room tem-
perature and dehydrated through a graded series of ethanol.
After critical point drying, cells were shadowed with gold
and photographed on an XL30 TMP environmental SEM
(FEI Company, the Netherlands) at 15 kV.
Phospho-receptor tyrosine kinase and phosphokinase
arrays
The phosphorylation level of receptor tyrosine kinases in
the fibroblasts was analyzed with Human Phospho-RTK
Array (R&D Systems, Abingdon, UK). 500,000 cells were
plated in 6-cm Petri dishes and grown for 48 h. Fresh
medium containing 1 % FBS (pre-incubation medium) was
changed 24 h prior to treatment. 30 kDa control medium
and 30 kDa CM were added for 5 and 15 min periods. The
cells were then solubilized at 1 9 107 cells/ml in lysis
buffer 6 provided in the kit and incubated on ice for
30 min, with intermittent gentle rocking, and centrifuged at
16,0009g for 5 min at 4 �C, after which the supernatant
was transferred to a clean tube. Protein concentration was
measured by the Bradford’s assay and the samples were
stored at -70 �C until the analysis. 100 lg of protein was
used for each assay performed according to the manufac-
turer’s instructions. The density of the spots in an exposed
film was analyzed by Image J software. The phosphokinase
array was done in the same way except that 30 min and 2 h
incubation periods were used.
Cytokine and angiogenesis array
To elucidate the cytokines and growth factors that the
C8161 melanoma cells secrete, Human Cytokine and
Angiogenesis arrays (R&D Systems, Abingdon, UK) were
performed according to the manufacturer’s instructions.
The positive spots and their intensities were estimated by
two independent observers visually from the array films.
Western blotting
After 2 and 24 h treatments, cellular proteins were solu-
bilized in RIPA lysis buffer. The samples (15 lg) were
electrophoresed on 10 % sodium dodecyl sulfate-poly-
acrylamide gels (SDS-PAGE), and transferred to Immobi-
lonTM-NC membranes (Millipore, Bedford, MA, USA) by
a constant current of 2 mA/cm2 in a Fastblot B43 semidry
blotter (Biometra GmbH, Gottingen, Germany). The blots
were blocked for 30 min at room temperature in 10 mM
Tris, 150 mM NaCl, pH 7.4 (Tris-saline blocking buffer)
containing 1–5 % BSA or 1–5 % fat-free milk powder and
Table 2 siRNA sequences
siRNA Sequence (50–30)
Has2 Sense-GCUGCUUAUAUUGUUGGCUtt
Antisense-AGCCAACAAUAUAAGCAGCtg
Sense-CCUAACUUAUGGACUGUUUtt
Antisense-AAACAGUCCAUAAGUUAGGtt
Sense-GGAAAAGUUCUUUCAACCUtt
Antisense-AGGUUGAAAGAACUUUUCCtt
Has3 Sense-CCUUCUCGUGCAUCAUGCAtt
Antisense-UGCAUGAUGCACGAGAAGGtg
Sense-CGGAAAAGCACUACCUGUCtt
Antisense-GACAGGUAGUGCUUUUCCGtg
Sense-CCAUCGAGAUGCUUCGAGUtt
Antisense-ACUCGAAGCAUCUCGAUGGtg
PDGFRa Sense-UAUAAUGGCAGAAUCAUCAtt
Antisense-UGAUGAUUCUGCCAUUAUAtt
PDGFRb Sense-UGUCACAGGAGAUGGUUGAtt
Antisense-UCAACCAUCUCCUGUGACAtt
Histochem Cell Biol (2012) 138:895–911 899
123
0.1 % Tween-20. Thereafter they were incubated with
primary antibodies overnight at 4 �C, using the following
antibody dilutions: anti-pAKT (ser 473) 1:200 (Cell Sig-
naling), anti-active p38 1:200 (Promega, Madison, WI,
USA), anti-pJNK 1:200 (Santa Cruz), anti-pERK1/2 1:500
(Santa Cruz), and anti-PDGFRb 1:250 (Cell Signaling).
After washing, the bound primary antibodies were detected
with DyLight TM 800/TM 680 secondary antibodies
(1:1,000 to 1:6000, ThermoScientific) in 10 mM Tris,
150 mM NaCl, pH 7.4, containing 2–5 % fat-free milk
powder for 1 h at room temperature. The blots were
washed four times with 0.1 % Tween-20 in Tris-saline
buffer and scanned for fluorescence with Odyssey� reader
(LI-COR�, Lincoln, NE, USA). After scanning, the bound
antibodies were removed by NaOH (0.2 M) treatment for
5 min at room temperature followed by incubation with an
antibody against actin (diluted 1:5,000, Sigma) in TBS
containing 2 % BSA overnight at 4 �C. After washing, the
blots were incubated with goat anti-rabbit IgG, DyLight
TM 680 secondary antibody 680 (1:4,000 dilution in TBS
containing 2 % BSA, ThermoScientific) for 1 h at room
temperature and rescanned.
