cGMP-phosphodiesterase 6, transducin and Wnt5a/Frizzled-2-signaling control cGMP and Ca2+ homeostasis in melanoma cells

Abstract

Malignant melanoma is one of the most aggressive human neoplasms which develop from the malignant transformation of normal
epithelial melanocytes and share the lineage with retinal cells. cGMP-phosphodiesterase 6 (PDE6) is one of the cancer-retina
antigens newly identified in melanoma cells. Normally, PDE6 hydrolyzes the photoreceptor second messenger cGMP allowing the
visual signal transduction in photoreceptor cells. cGMP also play an important signaling role in stimulating melanogenesis
in human melanocytes. Here, we present evidence that PDE6 is a key enzyme regulating the cGMP metabolism in melanoma cells.
Decrease in intracellular cGMP leads to calcium accumulation in melanoma cells. In these cells, cGMP-phosphodiesterase 6 can
be activated by another cancer-retina antigen, transducin, through Wnt5a–Frizzled-2 cascade, which leads to a lowering of
cGMP and an increase in intracellular calcium mobilization. Thus, the aberrant expression of PDE6 may control cGMP metabolism
and calcium homeostasis in melanoma cells.

KeywordscGMP-phosphodiesterase 6-Transducin-Cancer-retina antigens-Wnt-Frizzled-Melanoma

Full text

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RESEARCH ARTICLE
cGMP-phosphodiesterase 6, transducin and Wnt5a/Frizzled-2-
signaling control cGMP and Ca2+homeostasis in melanoma cells
Alexandr V. Bazhin•Vojtech Tambor•
Boyan Dikov•Pavel P. Philippov•Dirk Schadendorf•
Stefan B. Eichmu ¨ller
Received: 17 September 2009/Revised: 9 November 2009/Accepted: 10 November 2009
? Birkha ¨user Verlag, Basel/Switzerland 2009
Abstract
sive human neoplasms which develop from the malignant
transformation of normal epithelial melanocytes and share
the lineage with retinal cells. cGMP-phosphodiesterase 6
(PDE6) is one of the cancer-retina antigens newly identified
in melanoma cells. Normally, PDE6 hydrolyzes the photo-
receptor second messenger cGMP allowing the visual signal
transduction in photoreceptor cells. cGMP also play an
important signaling role in stimulating melanogenesis in
humanmelanocytes.Here,wepresentevidencethatPDE6is
akeyenzymeregulatingthecGMPmetabolisminmelanoma
cells. Decrease in intracellular cGMP leads to calcium
accumulation in melanoma cells. In these cells, cGMP-
phosphodiesterase 6 can be activated by another cancer-
retina antigen, transducin, through Wnt5a–Frizzled-2 cas-
cade, which leads to a lowering of cGMP and an increase in
intracellular calcium mobilization. Thus, the aberrant
expression of PDE6 may control cGMP metabolism and
calcium homeostasis in melanoma cells.
Malignant melanoma is one of the most aggres-
Keywords
Cancer-retina antigens ? Wnt ? Frizzled ? Melanoma
cGMP-phosphodiesterase 6 ? Transducin ?
Abbreviations
PDE6
NEM
FZ2
[Ca2?]i
ZAP
DIP
SIL
VAR
PKG
cGMP-phosphodiesterase 6
Melanocytes
Frizzled-2
Intracellular calcium concentration
Zaprinast
Dipyridamole
Sildenafil
Vardenafil
cGMP-dependent protein kinase
Introduction
Malignant melanoma is one of the most aggressive human
neoplasms which progresses through radial and vertical
growth phases and finally metastasizes [1]. The incidence of
this cancer increased worldwide over a quarter of a century
[2]. Having a very poor prognosis and being resistant to
conventional therapy like chemotherapy [3], melanoma
nevertheless responds eventually to a variety of immuno-
therapeutic approaches [4]. Sun exposure and genetic
susceptibility have been proposed as major etiological and
predisposing factors for melanoma development [5]. Mel-
anoma cells presumably develop from the malignant
transformation of normal epithelial melanocytes, thus
sharing the lineage with retinal cells [6].
Recently, we have shown that the key photoreceptor
proteins, that are normally restricted to retinal cells and the
pineal gland, function as cancer-retina antigens in mela-
noma cells [7]. The following photoreceptor proteins were
A. V. Bazhin (&) ? V. Tambor ? B. Dikov ? S. B. Eichmu ¨ller
Skin Cancer Unit, German Cancer Research Center,
69120 Heidelberg, Germany
e-mail: a.bazhin@dkfz.de
A. V. Bazhin ? S. B. Eichmu ¨ller
Department of Dermatology, University of Heidelberg,
University Hospital Mannheim, 68135 Mannheim, Germany
P. P. Philippov
Department of Cell Signalling,
A.N. Belozersky Institute of Physico-Chemical Biology, M.V.
Lomonosov Moscow State University, 119991 Moscow, Russia
D. Schadendorf
Department of Dermatology, University Hospital Essen,
45122 Essen, Germany
Cell. Mol. Life Sci.
DOI 10.1007/s00018-009-0214-0
Cellular and Molecular Life Sciences

