Báo cáo khoa học: Acidic extracellular pH increases calcium influx-triggered phospholipase D activity along with acidic sphingomyelinase activation to induce matrix metalloproteinase-9 expression in mouse metastatic melanoma pot

Acidic extracellular pH increases calcium influx-triggered
phospholipase D activity along with acidic
sphingomyelinase activation to induce matrix
metalloproteinase-9 expression in mouse metastatic
melanoma
Yasumasa Kato
1,2
, Shigeyuki Ozawa
1,3
, Mamoru Tsukuda
2
, Eiro Kubota
3
, Kaoru Miyazaki
4
,
Yves St-Pierre
5
and Ryu-Ichiro Hata
1
1 Department of Biochemistry and Molecular Biology, Kanagawa Dental College, Yokosuka, Japan
2 Department of Biology and Function in the Head and Neck, Yokohama City University Graduate School of Medicine, Japan
3 Department of Oral and Maxillofacial Surgery, Kanagawa Dental College, Yokosuka, Japan
4 Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Japan
5 INRS-Institut Armand-Frappier, Universite
´
du Que
´
bec, Laval, Que
´
bec, Canada

Keywords
acidic sphingomylinase; Ca
2+
influx;
extracellular acidic pH; MMP-9
Correspondence
Y. Kato, Department of Biochemistry and
Molecular Biology, Kanagawa Dental
College, Yokosuka 238-8580, Japan
Fax: +81 46 822 8839
Tel: +81 46 822 8840
E-mail:
(Received 23 January 2007, revised 23 April
2007, accepted 27 April 2007)
doi:10.1111/j.1742-4658.2007.05848.x
Acidic extracellular pH is a common feature of tumor tissues. We have
reported that culturing cells at acidic pH (5.4–6.5) induced matrix metallo-
proteinase-9 expression through phospholipase D, extracellular signal regu-
lated kinase 1 ⁄ 2 and p38 mitogen-activated protein kinases and nuclear
factor-jB. Here, we show that acidic extracellular pH signaling involves
both pathways of phospholipase D triggered by Ca
2+
influx and acidic
sphingomyelinase in mouse B16 melanoma cells. We found that BAPTA-
AM [1,2-bis(2-aminophenoxy)-ethane-N,N,N¢,N¢-tetraacetic acid tetrakis
(acetoxymethyl) ester], a chelator of intracellular free calcium, and the
voltage dependent Ca
2+
channel blockers, mibefradil (for T-type) and
nimodipine (for L-type), dose-dependently inhibited acidic extracellular

pH-induced matrix metalloproteinase-9 expression. Intracellular free cal-
cium concentration ([Ca
2+
]
i
) was transiently elevated by acidic extracellular
pH, and this [Ca
2+
]
i
elevation was repressed by EGTA and the voltage
dependent Ca
2+
channel blockers but not by phospholipase C inhibitor,
suggesting that acidic extracellular pH increased [Ca
2+
]
i
through voltage
dependent Ca
2+
channel. In contrast, SR33557, an L-type voltage depend-
ent Ca
2+
channel blocker and acidic sphingomyelinase inhibitor, attenu-
ated matrix metalloproteinase-9 induction but did not affect calcium influx.
We found that acidic sphingomyelinase activity was induced by acidic
extracellular pH and that the specific acidic sphingomyelinase inhibitors
(perhexiline and desipramine) and siRNA targeting aSMase ⁄ smpd1 could
inhibit acidic extracellular pH-induced matrix metalloproteinase-9 expres-

sion. BAPTA-AM reduced acidic extracellular pH-induced phospho-
lipase D but not acidic sphingomyelinase acitivity. The acidic
Abbreviations
aSMase, acidic sphingomyelinase; BAPTA-AM, 1,2-bis(2-aminophenoxy)-ethane-N,N,N¢,N¢-tetraacetic acid tetrakis (acetoxymethyl) ester; CM,
conditioned medium; [Ca
2+
]
i
, intracellular Ca
2+
concentration; DAG, diacylglycerol; ERK, extracellular signal regulated kinase; IL, interleukin;
IP
3
, inositol 1,4,5-triphosphate; JNK, c-Jun NH
2
-terminal kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase;
NF-jB, nuclear factor-jB; nSMase, neutral sphingomyelinase; PC, phosphatidylcholine; pH
e
, extracellular pH; PKCf, protein kinase Cf;
PLC, phospholipase C; PLD, phospholipase D; SM, sphingomyelin; SMase, sphingomyelinase; TNF-a, tumor necrosis factor a; TPA,
12-O-tetradecanoylphorbol 13-acetate; VDCC, voltage dependent Ca
2+
channel; VEGF, vascular endothelial growth factor.
FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3171
Acidic extracellular pH (pH
e
) has been frequently
observed in solid tumors, due to excess amounts of
anaerobic glucose metabolites. Acidic pH
e