Invasion/migration assay
Type I collagen (BD Biosciences, Two Oak Park, Bedford,
MA, USA) and Cultrex Basement Membrane Extract
(Trevigen, Gaithersburg, MD, USA) 1:1 gel was added on
top of confluent fibroblast cultures on 8-well Ibidi chamber
slides and the gel was allowed to polymerize at 37 �C for
1 h before the medium (30 kDa control or 30 kDa CM with
or without the PDGFR inhibitor) was added. Medium was
changed daily and the cells were allowed to invade for
2 days. The cultures were fixed with 4 % paraformalde-
hyde for 1.5 h at room temperature, washed with 0.1 M
phosphate buffer and incubated in glycine solution
(200 mM) for 20 min. Thereafter the cultures were
washed, blocked in 1 % BSA for 30 min and permeabili-
zed with 0.1 % Triton-X100-1 % BSA for 30 min at room
temperature. Fibroblasts were stained with 5 lg/ml Alexa
Fluor 594-Phalloidin (4 U/ml, Molecular Probes) for 2 h at
room temperature and washed with phosphate buffer.
Invasion was visualized using a Zeiss LSM700 micro-
scope. The wells were imaged at least from eight different
areas and image stacks were taken at 0.5 lm z intervals
using a 209 objective. ZEN 2009 software (Carl Zeiss
Microimaging GmbH) was used for image processing,
measurements and 3D rendering.
Proliferation assay
Fibroblasts (40,000 cells/well) were seeded in 24-well
plates and incubated for 24 h in their normal culture
medium. Thereafter, fresh medium, containing either
30 kDa control medium or 30 kDa CM, was changed
(duplicate wells). The cells were counted using a hemo-
cytometer 1, 2, 3 and 4 days after plating. The experiment
was repeated three times.
Statistical analyses
The data from hyaluronan ELSA measurements, RT-PCR,
morphometric measurements (axial ratio), proliferation and
invasion assay were analyzed by one-way analysis of
variance with LSD or Tukey as a post hoc test. A differ-
ence was considered statistically significant when the
p value was \0.05.
Results
Melanoma cell-derived factors stimulate fibroblast
hyaluronan synthesis by upregulating Has2 mRNA
and protein expression
Melanoma cell conditioned medium (CM) was collected
after a 48-h incubation and concentrated using a Millipore
ultrafiltration membrane with a 30-kDa cut-off, followed
by dilution with fresh fibroblast culture medium (DMEM,
1 % FBS, EuroClone). The unused melanoma culture
medium (1 % FBS-DMEM, Gibco) was processed in the
same way and used as control. After 24 h, the concentrated
30 kDa control medium (30 kDa control) stimulated hya-
luronan synthesis twofold compared to unconcentrated
control medium, while the concentrated melanoma CM
Fig. 1 Melanoma cell conditioned medium (=30 kDa CM) stimulates
fibroblast hyaluronan synthesis via Has2 upregulation. Fibroblasts
were treated for 24 h with either unconcentrated, unused melanoma
culture medium (control) or concentrated unused melanoma culture
medium (30 kDa control) or with unconcentrated (CM) or concen-
trated (30 kDa CM) conditioned medium, collected from C8161
melanoma cell cultures as described in the ‘‘Materials and methods’’.
The fibroblast culture media were collected and analyzed for
hyaluronan content using ELSA (a), or analyzed for the size
distribution of the secreted hyaluronan using Sephacryl S-1000 gel
chromatography. The data in a represent means ± SD of six separate
experiments and b represents one of two experiments with similar
results, the bars indicating the ranges of two parallel samples (b). The
mRNA expression levels of hyaluronan-producing enzymes (HAS1-3),
hyaluronan receptor (CD44) and hyaluronan-catabolizing enzyme
(Hyal2) were analyzed with qRT-PCR (c). The effects of Has2 and
Has3 siRNA on hyaluronan production in 30 kDa CM-treated
fibroblasts are shown in d. The data in d represent mean ± SD of
three individual experiments. *p \ 0.05, **p \ 0.01, ***p \ 0.001
one way analysis of variance, LSD post hoc test. Fibroblasts were
stained for HAS2 (e, f) with a specific HAS2 antibody. HAS2
immunoreactivity was more prominent in CM-treated (f) than control
cultures (e), localizing in intracellular vesicles (arrows) and at the
plasma membrane. Scale bars 40 lm in e and f
c
900 Histochem Cell Biol (2012) 138:895–911
123
(30 kDa CM) caused an approximately sevenfold increase
compared to the unconcentrated control medium (control)
and 3.5-fold as compared to the concentrated control
medium (30 kDa control) (Fig. 1a). The stimulatory effect
of melanoma cell CM (30 kDa CM) on fibroblast hyalu-
ronan synthesis was seen already after 5 h incubation. At
5- and 10-h time points, hyaluronan level in the culture
medium was increased by approximately 38 and 45 %,
respectively, (data not shown).
The melanoma CM passing through the 30 kDa cut-off
membrane had no effect on hyaluronan synthesis (data not
shown). Complete melanoma CM (which was not con-
centrated and diluted with the fresh medium) stimulated
fibroblast hyaluronan synthesis only about 1.7-fold
Hya
luro
nan
(% o
f co
ntro
l)
Contro
l
30 kD
a con
trol
30 kD
a CM CM
**
***
***
Gen
e ex
pres
sion
(%
of
cont
rol)
Has1Has2
Has3CD44
Hyal2
0
50
100
150
200
250
300
350
17 21 25 29 33 37 41 45
30 kDa Control
30 kDa CM
Hya
luro
nan/
frac
tion
(ng/
ml)
Fraction number
2500 kDa
500 kDa
150 kDa
400
0
50
100
150
200
250
Contr
siRNA
Has2 s
iRNA
Has3 s
iRNA
30 kD
a CM
+Contr
siRNA
30 kD
a CM
+Has2 s
iRNA
30 kD
a CM
+Has3 s
iRNA
Hya
luro
nan
(% o
f co
ntro
l)
a b
c d
200
400
600
800
1000
0
0
500
1000
1500
2000
2500***
*
***
30 kDa CM
30 kDa Control 30 kDa CM
e f
Has2 Has2
30 kD
a con
trol
Histochem Cell Biol (2012) 138:895–911 901
123
(Fig. 1a). This is likely due to the depletion of essential
nutrients in the complete melanoma cell CM.