Page 2

classified as cancer-retina antigens: rhodopsin, transducin,
cGMP-phosphodiesterase6
channels, guanylyl cyclase, rhodopsin kinase, recoverin,
and arrestin. Among these, PDE6 is expressed at a high
frequency. Such expression of cancer-retina antigens could
generate an autoimmune response [8], which in turn might
lead to development of a paraneoplastic neurological syn-
drome, melanoma-associated retinopathy [9]. Furthermore,
we demonstrated that expression of cancer-retina antigens
can be modulated by light as it proceeds in photoreceptor
cells [10]. Whereas the biochemistry and physiology of
visual transduction have been extensively studied, the role
of the photoreceptor proteins in melanoma cells still
remains to be resolved.
Photoreceptor PDE6 consists of two types of catalytic
subunits (a and b) and an inhibitory c subunit. In the retina,
PDE6 hydrolyzes the photoreceptor secondary messenger,
cGMP, allowing the closure of cGMP-gated channels
located in the plasma membrane of photoreceptors [11].
cGMP effector proteins also include cGMP-dependent
protein kinase (PKG) and phosphodiesterases themselves
[12]. In melanocytes, cGMP plays an important signaling
role and stimulates melanogenesis through the ultraviolet B
radiation [13].
In addition to photoreceptor and melanoma cells, PDE6
is expressed in zebrafish embryos and mouse F9 embryonic
teratocarcinoma cells [14]. There, PDE6 can be activated
through the cascade Wnt5a–Frizzled-2. Such activation of
PDE6 leads to a decrease of intracellular cGMP that
inhibits PKG and stimulates the intracellular calcium
mobilization [14]. This chain of events involves PKG
which is inhibited at a low cGMP concentration [15]. As a
consequence, a high calcium concentration activates the
phosphoprotein phosphatase calcineurin and calcium/cal-
modulin protein kinase II, and finally stimulates the gene
expression through NF-AT [16].
Here, we present evidence that PDE6 is a key enzyme
regulating the cGMP metabolism in melanoma cells.
Decrease in the intracellular cGMP as a result of its
hydrolysis by PDE6 leads to an accumulation of intracel-
lular calcium. In melanoma cells, PDE6 can be activated
through Wnt5a–Frizzled-2 by another cancer-retina antigen
transducin, leading to a decrease of intracellular cGMP
concentration and an increase of intracellular calcium
mobilization.
(PDE6),cGMP-dependent
Materials and methods
Materials
Pertussis toxin, zaprinast, Fura-2/AM, and polyclonal
(monospecific) rabbit antibodies against PDE6b and
Frizzled-2 were purchased from Sigma-Aldrich (Sch-
nelldorf, Germany). Sildenafil (Viagra) was obtained
from Pfizer Pharma (Karsruhe, Germany). Wnt5a purified
proteinwaspurchasedfrom
Germany). The Apo-BrdU-IHCTMKit was from Biovi-
sion (Mountain View, CA, USA). SureSilencingTM
shRNA plasmids for a, b, and c subunits of human PDE6
were purchased from SABiosciences (Frederic, MD,
USA). A polyclonal (monospecific) rabbit antibody
against transducin was obtained as described in [7]. A
polyclonal (monospecific) rabbit antibody against PDE6c
was purchased from Affinity BioReagents (Golden, CO,
USA), a polyclonal (monospecific) rabbit antibody
against Wnt5a was obtained from Cell Signaling Tech-
nology (Danvers, MA), and a monoclonal antibody
against poly(ADP-ribose) was purchased from Alexis
(Axxora, LCC San Diego, CA, USA).
Abnova(Heidelberg,
Cell cultures
Established melanoma cell lines (Ma-Mel-04, -05, -11, -12,
-17, -21, UKRV-Mel-02, -06, -14a, -21, -31, MeWo, and
SK-Mel-023) from the Skin Cancer Unit at the German
Cancer Research Center, Heidelberg, Germany were cul-
tivated in RPMI-1640 medium. The keratinocyte cell
line HaCaT [17] was cultivated in DMEM High Glucose
(4.5 g/l) medium. Melanocytes were derived from foreskin
and are from a commercial source (batches 4121602,
0100402.1,and 6030102.2;
Germany). Normal epithelial melanocytes (NEM) were
cultivated in MEM Alpha Medium with ribo- and deoxy-
ribonucleosides (Promocell). All media were supplemented
with 10% fetal calf serum, L-glutamine (2 mM) and peni-
cillin/streptomycin solution (5 U/ml), and were purchased
from PAA Laboratories (Coelbe, Germany). Cell lines
were cultivated at 37?C and 5% CO2. For drug treatments,
cells (1 9 105per Petri dish (35 mm)) were cultivated in
complete medium for 24 h and then supplemented with
0.3 lM zaprinast (ZAP) or 500 ng/ml pertussis toxin and
cultivated for 24 h. Subsequently, cells were washed,
counted, and equal amounts of cells were used for PDE
activity, cGMP, and calcium measurement.
Promocell, Heidelberg,
shRNA knockdown of PDE6 subunit expression
Plasmid-based
described by the manufacturer. Briefly, competent E. coli
cells were transformed with the provided plasmids for
shRNA for PDE6-a, PDE6-b, and PDE6-c following the
manufacturer’s protocol. Subsequently, plasmids were
isolated, purified, and transfected separately to melanoma
cell lines with replicates. Transfected cells were enriched
by selection for neomycin resistance. Knock-down effects
RNAinterferencewasperformed as
A. V. Bazhin et al.