has been
reported to affect the efficacy of chemotherapy, inclu-
ding reducing the cytotoxicity of bleomycin, doxorubi-
cin, daunorubicin, epirubicin, mitoxantrone, and vinca
alkaloids, but potentiating 5-fluorouracil [1]. A recent
study demonstrated that acidic pH
e
is a predictor of
metastasis-free survival in canine soft tissue sarcomas
treated with thermoradiotherapy [2]. The acidic micro-
environment may also regulate tumor angiogenesis
using a signal pathway different from that of hypoxia
[3–7]. Hypoxia was recently reported to affect expres-
sion of matrix metalloproteinases (MMPs) [8], which
are important in inflammation, tumor invasion, and
metastasis.
We have reported that acidic pH
e
induced the expres-
sion of MMP-9 ⁄ gelatinase B (EC 3.4.24.35) in highly
metastatic mouse B16 melanoma cell lines, while not
affecting the expression of MMP-2 ⁄ gelatinase A [9].
We have also reported that acidic pH
e
-induced MMP-9
expression was mediated via the phospholipase D
(PLD)–mitogen-activated protein kinase (MAPK)
[extracellular signal regulated kinase (ERK)1 ⁄ 2 and
p38] pathway, at least in part through acidic pH
e

signa-
ling through nuclear factor-jB (NF-jB) [10].
Acidic pH
e
has been shown to increase intracellular
Ca
2+
concentration ([Ca
2+
]
i
) in fibroblasts [11], endo-
thelial cells [12], and smooth muscle cells [13–15]. In
addition, increased [Ca
2+
]
i
has been found to activate
PLD [16,17], which is also involved in the acidic pH
e
induction of MMP-9 expression [10]. [Ca
2+
]
i
elevation
can be divided into major two pathways: Ca
2+
influx
through specific channels and release of Ca
2+

from the
endoplasmic reticulum by inositol 1,4,5-triphosphate
(IP
3
), a product of phospholipase C (PLC). Voltage
dependent Ca
2+
channels (VDCC) have been classified
into low (T-type) and high (L-type) voltage types,
which can be blocked by mibefradil and nimodipine,
respectively. SR33557, which is another type of the
L-type VDCC blocker, can also inhibit mRNA expres-
sion of acidic sphingomyelinase (aSMase) ⁄ acid lyso-
somal sphingomyelin phosphodiesterase 1 (smpd1)in
the signal transduction pathways of interleukin (IL)-1
and tumor necrosis factor a (TNF-a) [18,19].
MMP-9 can be up-regulated by various stimuli,
including IL-1 and TNF-a, which trigger the ceramide-
signaling pathway [20]. Ceramide, which is generated
by the hydrolysis of sphingomyelin (SM) acts as a lipid
second messenger for apoptotic signaling [21]. Both
aSMase and neutral sphingomyelinase (nSMase) can
activate MAPKs, such as ERK1 ⁄ 2, Jun-N-terminal kin-
ase (JNK), and p38, in various cell types [22–25]. More-
over, ceramide can induce MMP expression [26,27].
Here, we report that PLD, w hich is activated by Ca
2+
influx and aSMase, mediates the aci dic pH
e
induction of

MMP-9, at least in part through NF-jB activation.
Results
Acidic pH
e
increases Ca
2+
influx through VDCC
Increased [Ca
2+
]
i
has been shown to activate PLD
[16,17] and acidic pH
e
has been shown to elevate
[Ca
2+
]
i
in fibroblasts [11], endothelial cells [12], and
smooth muscle cells [13–15]. To determine the involve-
ment of [Ca
2+
]
i
, in acidic pH
e
signaling, we treated
cells with the calcium chelator BAPTA-AM [1,2-
bis(2-aminophenoxy)-ethane-N,N,N