To study whether 30 kDa CM affects the size distribu-
tion of the secreted hyaluronan, medium samples were
analyzed using Sephacryl S-1000 gel chromatography.
Relatively high proportion of the secreted hyaluronan was
of high molecular mass (*2.5 9 106 kDa) in both control
and melanoma cell CM-treated cultures, with no apparent
size difference (Fig. 1b).
To elucidate whether increased Has mRNA expression
is responsible for the elevated hyaluronan secretion, RNA
was isolated from 30 kDa control medium and 30 kDa
CM-treated fibroblast cultures, and subjected to qRT-PCR.
The Ct values for Has1, Has2 and Has3 were approxi-
mately 33, 24 and 29, respectively, (data not shown)
indicating that Has2 is the main Has isoform in fibroblasts,
in line with previous publications (Averbeck et al. 2007).
The expression of Has2 was upregulated by 20-fold after
24 h treatment with melanoma cell CM (Fig. 1c), while the
levels of Has1 and Has3 mRNA showed a more modest
twofold increase (Fig. 1c). As the expression level of Has1
is very low in fibroblasts, its contribution to total hyalu-
ronan synthesis is probably marginal. The expressions of
hyaluronidase 2 (Hyal2) and hyaluronan receptor CD44
were not affected by melanoma cell CM (Fig. 1c). Immu-
nostaining with HAS2-specific antibody revealed a more
intense staining in fibroblasts treated with melanoma cell
CM (Fig. 1f) than in the control cells (Fig. 1e). The HAS2
immunostaining was most concentrated in small intracel-
lular vesicles (arrows), nuclear membrane (giving an
impression of nuclear staining), and plasma membrane.
To verify the relative contributions of Has2 and Has3 to
the hyaluronan response induced by melanoma CM, we
suppressed their expression with siRNAs. Fibroblasts were
transiently transfected with Has2 and Has3 siRNAs and
treated with control and melanoma cell CM. Has2 siRNA
transfection caused a significant downregulation in the
basal hyaluronan secretion level and reversed the effect of
melanoma cell CM on fibroblast hyaluronan synthesis,
while Has3 siRNA transfection had only a minor effect
(Fig. 1d). These experiments support the conclusion that
the hyaluronan production induced in fibroblasts treated
with melanoma CM is due to Has2 upregulation.
Melanoma cell CM induces a thick pericellular
hyaluronan coat and hyaluronan cables in fibroblasts
We performed histochemical stainings to study the amount
and localization of cell-associated hyaluronan (Fig. 2). In
the control, untreated cultures the intensity of hyaluronan
staining varied, being usually either weak or modest, with
occasional intensely hyaluronan positive cells (Fig. 2a, c).
In contrast, fibroblast cultures treated with the melanoma
cell CM showed uniform, intense staining for hyaluronan
(Fig. 2b, d). Hyaluronan staining was partially colocalized
with CD44 (Fig. 2d, yellow). To visualize the hyaluronan
coat in its natural form, we also applied fluorescently
labeled HABR on unfixed cells (Fig. 2g, h). Pericellular
hyaluronan formed a thick hyaluronan coat around the
melanoma cell CM-treated fibroblasts (Fig. 2h), while in
control medium the coat was thin and less intensely fluo-
rescent (Fig. 2g). In addition to the pericellular hyaluronan
associated tightly with the cell surface, long hyaluronan
positive, cable-like structures extending on top of several
cells could be seen in cultures treated with melanoma cell
CM (Fig. 2d arrows, insert), but not in control cultures
(Fig. 2c).
Digestion of fixed but unpermeabilized cells with
Streptomyces hyaluronidase removed most of the hyalu-
ronan staining (Fig. 2i, j), confirming that it was localized
on cell surface. However, part of hyaluronan was resistant
to hyaluronidase treatment, indicating intracellular loca-
tion, as also confirmed by the confocal analyses (Fig. 2e,
f). Treatment of the fibroblasts with the melanoma cell CM
increased the number of cells containing intracellular
hyaluronan (Fig. 2j, asterisks).
Induced hyaluronan synthesis is due
to PDGFR-PI3K-AKT and p38 signaling
Cytokine and angiogenesis arrays revealed that C8161
melanoma cells produce significant amounts of various
cytokines and growth factors like GM-CSF, IL-8, IL-6,
IL-1a and b, PDGF-AA, PDGF-AB/PDGF-BB, uPA and
MIF (Supplementary Table I and II).