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were checked by western blotting using specific antibodies
against PDE6-a, PDE6-b, and PDE6-c.
Protein analysis
Western blot analysis and ELISA were performed as
described elsewhere [10].
cGMP measurement
cGMP concentration was measured with the HitHunter
cGMP Assay (Amersham Bioscience Europe, Freiburg,
Germany) as described by the manufacturer. Briefly, har-
vested cells were resuspended in PBS at the concentration
of 40,000 per 10 ll and seeded on 96 half-well white
plates. After incubation with different reagents from the kit
at room temperature, the plates were read on a luminescent
reader at 1 s/well. Standard concentrations of cGMP (from
10-4to 10 lM) were simultaneously proceeded to estimate
cGMP concentration in the probes of interest. Each cell
line was measured in triplicate, and the final concentration
of cGMP was determined from at least three independent
experiments.
Measurement of PDE-activity
PDE activity was measured with the HitHunter cGMP
Assay as a decrease of cGMP concentration after incu-
bation with protein extracts from the cell lines. Briefly,
the proteins were extracted with TRIS buffer (10 mM
TRIS pH 7.4, containing 50 mM NaCl, 1 mM DTT, and
proteinase inhibitor cocktail). For each cell line, the
HitHunter cGMP Assay was performed using standard
concentrations of cGMP (from 10-4to 10 lM) with or
without protein extract from the cells using different
protein concentrations. PDE activity was calculated as a
decrease of cGMP concentration during 1 min at 37?C
per 1 mg of protein. PDE activity was measured in trip-
licate, and three cGMP concentration points were used for
the activity calculation. The final activity was calculated
1 2 3 4 5 6 7 8 9
kD
99
10
47
PDE6β
PDE6γ
PDE6α
β-actin
99
99
99
10
47
47
47
PDE6α
PDE6β
PDE6γ
β-actin
β-actin
β-actin
1 2 1 2 1 2 1 2
kD
MaMel 04 MaMel 11 MaMel 12 UKRV-Mel-31
A
B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
100
47
β-actin
PDE5
kD
C
Fig. 1 Protein expression of
PDE6 and PDE5. a Expression
of PDE6 subunits in NEM (1),
HaCaT (2) and melanoma cell
lines Ma-Mel-04 (3), Ma-Mel-
12 (4), UKRV-Mel-31 (5),
Ma-Mel-11 (6), Ma-Mel-21 (7),
and MeWo (8), as well as Y79
retinoblastoma cell line as a
positive control (9);
b expression of PDE6 subunits
in untransfected controls (1) or
in cell lines transfected with
shDNA plasmids (2) as
indicated on the right side;
c expression of PDE5 in heart as
a positive control (1, 9), NEM
(2), HaCaT (3), and melanoma
cell lines Ma-Mel-04 (4),
Ma-Mel-12 (5), UKRV-Mel-31
(6), Ma-Mel-11 (7), Ma-Mel-21
(8), MeWo (10), UKRV-Mel-06
(11), UKRV-Mel-14a (12),
UKRV-Mel-21 (13), SK-Mel23
(14), Ma-Mel-05 (15), and
Ma-Mel-21 (16)
Function of PDE6 in melanoma cells

Page 4

from three independent experiments with different protein
concentrations added.
Calcium measurement
The free intracellular calcium concentration ([Ca2?]i) was
measured with fura-2/AM. 1 9 105cells were incubated
with 3 lM of fura-2/AM for 30 min at 37?C in the dark.
After sedimentation, cells were resuspended in Krebs/
HEPES buffer (143.3 mM Na?, 4.7 mM K?, 2.5 mM
Ca2?, 1.3 mM Mg2?, 125.6 mM Cl-, 25 mM HCO3-,
1.2 mM H2PO4-, 1.2 mM SO42-, 11.7 mM glucose, and
10 mM HEPES, pH 7.4), and then the cells were incubated
for 15 min at room temperature in the dark. After washing
with the Krebs/HEPES buffer, the fluorescence was mea-
sured by 340 and 380 nm excitation, and by 510 nm
emission. The [Ca2?]i was calculated according to the
Grynkewicz equation [18]:
½Ca2þ?iðnMÞ ¼ Kd? ½ðR ? RminÞ=ðRmax? RÞ? ? Sfb
where Kd(for Ca2?binding to fura-2 at 37?C) = 225 nM,
R = 340/380 ratio, Rmax= 340/380 ratio under Ca2?-sat-
urating conditions (after adding 0.1% Triton X 100),
Rmin= 340/380 ratio under Ca2?-free conditions (after
adding 4.5 mM EGTA), and Sfb = ratio of baseline fluo-
rescence (380 nm) under Ca2?-free and -bound conditions.
For measurement of [Ca2?]ichanging, fura-2/AM-loaded
cells were treated with Wnt5a (20 ng/ml) or buffer alone.
Measurement of fluorescence by 340 and 380 nm excita-
tion was performed with TECAN.SPECTRAFluor Plus
each 60 s during 15 min, and the ratio 340/380 was
calculated.
Statistical analysis
Statistical analysis was performed by Student’s t test with
significant differences determined as P\0.05.
Results
Expression of PDE6 subunits in melanoma cell lines
Since the a subunit of PDE6 (PDE6-a) was frequently
detected in melanoma cells [7] (Fig. 1a), we wanted to
understand whether other PDE6 subunits, namely b and c
(PDE6-b and PDE6-c, respectively), are also expressed in
melanoma cell lines. Western blot analysis revealed the
expression of both PDE6-b and -c in some melanoma cell
lines, but not in keratinocytes nor in melanocytes (Fig. 1a;
Table 1). To confirm that the positive western blotting
signals indeed correspond to subunits of PDE6, we per-
formed stable transfections of four melanoma cell lines
(Ma-Mel-04, Ma-Mel-11, Ma-Mel-12, and UKRV-Mel-31)
with shRNA plasmids for PDE6-a, PDE6-b, and PDE6-c.
Transfection of the melanoma cell lines with the PDE6-a
Table 1 Expression of PDE6 subunits, PDE activity, and concentrations of cGMP and Ca2?in melanoma and normal skin cell lines
Cell line Expression of PDE6 subunits Total PDE activity,
[U/mg 9 10-6]a
[cGMP], [nM]a
[Ca2?]i, [nM]a
abc
NEMb
–––0.083 ± 0.009821.2 ± 3.62.9 ± 1.5
HaCaT––– 3.13 ± 1.558.93 ± 5.051.9 ± 0.5
Ma-Mel-17–––0 50.3 ± 7.62.9 ± 0.6
Ma-Mel-37–––025.7 ± 6.67.6 ± 1.8
UKRV-Mel-06––?0.14 ± 0.1143.4 ± 5.8 2.1 ± 0.66
UKRV-Mel-14a––? 0.46 ± 0.18163.7 ± 10.11.4 ± 0.7
Ma-Mel-04???3.8 ± 1.99.3 ± 4.9 4.2 ± 0.3
Ma-Mel-12?–? 1.56 ± 0.5618.2(± 8.3) 1.6 ± 0.02
UKRV-Mel-31??–15.9 ± 1.110.7 ± 1.8 24.5 ± 0.8
Ma-Mel-11??–24.3 ± 9.45 11.9 ± 6.611.2 ± 2
UKRV-Mel-21?–– 23.9 ± 0.66 15 ± 0.231.8 ± 6.3
SK-Mel23?––35.9 ± 1.7 4.5 ± 2.7 21.9 ± 5.6
Ma-Mel-05?––14.4 ± 6.49.5 ± 2.1 34.2 ± 7.6
Ma-Mel-21–?–25.8 ± 0.295 ± 0.811.2 ± 1
MeWo?–– 44.2 ± 15.6 2.1 ± 0.2 10 ± 2.8
aMean of at least three independent experiments
bPooled NEM from three healthy donors
A. V. Bazhin et al.

Page 5

shRNA plasmid led to the disappearance of the PDE6-a
band (Fig. 1b). The same knock-down effects were seen
after transfection of Ma-Mel-04, Ma-Mel-11, and UKRV-
Mel-31 with the PDE6-b shRNA plasmid, and after
transfection of Ma-Mel-04 and Ma-Mel-12 with the
PDE6-c shRNA plasmid (Fig. 1b). Thus, PDE6-a, PDE6-b,
and PDE6-c subunits are indeed expressed in melanoma
cell lines.
PDE6 is functional in melanoma cell lines
To understand whether PDE6 is functional in melanoma
cells, measurement of the general PDE activity in various
melanoma cell lines was conducted (Fig. 2a). The analyzed
cell lines can be divided in two groups according to their
PDE activity (Table 1; Fig. 2b): The first group has a low
PDE activity and consists of cell lines which (1) do not
express any PDE6 subunit (NEM, HaCaT, Ma-Mel-17 and
-37) or (2) possess PDE6-c (UKRV-Mel-06, -14a, Ma-Mel-
04 and 12). The second group (Ma-Mel-05, -11, -21,
UKRV-Mel-21, -31, Sk-Mel-231, and MeWo) has a high
PDE activity and comprises cell lines, containing PDE6-a
and/or PDE6-b but have no PDE6-c. Thus, the data of
western blot analysis of PDE6 subunits correlates well with
the PDE activity. We also compare the PDE activity with
the level of cGMP in the cell lines tested and detected the
nM range of cGMP concentration in those cell lines which
show a low PDE activity (Table 1). However, the PDE
activity in Ma-Mel-04, Ma-Mel-05, UKRV-Mel-21, and
UKRV-Mel-31 cell lines does not show a good correlation
with the level of cGMP. This discrepancy could be
attributed to the functional activity of guanylyl cyclase 1
which has been earlier found to be expressed in these cell
lines [7].
Further, we analyzed whether the PDE activity revealed
in melanoma cells can be attributed to the retinal-specific
PDE6, but not to other members of the PDE family. For
this aim, we performed a stable transfection of melanoma
cell lines with shRNA plasmids for PDE6-a, PDE6-b, and
PDE6-c. Transfection of melanoma cells with shRNA
plasmids for PDE6-a or PDE6-b led to inhibition of PDE
activity between 55 and 85% (Fig. 2c). Furthermore,
transfection of Ma-Mel-04 and Ma-Mel-12 with shRNA
plasmids for PDE6-c led to upregulation of PDE activity in
these cell lines (Fig. 2c).
A confirmatory result was obtained in the cell culture
system. In this case, melanoma cell lines and kerati-
nocyte celllineHaCaT
inhibitor of PDE6 ZAP (300 nM) for 24 h, and then the
amount of cGMP accumulated in the cells was mea-
sured (Fig. 3a). Melanoma cell lines positive for PDE6-a
and/or PDE6-b and negative for PDE6-c accumulated
intracellular cGMP after cultivation with ZAP (Fig. 3a).
were cultivated withan
However, the inhibitors had no effect upon cGMP
accumulation in the cells positive for PDE6-c or nega-
tive for PDE6-a and/or PDE6-b. Cultivation of Ma-
Mel-11 and UKRV-Mel-31 transfected with shRNA
plasmids for either PDE6-a or PDE6-b led to cGMP
accumulation in comparison with non-transfected cell
0
0.0000
1000
2000
3000
4000
5000
0.05000.1000 0.1500 0.20000.2500
[cGMP], µM
RLU
∆[cGMP]
0.00E+00
1.00E-05
2.00E-05
3.00E-05
4.00E-05
5.00E-05
PDE activity, U/mg
Ma-Mel 05, 11, 21,
UKRV-Mel-21, -31,
SK-Mel23, MeWo
NHEM, HaCat,
Ma-Mel 04, 12, 17, 37,
UKRV-Mel-06, -14a
A
B
PDE activity
0.00E+00
1.00E-05
2.00E-05
3.00E-05
co
shRNA
PDE6α
shRNA
PDE6β
co
shRNA
PDE6α
shRNA
PDE6β
co
shRNA
PDE6γ
co
shRNA
PDE6γ
Ma-Mel 11 UKRV-Mel-31Ma-Mel 04 Ma-Mel 12
U/mg
C
Fig. 2 PDE activity in melanoma cell lines is attributed to PDE6.
a Exemplary determination of PDE activity in melanoma cell line
Ma-Mel-05. Luminescence signal (y axis) in relative luminescence
unit (RLU) which is proportional to the cGMP concentration was
measured at different concentrations of cGMP (x axis) without
(broken line) or with (solid line) whole protein extract of the cell line
(0.35 mg/ml). For details, see ‘‘Materials and methods’’. b Dispersion
of PDE activity in melanoma cell lines, HaCat and NEM, depends on
the pattern of PDE6 expression. c PDE activity was measured in
melanoma cell lines transfected with shDNA plasmids. Statistically
significant differences (P\0.05) are marked with an asterisk
Function of PDE6 in melanoma cells