To examine the signaling routes involved in melanoma
cell CM-induced Has2 upregulation in fibroblasts, we
screened the phosphorylation profile of different tyrosine
kinase receptors and intracellular kinases and their sub-
strates. After 10 min treatment, melanoma cell CM
strongly activated the PDGF receptors a and b in fibro-
blasts. Furthermore, the EGF receptor was slightly upreg-
ulated in melanoma cell CM-treated fibroblasts, while
control medium was unable to induce phosphorylation in
any of the receptors in the arrays (Fig. 3a). Of the signaling
proteins, treatment of the fibroblasts for 30 min with
melanoma cell CM markedly increased the phosphoryla-
tion levels of p38, JNK, AKT, CREB, HSP27, STAT3 and
cJun (Fig. 3b, c). After 2 h treatment, the phosphorylation
of HSP27 and STAT3 had completely disappeared and the
phosphorylation of JNK also returned close to control
level, and of AktT308 below control (Fig. 3c). However, at
24-h time point AKT showed a tenfold increase in phos-
phorylation by melanoma cell CM (Fig. 3e). Also, p38 and
extracellular signal-regulated kinase 1/2 (ERK1/2) were
significantly activated at this time point (Fig. 3d), while
902 Histochem Cell Biol (2012) 138:895–911
123
Fig. 2 Melanoma cell
conditioned medium (=30 kDa
CM) increases hyaluronan
staining intensity and the size of
hyaluronan coat. Fixed and
permeabilized fibroblast
cultures were stained for
hyaluronan using DAB as a
chromogen (a, b). In c–f and i, j,fibroblast cultures were stained
for hyaluronan (red) and its cell
surface receptor CD44 (green),
as described in ‘‘Materials and
methods’’. To visualize the
intact pericellular hyaluronan
coat, unfixed cells were exposed
to fHABC and viewed with the
confocal microscopy (g, h).
Fibroblasts exposed to
melanoma cell secreted factors
(30 kDa CM) showed more
intracellular hyaluronan (f) as
well as more intense overall
hyaluronan staining (b, d) than
the control cultures (a, c, e).
Furthermore, hyaluronan cables
were observed in melanoma cell
CM-treated fibroblasts (arrowsin d and insert). e and f are
Z-sections created from stacks
and c, d, g and h represent
single optical sections.
i, j Intracellular hyaluronan
(red) as compressed stacks of
confocal optical sections.
In these cultures (i, j),Streptomyces hyaluronidasetreatment after fixation was used
to remove pericellular
hyaluronan before
permeabilization. Scale bars10 lm in e and f, 40 lm in c, d,
g, h, i and j, and 80 lm in a, b
Histochem Cell Biol (2012) 138:895–911 903
123
Spot
Int
ensi
ty (
% o
f co
ntro
l)
a c
30 kDa CM
30 kDa Control
1) p382) JNK pan3) AKT (S473)4) AKT (T308)
5) CREB6) HSP277) STAT38) cJUN
1 23 4
5 6
7 8
p38a
Akt
(S47
3)A
kt (T
308)
CR
EBH
SP27
STAT
3c-
Jun
JNK
pan
b
Human Phospho-RTK Array Human Phospho Kinase Array
0
100
200
300
400
500
600 30 min2 h
pERK1/2
actin
actin
p38
actin
30 kD
a
Contro
l30
kDa
CM
0
200
400
600
800
1000
1200
1400
1600
pAKT
30 kD
a CM
30 kD
a CM
+
AKT inhib
itor
30 kD
a CM
+
p38 i
nhibi
tor
30 kD
a CM
+
PDGFR inhib
itor
30 kD
a CM
30 kD
a CM
+
AKT inhib
itor
30 kD
a CM
+
p38 i
nhibi
tor
30 kD
a CM
+
PDGFR inhib
itor
30 kD
a Con
trol
actin
pAK
T I
nten
sity
(%
of
cont
rol)
0
50
100
150
200
250
300
350
pERK
1
pERK
2p3
8
pJN
K
Inte
nsity
(% o
f con
trol)
pJNK
1) EGFR2) PDGFRα3) PDGFRβ
1 2 3
d
e
1 23
45 6
7 8
Fig. 3 Factors secreted by melanoma cells activate EGF and PDGF
receptors and AKT, ERK1/2 and p38 kinases in fibroblasts.
Phosphorylation level of several phospho tyrosine kinase receptors
and signaling proteins was analyzed with Human Phospho-Receptor
Tyrosine Kinase Array Kit and Human Phospho Kinase Array Kit.
Fibroblasts were treated with melanoma cell CM for 15 min and
protein samples were collected for Phospho-RTK array. The phos-
phorylation of EGFR and PDGFR was induced after melanoma cell
CM treatment (a). In Human Phospho Kinase Array, the results of the
control array (above) and one after 30 min treatment with melanoma
cell CM (below) are shown (b). The dots encircled represent the
activated kinases listed below the image. The means and ranges of dot
densities collected from the two spots are shown in c. Fibroblast
cultures were treated with melanoma cell CM with or without the
inhibitors for 24 h. Cell lysates were analyzed by Western blotting
with phospho-specific antibodies against pERK1/2, pJNK, p38
(d) and pAKT (e). Actin-normalized band intensities are shown as
mean ± SEM of three separate experiments
904 Histochem Cell Biol (2012) 138:895–911
123
JNK was below the control level (Fig. 3d). The activation
of AKT was blocked with the AKT kinase (1 lM) and
PDGFR (5 lM) inhibitors, while p38 inhibitor had smaller
effect on AKT phosphorylation (Fig. 3e).