Page 6

lines (Fig. 3b). In Ma-Mel-04 and Ma-Mel-12 transfected
with shRNA plasmids for PDE6-c, we observed a
reduction of cGMP (Fig. 3b).
In total, the data of this section confirm that the PDE
activity detected in melanoma cell lines belongs to PDE6
but not to other members of the PDE family.
cGMP accumulation
0
50
100
150
200
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
ZAP
HaCat Ma-Mel
17
Ma-Mel
37
UKRV-
Mel-06
UKRV-
Mel-14a
Ma-Mel
04
Ma-Mel
12
UKRV-
Mel-31
Ma-Mel
11
UKRV-
Mel-21
SK-
Mel23
Ma-Mel
05
MaMel
21
MeWo
%
*
*
*
*
*
*
*
A
cGMP accumulation
0
100
200
300
400
co
shRNA
PDE6α
shRNA
PDE6β
co
shRNA
PDE6α
shRNA
PDE6β
co
shRNA
PDE6γ
co
shRNA
PDE6γ
Ma-Mel 11UKRV-Mel-31Ma-Mel 04Ma-Mel 12
%
Calcium accumulation
0
50
100
150
200
250
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
ZAP
co
shRNAPDE6α
shRNAPDE6β
co
shRNAPDE6α
shRNAPDE6β
co
shRNAPDE6γ
co
shRNAPDE6γ
HaCat UKRV-
14a
Ma-Mel
04
Ma-Mel
05
Ma-Mel
11
Ma-Mel
21
Ma-Mel 11 UKRV-Mel-
31
Ma-Mel
04
Ma-Mel
12
%
**
*
*
C
***
*
*
*
*
B
Fig. 3 cGMP accumulation and [Ca2?]i mobilization depend on
PDE6. Melanoma cell lines with different patterns of PDE6 expres-
sion and the HaCaT cell line were cultivated for 24 h with 300 nM
ZAP (a) or melanoma cell lines were transfected with shRNA coding
plasmids (b) and then the accumulation of cGMP in the cells was
measured. c Melanoma cell lines and the HaCaT cell line were
cultivated with 300 nM ZAP or transfected with shRNA coding
plasmids, and then the [Ca2?]iwas measured. Statistically significant
differences (P\0.05) are marked with an asterisk
A. V. Bazhin et al.