To further elucidate the involvement of the activated
kinases in the stimulation of hyaluronan synthesis, we
exposed fibroblasts to melanoma cell CM (30 kDa CM)
with different kinase inhibitors and studied their effect on
fibroblast hyaluronan synthesis. The AKT kinase inhibitor
(1 lM) and the PI3K inhibitor Wortmannin (2 lM) cut the
stimulation of melanoma cell CM on fibroblast hyaluronan
synthesis to about half (Fig. 4a). The p38 kinase inhibitor
SB203580 (15 lM) had even stronger effect and the
PDGFR inhibitor AG1296 (5 lM) totally reversed the
stimulatory effect of melanoma cell CM on fibroblast
hyaluronan synthesis, suggesting that these signaling routes
are involved in the surge of hyaluronan synthesis. Instead,
the EGFR inhibitor, AG1478 (0.1 lM) and MEK kinase
inhibitor, UO126 (5 lM) had only slight effects on fibro-
blast hyaluronan synthesis, suggesting that activation of the
EGFR-initiated pathway has a minor role in the induction
of hyaluronan production by melanoma cell CM.
To confirm the role of PDGFR in the melanoma cell
conditioned medium-induced hyaluronan response, we
used RNA interference technology with PDGFR a and bspecific siRNAs and analyzed the mRNA levels of Has
isoenzymes, CD44 and Hyal2 (Fig. 4b). While the other
genes analyzed showed just slight tendencies toward
reduced expression in PDGFR siRNA-treated cells com-
pared to control siRNA-treated cells, the PDGFR knock-
down significantly reduced the melanoma cell CM-induced
Has2 expression (Fig. 4b).
The morphology of fibroblasts is changed
after melanoma cell CM treatment
Microscopy of the cells subjected to melanoma cell CM
revealed that the fibroblasts acquired a spindle shape
morphology, clearly different from that in control cells
(Fig. 2a, b). Analyses with scanning electron microscopy
(SEM) supported this finding. Control cells were usually
flattened and more spread (Fig. 5a, c, e), with broad and
short cellular extensions, while melanoma cell CM-treated
fibroblasts were extremely elongated (Fig. 5b, d, f). Cell
AKT-inhibitor
p38 inhibitorPDGFR inhibitor
EGFR inhibitorMEK inhibitor
+
++
+
+PI3K inhibitor +
++
++
30 kDa Control 30 kDa CM
Hya
luro
nan
(% o
f co
ntro
l)
***
****
0
100
200
300
400
500
600
Has1 Has2 Has3 CD44 Hyal2
30 kDa Control (Contr siRNA)30 kDa Control (PDGFRα/β siRNA)30 kDa CM (Contr siRNA)30 kDa CM + PDGFRα/β siRNA
Gen
e ex
pres
sion
(%
of
cont
rol)
a b
100
200
300
0
50
150
250
350
*
*** **
**
*** ***
Fig. 4 Hyaluronan accumulation induced by melanoma cell CM is
counteracted by inhibitors of AKT, PI3K, PDGFR and p38. Fibro-
blasts were treated with inhibitors of signaling proteins with and
without melanoma cell conditioned medium (30 kDa CM), and the
culture media collected after 24 h incubation were analyzed for
hyaluronan secretion (a). The results are expressed as percentages of
the untreated control cultures (mean ± SD of six experiments). In b,
fibroblasts were transfected either with control siRNA or PDGFRa/b
siRNAs and thereafter the effect of melanoma cell CM or control
medium on the mRNA expression levels of hyaluronan-producing
enzymes (HAS1-3), hyaluronan receptor (CD44) and hyaluronan-
catabolizing enzyme (Hyal2) was studied with qRT-PCR. The data
represent mean ± SEM of three independent experiments. The
statistical significance of the differences was tested using ANOVA
with LSD post hoc test
Histochem Cell Biol (2012) 138:895–911 905
123
elongation was also confirmed with image-analysis pro-
gram as described in ‘‘Materials and methods’’. The axial
ratios of melanoma cell CM-treated fibroblasts were sig-
nificantly higher than those of control cells (Fig. 5g). In
addition, these melanoma cell CM-treated fibroblasts
contained numerous, thin protrusions (Fig. 5b, arrows).
Staining with phalloidin showed that protrusions were rich
in filamentous actin (Fig. 5f), directed laterally from the
cell body and attached to the substratum (Fig. 5b, arrows; f
insert). These protrusions in melanoma cell CM-treated
0
1
2
3
4
5
6
30 kD
a Con
trol
30 kD
a CM
Axi
al r
atio
(C
ell e
long
atio
n)
**
30 kDa Control 30 kDa CM
g
Fig. 5 Melanoma cell
conditioned medium (=30 kDa
CM) changes the morphology of
fibroblasts. Fibroblast cultures
were processed for scanning
electron microscopy as
described in ‘‘Materials and
methods’’. Fibroblasts treated
with 30 kDa CM (b, d) were
more elongated than control
cells (a, c) and contained long,
thin plasma membrane
protrusions (arrows in b). In
e and f, fibroblast cultures were
stained for actin (red) and CD44
(green), to visualize plasma
membrane protrusions (arrowsin f). Scale bars 20 lm in a, b,
e and f; 10 lm in f insert;500 lm in c and
d. Morphometric measurement
of the axial ratios of control and
melanoma cell CM-treated
fibroblasts (g). Data represent
mean ± SEM of 50 cells from
three independent experiments.
The statistical significance of
the differences was tested using
ANOVA with LSD post hoc test
(**p \ 0.01)
906 Histochem Cell Biol (2012) 138:895–911
123
fibroblasts may be a hallmark of their reduced adhesion and
induced motility.
Melanoma cells secrete factors that stimulate fibroblast
proliferation and invasion
As hyaluronan synthesis is often associated with cell pro-
liferation (Liu et al. 2001; Kozlova et al. 2012) and inva-
sion (Kim et al. 2004), we assayed the effect of melanoma
cell CM on fibroblast proliferation by counting cell num-
bers and on their migration/invasion by analyzing the
ability to spread into type I collagen-Cultrex (1:1) gel
(Figs. 6, 7). After 3 days the cell number was increased
approximately 40 % in melanoma cell CM-treated fibro-
blasts compared to control cells (Fig. 6).
The limited ability of fibroblasts in control medium to
invade the matrix was significantly stimulated by melanoma
cell CM (Fig. 7a, b). In control cultures there were wide
areas without any invading cells, while the cultures treated
with melanoma cell CM were uniformly filled by the
invading cells (Fig. 7a). AG1296 (5 lM), a PDGFR inhib-
itor, and knocking down of PDGFRa and b with specific
siRNAs completely reversed the effect of melanoma cell
CM stimulation on fibroblast invasion (Fig. 7a, b, c), indi-
cating that the invasion was dependent on signaling through
this pathway. Since matrix metalloproteinases (MMPs) are
known to be important for cell invasion (Gaggioli et al.
2007), and their expression in fibroblasts has been shown to
be stimulated with melanoma cell conditioned medium
(Wandel et al. 2000), we screened the mRNA expression
levels of MMP-1, MMP-2, MMP-9 and MMP-14 (MT1-
MMP) (Fig. 7e, Supplementary Fig. 1). We found that
C8161 melanoma cell CM stimulated the expression of
MMP-1 by approximately 340-fold and the expression
of MMP-9 100-fold. Furthermore, the expression level of
MMP-14 was slightly increased (Fig. 7e). Of these, only
MMP9 appeared to be downstream of PDGFR as indicated
by the experiments using the PDGFR inhibitor and PDGFRaand b siRNAs (Fig. 7e, Supplementary Fig. 2).
As the increased expression of MMPs could explain the
increased invasion induced by melanoma cell conditioned
medium, we wanted to see if hyaluronan response played
any role in it. To this end we used Has2 siRNA. It had an
inhibitory effect, preventing the melanoma cell CM-
induced invasion, indicating that hyaluronan is essential for
the migratory response of fibroblasts (Fig. 7d).
Discussion
There is growing evidence that the interaction between
tumor and stromal cells is essential for cancer progression.
In the present work, we show that melanoma cell condi-
tioned medium (CM) activates hyaluronan synthesis in
fibroblasts, mainly through increased expression of Has2.
Of the various cytokines and growth factors in the mela-
noma cell conditioned medium and the signaling pathways
activated by it, particularly PDGF and the activation of
PDGFR, PI3K-AKT and p38 signaling pathways were
found to be important for the enhanced hyaluronan pro-
duction. Furthermore, our data show that melanoma cell-
derived factors induce the fibroblasts to adapt an activated
phenotype with spindle shape morphology, formation of
long cellular protrusions, upregulation of MMP1, MMP9
and MMP14 expressions, enhanced cell proliferation and
increased invasion into the matrix. Our results demonstrate
that both PDGFR signaling and upregulation of Has2
expression are essential for the activation of fibroblast
invasion.
In line with the previous results (Edward et al. 2005,
2010) we found that the C8161 cells secreted soluble fac-
tors, which stimulate fibroblast hyaluronan synthesis and
Has expression. Although fibroblasts expressed all hyalu-
ronan synthase isoenzymes (Has1-3) as also reported pre-
viously (Averbeck et al. 2007), and all of them responded
to the melanoma CM treatment, Has2 was the isoenzyme
most probably responsible for the increased hyaluronan
production. Thus, its basal expression level was highest of
all Has isoforms in fibroblasts, and it showed the highest
fold change after melanoma cell CM treatment. Confirming
the dominant role of Has2 in the CM-induced hyaluronan
response, knocking down of Has2 expression with siRNA
completely blocked the stimulation. The data indicate that
melanoma cell CM regulates Has2 activity in fibroblasts at
the transcriptional level, and this results in a higher HAS2
2
2.5
3
0 1 2 3 40.5
1
1.5
Cel
l Num
ber
(10
cells
/wel
l)5
Days
30 kDa Control
30 kDa CM
p= 0.028 (*)
Fig. 6 Melanoma cell-secreted factors activate fibroblast prolifera-
tion. Subconfluent fibroblast cultures were exposed to melanoma cell
CM and cell number was counted 1, 2 and 3 days thereafter. The
statistical significance of the differences was tested using ANOVA
with LSD post hoc test (*p \ 0.05). The data represent mean ± SEM
of three independent experiments
Histochem Cell Biol (2012) 138:895–911 907
123
protein level as indicated by immunohistochemical stain-
ings (Fig. 1f).
Has2 mRNA expression is known to be regulated by
both cytokines and growth factors (Chow et al. 2010;
Karvinen et al. 2003; Pasonen-Seppanen et al. 2003). To
get an overview of the possible factors involved in the
hyaluronan synthesis activation by melanoma CM in
fibroblasts, we utilized two commercial cytokine arrays
which revealed that C8161 melanoma cells produced many
cytokines and growth factors including GM-CSF, Il-8,
IL-6, IL-1a and 1b, TNF-a, MIF, PDGF-AA, PDGF-AB/
PDGF-BB, HB-EGF, and uPA (Supplementary Table I and
II). From these at least IL-1b, IL-6, TNF-a and PDGF are
known to stimulate hyaluronan synthesis in fibroblasts
(Duncan and Berman 1991; Sampson et al. 1992; Li et al.