Page 7

Changes in calcium mobilization in melanoma cell
lines correlates with cGMP levels and PDE6 activity
Recently, it has been shown that reduction of the cGMP
concentration through down-regulation of PKG leads to the
intracellular calcium ([Ca2?]i) mobilization in the mouse
totipotent F9 teratocarcinoma cells [15]. We were curious
to know whether the melanoma cell lines with different
intracellular cGMP concentrations have various [Ca2?]i
concentrations. Indeed, differences in [Ca2?]idependent on
cGMP concentrations were detected: melanoma cell lines
with high levels of cGMP concentration had increased
(above 10 nM) [Ca2?]i(Table 1).
To further confirm the interrelationship between intra-
cellular [Ca2?]iand cGMP concentration, we cultivated the
cell lines with PDE6 inhibitor zaprinast (ZAP) and mea-
sured the [Ca2?]iconcentration. Cultivation with ZAP led
to a decrease of the [Ca2?]imobilization in the cell lines
positive for PDE6 but had no influence on the [Ca2?]iin
the cell lines negative for the catalytic subunits of PDE6
(Fig. 3c). These data were confirmed in experiments with
stable transfected melanoma cell lines with shRNA plas-
mids for PDE6-a, PDE6-b, or PDE6-c. Cultivation of Ma-
Mel-11 and UKRV-Mel-31 transfected with shRNA plas-
mids for either PDE6-a or PDE6-b led to a decrease of the
[Ca2?]imobilization in comparison with non-transfected
cell lines (Fig. 3c). In addition, we found an increase of the
[Ca2?]imobilization in Ma-Mel-04 and Ma-Mel-12 trans-
fected with shRNA plasmids for PDE6-c (Fig. 3c). These
results suggest that PDE6 participates in the calcium
homeostasis in melanoma cells.
Wnt5a activates PDE6 through Frizzled-2 and
transducin and stimulates calcium mobilization
From the elegant work of Ahumada et al. [14], we learned
that in F9 and in Chinese hamster ovary cells the acti-
vation of Frizzled-2 (FZ2) receptor after binding of
Wnt5a leads to activation of the G protein transducin,
which in turn binds to PDE6-c and thus activates PDE6
[14]. We supposed that in the melanoma cell lines, which
also possess PDE6-c, the sequence of events could be the
same.
First, we checked the protein expression of transducin,
Wnt5a, and FZ2 in melanoma cell lines, keratinocytes and
melanocytes (Table 2 ; Fig. 4). It has been found that
transducin is expressed in three melanoma cell lines
(Ma-Mel-04, -12 and -37). Wnt5a is detected in half of
melanoma cell lines tested and in HaCaT, but not in
melanocytes. FZ2 expression is present in all melanoma
cell lines, but neither in keratinocytes nor in NEM.
Using two melanoma cell lines (Ma-Mel-04 and -12)
positive for PDE6, transducin and FZ2, we studied the
effects of Wnt5 on PDE6 activation. Incubation of the cells
with the purified recombinant Wnt5a led to an increase of
PDE6 activity. Other cell lines with different expression
Table 2 Protein expression of transducin, Wnt5a and Frizzled-2 in
melanoma and normal skin cell lines
Cell linesTransducinWnt5aFrizzled-2
NEM–––
HaCaT–?–
UKRV-Mel-06–??
UKRV-Mel-14a–??
UKRV-Mel-21–??
UKRV-Mel-31–??
SK-Mel23–??
Ma-Mel-04?–?
Ma-Mel-05––?
Ma-Mel-11––?
Ma-Mel-12???
Ma-Mel-17–??
Ma-Mel-21––?
Ma-Mel-37?–?
Ma-Mel-52–??
MeWo–??
Transducin
Wnt5a
Frizzled-2
β-actin
kD
43
37
47
47
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Fig. 4 Protein expression of
transducin, Wnt5a, and FZ2.
Protein expression in Y79 (1),
NHEM (2), HaCat (3), UKRV-
Mel-06 (4), -14a (5), -21 (6), -31
(7), SK-Mel-23 (8), Ma-Mel-04
(9), -05 (10), -11 (11), -12 (12),
-17 (13), -21 (14), -37 (15), -52
(16), and MeWo (17)
Function of PDE6 in melanoma cells

Page 8

PDE6 activity PDE6 activity
00
50 50
100100
150 150
200200
250250
300300
350 350
cocoWnt5a Wnt5acocoWnt5aWnt5a cocoWnt5aWnt5acocoWnt5aWnt5acoco Wnt5a Wnt5acoco Wnt5aWnt5a
Ma-Mel 04 Ma-Mel 04 Ma-Mel 12Ma-Mel 12Ma-Mel 37 Ma-Mel 37 UKRV-Mel-14a UKRV-Mel-31 UKRV-Mel-14a UKRV-Mel-31 HaCat HaCat
%%
**
**
AA
PDE6 activity PDE6 activity
00
50 50
100100
150150
200200
250250
300300
350350
cocoWnt5aWnt5a FZ3
ABAB
FZ3
AB +
Wnt5aWnt5a
coco Wnt5aWnt5a FZ3
AB AB
FZ3
AB +
Wnt5aWnt5a
Ma-Mel 0Ma-Mel 0
MM44
a-Mel 12a-Mel 12
%%
**
**
BB
PDE6 activity PDE6 activity
00
5050
100100
150 150
200200
250 250
300300
350 350
co coWnt5a Wnt5aPTPTPT +
Wnt5a Wnt5a
coco Wnt5a Wnt5aPTPTPT +
Wnt5aWnt5a
Ma-Mel 0 Ma-Mel 0
MM44
a-Mel 12a-Mel 12
CC
**
**
PDE6 activityPDE6 activity
00
5050
100100
150150
200 200
250250
untransfected
co co
shRNAPDE6γ untransfectedshRNAPDE6γ untransfected
co + Wnt5a co + Wnt5a
shRNAPDE6γ
+ Wnt5a+ Wnt5a
untransfected
co + PT co + PT
shRNAPDE6γ
+ PT + PT
%%
**
**
**
**
**
DD
FZ3 FZ3
AB +
FZ3 FZ3
AB +
PT + PT +
%
untransfectedshRNAPDE6γ
untransfectedshRNAPDE6γ
A. V. Bazhin et al.

Page 9

profiles of PDE6, transducin, and FZ2 did not respond to
Wnt5a (Fig. 5a). To show that Wnt5a acts through FZ2
receptor, the cells were pretreated with antibody against
FZ2 before the incubation with Wnt5a. This pretreatment
abolished the effect of Wnt5a on the PDE6 activation
(Fig. 5b). Also, this effect was prevented by pertussis
toxin, a specific inhibitor of Giproteins and transducin
(Fig. 5c). Thus, Wnt5a activates PDE6 through FZ2
receptor and G protein transducin. We found additional
support for the hypothesis that the Wnt5a activates PDE6
through influence on PDE6-c in experiments with stable
transfection of the melanoma cell line Ma-Mel-04 with
shRNA plasmids for PDE6-c (Fig. 5d). Transfection of
melanoma cells with shRNA plasmids for PDE6-c led to
the same effect as after incubation with Wnt5a-upregula-
tion of PDE activity in this cell line (Fig. 5d). At the same
time, an addition of Wnt5a to the transfected cells did not
affect the PDE activity. An incubation of the transfected
Ma-Mel-04 with PT did not lead to the abolishment of high
PDE activity induced by PDE6-c transfection (Fig. 5d).
This indicates that PDE6 is fully activated and does not
need an additional activation by the Wnt5a–FZ2–trans-
ducin cascade.
To understand whether PDE6 activation by Wnt5a
would lead to an increase of [Ca2?]i mobilization, we
measured kinetics of the [Ca2?]imobilization in Ma-Mel-
04 and Ma-Mel-12 after adding of Wnt5a (Fig. 6). Indeed,
Wnt5a stimulated the [Ca2?]imobilization in these mela-
noma cell lines, with a maximum seen 5 min after
incubation, but this stimulation had no effect in the case of
keratinocytes even after 20 min of incubation. The Wnt5a-
dependent [Ca2?]imobilization was blocked by the culti-
vation of the cells with ZAP or pertussis toxin (Fig. 6).
Thus, one may conclude that Wnt5a regulates the [Ca2?]i
mobilization through the cascade FZ2 receptor–G protein–
transducin–PDE6.
Discussion
The main aim of this study was to investigate whether the
aberrantly expressed PDE6 is functional in cancer cells.
We have shown that: (1) PDE6 is active and hydrolyses
cGMP in melanoma cell lines; (2) a decrease in intracel-
lular cGMP concentration leads to an increase of
intracellular calcium mobilization in the cells; and (3)
PDE6 can be activated through the cascade Wnt5a–Friz-
zled-2–transducin–PDE6.
From the biochemical point of view, PDE6 (as almost
all PDEs) is a dimer and composed of two large homolo-
gous catalytic subunits PDE6-ab in rod or PDE6-a in cone,
and two small inhibitory subunits PDE6-c [19]. In this
work, we identified different patterns of the PDE6 subunit
expression: PDE6-abc, PDE6-aa, PDE6-ab, PDE6-b, and
PDE6-c. Although the dominant catalytic unit of rod PDE6
is a heterodimer PDE6-ab [19], this does not exclude the
existence of functional PDE6-aa and PDE6-bb [20].
However, the role of dimerization of the PDE6 subunits is
not fully understood, neither in photoreceptors nor in
cancer cells, and needs future investigations. Another
HaCat
1
1.5
2
0 1.53 4.567.59
time, min
10.5 1213.5 1516.5 1819.5 21
A340/A380
co
ZAP
PT
Ma-Mel 04
0.8
1.2
1.6
0 1.53 4.56 7.59
A340/A380
co
ZAP
PT
Ma-Mel 12
1
1.5
2
01.534.56 7.59
A340/A380
co
ZAP
PT
time, min
time, min
Fig. 6 Wnt5a stimulates the increase of the [Ca2?]imobilization in
Ma-Mel-04 and Ma-Mel-12 melanoma cells, but not in HaCaT cell
line. Untreated and ZAP- or pertussis toxin-treated cells were loaded
with fura-2/AM and then 20 ng/ml of Wnt5a was added to start the
reaction. Measurement of fluorescence by 340 and 380 nm excitation
was performed each 60 s during 15 min, and the ratio 340/380 was
calculated. For details, see ‘‘Materials and methods’’
Fig. 5 Wnt5a activates PDE6 through the FZ2 and transducin
cascade. a PDE6 activity was measured in melanoma cell lines with
different pattern of PDE6 expression and in HaCaT cell line incubated
with or without 20 ng/ml Wnt5a for 1 h. b PDE6 activity after
stimulation with Wnt5a was measured in Ma-Mel-04 and Ma-Mel-12
melanoma cells pretreated with 50 ng/ll antibody against FZ2 for
30 min. c PDE6 activity after stimulation with Wnt5a was measured
in melanoma cells cultivated with 50 ng/ml of pertussis toxin for
24 h. d PDE6 activity in untrasfected and transfected with shRNA
coding plasmids for PDE6-c, Ma-Mel-04 with and without additional
stimulation/inhibition with Wnt5a or PT. Statistically significant
differences (P\0.05) are marked with an asterisk
b
Function of PDE6 in melanoma cells