2007; Yamada et al. 2004) as well as in some other cell
types (Chow et al. 2010; David-Raoudi et al. 2009;
Jacobson et al. 2000; Vigetti et al. 2010). Which of the Has
isoforms is targeted by each cytokine, may depend on the
cell type. In fibroblasts, Has2 has been shown to be
30 kDa Control
30 kDa CM
30 kDa CM + PDGFR inhibitor
a
0
1
2
3
4
5
6
0
1
2
3
4
5
6
7
8
Inva
sion
are
a (x
10 ,
pixe
ls)
430
kDa C
ontro
l
30 kD
a CM
30 kD
a CM
+
PDGFR inhib
*
Contr
siRNA
Has2 s
iRNA
Contr
siRNA
Has2 s
iRNA
30 kDa Control
30 kDa CM
Contr
siRNA
PDGFRα/β
siRNA
Contr
siRNA
PDGFRα/β
siRNA
30 kDa Control
30 kDa CM
30 kDa Control (Contr siRNA)30 kDa Control (PDGFRα/β siRNA)30 kDa CM (Contr siRNA)30 kDa CM + PDGFRα/β siRNA
MMP2 MMP9 MMP14MMP10
1
2
3
4
5
Gen
e ex
pres
sion
(%
of
cont
rol x
10
, MM
P1)
4
0
2000
4000
6000
8000
10 000
12 000
Gene expression (%
of control, MM
P2, MM
P9, MM
P14)
b c d
*******
***
**
**
**
******
****** ***
******
e
0
1
2
3
4
5
6
7
***
****
**
Fig. 7 Melanoma cell-secreted factors stimulate fibroblast invasion
and MMP expression. Representative images of control fibroblasts
and cells treated with melanoma cell CM, and melanoma cell
CM ? PDGFR inhibitor after 3 days invasion in type I collagen-
Cultrex gel. a The cells were stained with Alexa Fluor 594-phalloidin
and the images were constructed from stacks of optical sections to
measure the invasion area (b). The extent of invasion was quantified
from three independent experiments (mean ± SEM, *p \ 0.05).
PDGFR inhibitor reversed the stimulatory effect of 30 kDa CM on
fibroblast invasion. (c) The effect of PDGFRa/b and Has2
(d) silencing with siRNAs on melanoma cell CM-induced invasion.
The data represent mean ± SEM of three independent experiments.
In e, fibroblasts were transfected either with control siRNA or
PDGFRa/b siRNAs and thereafter the effect of melanoma cell CM or
control medium on the mRNA expression levels of MMP1, MMP2,
MMP9 and MMP14 was analyzed with qRT-PCR. The data represent
mean ± SEM of three independent experiments. The statistical
significance of the differences was tested using ANOVA with LSD
post hoc test
908 Histochem Cell Biol (2012) 138:895–911
123
upregulated by PDGF-BB (Li et al. 2007), IL-1b and IL-6
(Sampson et al. 1992; Ducale et al. 2005; Wood et al. 2011;
Yamada et al. 2004), and EGF (Yamada et al. 2004). EGF
also upregulates Has1 expression in fibroblasts (Yamada
et al. 2004).
The present data indicated strong activation of both
EGFR and PDGFR. A specific inhibitor of EGFR failed to
have any effect on melanoma CM-induced hyaluronan
accumulation, while inhibition of PDGFR activation
almost totally reversed it (Fig. 4a), suggesting that PDGF
was the main inducer of hyaluronan synthesis in C8161
melanoma cell CM. Furthermore, knocking down of
PDGFRa and b with siRNA reversed the effect of mela-
noma cell CM on fibroblast Has2 expression (Fig. 4b),
indicating that the activation of this signaling pathway
results in upregulation of Has2 expression in melanoma
cell CM-treated fibroblasts. Similarly, CM from two other
melanoma cell lines, Bro and HT144 has been shown to
activate hyaluronan synthesis in fibroblasts via PDGF-
dependent mechanism, while another line, A375, failed to
do it (Willenberg et al. 2012).
PDGF can activate several downward signaling path-
ways, including PI3K-AKT, and MAPK (Li et al. 2007).
Fitting with the finding that melanoma CM activated
PDGFR, we found enhanced phosphorylation levels of
AKT, ERK1/2 and p38 in dermal fibroblasts after mela-
noma cell CM exposure (Fig. 3). The effect of PDGF-BB
on Has2 expression has been reported to be mediated via
PI3K-AKT and ERK signaling pathways, while inhibition
of p38 had no effect (Li et al. 2007). In the present work,
the melanoma CM-induced hyaluronan response was par-
tially inhibited with PI3K, AKT and p38 inhibitors, while
the inhibition of MEK had only minor effect on melanoma
cell CM-induced fibroblast hyaluronan synthesis (Fig. 4a).
It is possible that PDGFR activation in melanoma CM may
use other signaling routes in addition to PI3K to activate
hyaluronan synthesis, perhaps involving p38 (Yamaguchi
et al. 2004), or that p38 is activated by other cytokines, like
IL-1b or IL6 (David-Raoudi et al. 2009), present in the
C8161 melanoma cell CM.