Page 10

interesting point is the expression of PDE6-c alone. PDE6-c
belongs to the group of so-called intrinsically disordered
proteins which represent a number of natively unfolded or
intrinsically unstructed proteins [21]. Such proteins have
little or no ordered structure under physiological conditions
and, due to their structural plasticity, can bind to different
targets and thus fulfill diverse functions [22]. Hence, the
expression of PDE6-c without expression of the catalytic
subunits of PDE6 could be explained by different functions
and other molecular targets of PDE6-c in melanoma cells.
Depending on the PDE6 subunits expression and the
level of phosphodiesterase activity, the melanoma cell lines
tested can be classified into three groups (Fig. 7). The first
group consists of melanoma cell lines which do not express
PDE6 and have a low, or even no, phosphodiesterase
activity (Fig. 7: type I). Type II comprises cells with a high
phosphodiesterase activity, these cells express PDE6-a
and/or PDE6-b, are negative for PDE6-c and can be
inhibited by phosphodiesterase inhibitors (Fig. 7). Type III
represents cells expressing both catalytic (PDE6-a and
PDE6-b) and inhibitory (PDE6-c) subunits, show a low
phosphodiesterase activity and express transducin (Fig. 7).
The cascade Wnt5a–Frizzled-2–transducin–PDE6 is oper-
ational in the melanoma cell lines of type III, consequently
these cells posses a high phosphodiesterase activity upon
activation (see Fig. 7).
Wnt5a is a secreted regulatory protein serving as a
ligand for the FZ-2 receptor [23]. Since the FZ-2 receptor
is structurally similar to visual rhodopsin [24], the Wnt5a–
FZ-2 downstream cascade in the embryonic development
seems to have the same molecular partners as rhodopsin
has in photoreceptor cells, i.e. transducin and PDE6 [14].
Thus, the Wnt5a–FZ-2 cascade shares the same signaling
pathway as does the visual cascade [25]. In this work, we
present evidence that the same signaling paradigm also
takes place in melanoma cells (see Fig. 7): Wnt5a binds to
FZ-2 receptor which activates G protein transducin. Acti-
vated transducin, in turn, binds to the inhibitory c subunit
of the PDE6 holoenzyme and makes PDE6 active. Finally,
active PDE6 hydrolyses cGMP.
In zebrafish embryos and mouse F9 embryonic terato-
carcinoma cells, signal transduction in the cascade Wnt5a–
FZ-2–transducin–PDE6 leads to a decrease of intracellular
cGMP, thus stimulating the intracellular calcium mobili-
zation [14, 26]. This chain of events involves PKG which is
inhibited at a low cGMP concentration [15]. As a conse-
quence, a high calcium concentration activates the
phosphoprotein phosphatase calcineurin and calcium/cal-
modulin protein kinase II, and finally stimulates the gene
expression through NF-AT [16]. Do the same signaling
proteins serve as molecular targets for calcium in mela-
noma cells? This question should be resolved in future
investigations.
In zebrafish development, the cascade Wnt5a–FZ-2–
transducin–PDE6 canbe
movements [14, 27]. As in development, tumorigenesis
involves morphogenesis, movement, and proliferation.
Wnt5a signaling can directly affect cell motility and
invasion of metastatic melanoma through the activation of
protein kinase C by a high calcium concentration [28]. It
has also been shown that the Wnt5a gene expression can be
used for separation of highly aggressive melanomas from
manifestedingastrulation
PDE6
[cGMP]
X
[Ca2+]
XX
proliferation?
FZ2
γ
Wnt5a
PDE6
[cGMP]
X
[Ca2+]
XX
FZ2
γ
?
Tr
PDE6
*
[cGMP]
X
[Ca2+]
XX
FZ2
γ
?
Wnt5a
*
Tr
*
PDE6
*
[cGMP]
X
[Ca2+]
XX
FZ2
γ
?
PDE6
inhibitors
Low PDE6 activity
High PDE6 activity
Type I
Type II
Type III
Fig. 7 A schematic
presentation of three types of
melanoma cells in regard to
PDE6. See text for details
A. V. Bazhin et al.

Page 11

their less invasive counterparts [29]. The usage of PDE6
inhibitors could simulate a reverse effect of Wnt5a by
reduction of PDE6 activity. Serafini et al. [30] have shown
that PDE inhibitors (sildenafil, tadalafil, and vardenafil)
modulate the antitumor immune response by reducing the
myeloid-derived suppressor cell function. The authors
discuss a potential use of these inhibitors as supplement to
the tumor-specific immune therapy.
Why do not all melanoma cell lines express the com-
ponents ofWnt5a–FZ–2–transducin–PDE6
(instead of FZ-2)? It is well known that cancer cell lines in
comparison to cancer tissues lose the ability to express
some proteins after their isolation from the tumors and
cultivation in vivo [31]. How frequently are PDE6 subunits
and transducin expressed in melanoma tissues? Does this
expression correlate with clinical data of melanoma
patients? Is the cascade Wnt5a–FZ-2–transducin–PDE6
really involved in motility and invasion of melanoma cells?
These questions should be answered in future studies.
cascade
Acknowledgments
excellent technical assistance and Dr. M. Rogers for the English
correction. This work was supported by the Intramural Program of
DKFZ to A.V.B., and by the Russian Foundation for Basic Research
(09-04-00395-a) and Hanse Wissenschaftskolleg, Germany to P.P.P.
We are grateful to Mrs. A. Heinzelmann for her
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