The increased hyaluronan production by the melanoma
cell CM-treated fibroblasts may influence the behavior and
the phenotype of the fibroblasts themselves, or it may
modify the ECM to support growth/invasion of neighbor-
ing cells like the cancer cells, endothelial cells or inflam-
matory cells. Melanoma cell CM-treated fibroblasts
showed enhanced Has2 and MMP expression, acquired
elongated shape, wide pericellular hyaluronan coats and
increased intracellular hyaluronan deposits, characteristic
to activated myofibroblasts found during wound healing
and in cancer (Webber et al. 2009a, b). Although, Has2
upregulation and hyaluronan production have been linked
to myofibroblast differentiation (Webber et al. 2009a, b),
we were unable to see changes in the expression of myo-
fibroblast marker, a-smooth muscle actin, in conditioned
medium-treated fibroblasts. This may be due to low level
of TGF-beta in melanoma cell CM, which is known to
induce myofibroblast differentiation (Webber et al. 2009a).
In line with the activated phenotype, the melanoma cell
CM-treated fibroblasts invaded into type I collagen-Cultrex
gel more avidly than the control cells, and this was
reversed with the PDGFR inhibitor and knock-down of
PDGFRa and b with siRNA. Since PDGFR silencing
inhibited also Has2 expression, we tested whether Has2
siRNA transfection is able to reverse the effect of mela-
noma cell CM on fibroblast invasion. Although the knock
down of Has2 blocked the stimulatory effect of melanoma
cell CM on fibroblast invasion, the finding that several
MMPs, found to be essential for fibroblast invasion
(Gaggioli et al. 2007), were also increased by the mela-
noma cell CM, suggest that these probably are also
involved in the activation of fibroblast invasion together
with hyaluronan. We do not know, at the moment, the
mechanism how HAS2/hyaluronan influences fibroblast
invasion, if it is through MMP-dependent or independent
mechanism. However, it was recently shown that HAS2
regulates the ability of breast carcinoma cells to invade in
Matrigel through regulating the expression of TIMP1, a
regulator of MMP activity (Bernert et al. 2011).
Cancer cell-associated fibroblasts (CAFs) are supposed
to remodel the extracellular matrix with MMPs creating
microtracks for cancer cell migration (Che et al. 2006;
Gaggioli et al. 2007). Hyaluronan secreted by the activated
fibroblasts may keep these tracks open, facilitating the
motility of cancer cells. Stromal hyaluronan has been
shown to facilitate tumor cell migration and invasion by
diminishing intercellular adhesion (Itano et al. 2002) and
forming a hydrated matrix suitable for cell locomotion
(Sironen et al. 2011). In the invasive melanoma, the tumor
stroma is intensely hyaluronan positive (our unpublished
data and (Karjalainen et al. 2000).
Melanoma cell CM-treated fibroblast cultures showed a
special conformational change in the organization of hya-
luronan, i.e. formation of so called hyaluronan cables.
These structures are formed in cultured cells exposed to
inflammatory cytokines, hyperglycemia and ER-stress (de
La Motte et al. 1999). These hyaluronan aggregates bind
avidly CD44-bearing leukocytes (Lauer et al. 2008), a
feature which cannot be obtained by solely increasing
hyaluronan synthesis (Jokela et al. 2008). Our finding that
melanoma cell CM induces this conformational change
suggests that activated fibroblasts in melanoma stroma
adopt similar conformation as seen in inflamed colon (de la
Motte et al. 2003), and that it may operate in the monocyte
and macrophage recruitment to the tumor area as previ-
ously suggested (Itano et al. 2008). This hyaluronan-rich
Histochem Cell Biol (2012) 138:895–911 909
123
tumor microenvironment has been shown to induce neo-
vascularization and lymphangiogenesis, both important for
tumor progression (Koyama et al. 2007; Koyama et al.
2008). The enhanced vascularization has been attributed to
the hyaluronan degradation products (Pardue et al. 2008)
generated either by the action of reactive oxygen species
(ROS) or hyaluronidase 2 (Hyal-2) (Andre et al. 2011;
Monzon et al. 2010). We did not, however, find any signs
of hyaluronan size reduction in fibroblast culture medium
after the melanoma cell CM treatment (Fig. 1b). Consistent
with this the expression level of Hyal-2 in fibroblasts was
not influenced by the melanoma cell CM treatment
(Fig. 1c). The hyaluronan fragmentation may require the
contribution of other cell types like the tumor-associated
inflammatory cells attracted and bound by the hyaluronan
cables.
The present study demonstrates that melanoma cells
activate PDGFR-PI3K-AKT and p38 signaling pathways in
fibroblasts, leading to Has2 upregulation and enhanced
hyaluronan production. Upregulation of Has2 expression is
associated with increased ability of the fibroblasts to invade
into the matrix. Altered hyaluronan metabolism in the
stromal fibroblasts together with induced MMP expression
may have profound influence on melanoma progression.
Acknowledgments We acknowledge expert technical help from
Eija Kettunen, Arja Venalainen, Eija Rahunen and Tuula Venalainen.
Academy of Finland (S.P-S.), Paavo Koistinen Foundation (S.P-S.),
The North Savo Cancer Fund (S.P-S), Cancer Center of Eastern
Finland (M.T. and R.T.) and Sigrid Juselius Foundation (R.T. and
M.T.) supported this work financially.
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