1
,N
1
-tetraacetic acid
tetrakis (acetoxymethyl) ester]. We found that
BAPTA-AM dose-dependently attenuated the acidic
pH
e
-induced MMP-9 expression with an IC
50
of
5.1 lm (Fig. 1A). When we tested the effects of VDCC
blockers on acidic pH
e
-induced MMP-9 expression, we
found that the L-type VDCC blockers SR33557 [28,29]
and nimodipine and the T-type blocker mibefradil
dose-dependently inhibited acidic pH
e
-induced MMP-9
expression, with an IC
50
of 13.7 lm, 3.0 lm, and
1.0 lm, respectively (Fig. 1B,C). These agents at the
same concentrations showed neither cellular toxicity
nor any other gelatinolytic activity.
Using Fluo4-AM, a fluorescent probe used to meas-
ure [Ca
2+
]

i
, we observed a transient increase in [Ca
2+
]
i
in the presence, but not in the absence, of extracellular
Ca
2+
(Fig. 2A). The calcium chelator, EGTA, but not
the broad PLC inhibitor U73122, attenuated the acidic
pH
e
-induced transient increase in [Ca
2+
]
i
, suggesting
that [Ca
2+
]
i
is increased by Ca
2+
entry not by inositol
1,4,5-triphosphate (IP
3
)-induced Ca
2+
release from
the endoplasmic reticulum (Fig. 2B). Mibefradil and

sphingomyelinase inhibitors did not affect the phosphorylation of extracel-
lular signal regulated kinase 1 ⁄ 2 and p38, but they suppressed nuclear
factor-jB activity. These data suggest that the calcium influx-triggered
phospholipase D and acidic sphingomyelinase pathways of acidic extracel-
lular pH induced matrix metalloproteinase-9 expression, at least in part,
through nuclear factor-jB activation.
aSMase and Ca
2+
influx in acid induction of MMP-9 Y. Kato et al.
3172 FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS
nimodipine prevented acidic pH
e
-induced Ca
2+
influx
(Fig. 2B), suggesting that Ca
2+
influx, which occurred
through T-type and L-type VDCCs, triggered acidic
pH
e
-induced MMP-9 expression. SR33557 (25 lm) did
not affect acidic pH
e
-induced Ca
2+
influx (Fig. 2B) but
suppressed MMP-9 expression (Fig. 1B), suggesting
that aSMase may be involved in acidic pH
e

signaling.
aSMase mediates acidic pH
e
-induced MMP-9
expression
To investigate the involvement of aSMase in acidic
pH
e
signaling, we tested the effects of the aSMase
specific inhibitors perhexiline [30–32] and desipramine
[32–35]. Both dose-dependently inhibited acidic pH
e
-
induced MMP-9 expression, with an IC
50
of 0.5 lm
A
B
Fig. 2. [Ca
2+
]
i
is increased through VDCC but not from the endo-
plasmic reticulum. Cells (40 000) cells were incubated overnight
with serum-containing growing medium and with serum-free med-
ium (pH 7.3) for 4 h and loaded with Fluo-4-AM (0.9 l
M) in NaCl ⁄ Pi
containing 0.495 m
M MgCl
2

for 30 min at room temperature. (A)
After washing, the cells were simulated by overlaying an acidic pH
buffer [horizontal gray bar; NaCl ⁄ Pi (pH 5.9) supplemented with
15 m
M Hepes, 4 mM phosphoric acid, and 0.495 mM MgCl
2
] in the
presence (open circle) or absence (closed circle) of 0.901 m
M
CaCl
2
. [Ca
2+
]
i
was measured at 490 nm excitation and 535 nm
emission wavelengths, at 0.26 s intervals. (B) Cells were treated
with EGTA (5 m
M), mibefradil (2.5 lM), nimodipine (5 lM), SR33557
(25 l
M), and U73122 (50 lM) on [Ca
2+
]
i
for 15 min and stimulated
as above. Bars indicate SD.
A
B
C
Fig. 1. Intracellular Ca

2+
chelator and VDCC blockers reduce acidic
pH
e
-induced MMP-9 expression. Nearly confluent cells in a 24-well
culture plate were serum-starved overnight and cultured with acidic
medium (pH 5.9) in the presence of the indicated concentrations of
(A) BAPTA-AM, or (B) SR33557 for 48 h, or (C) mibefradil or nimodi-
pine for 24 h. Proteins in the medium were ethanol concentrated,
and gelatinolytic activity was detected by gelatin zymography.
Experiments were performed three times; one representative
experiment is shown accompanied with the induction rate, which
was estimated by densitometry. Concentration dependent reduc-
tion was seen with P-values less than 0.01 for SR33557, mibefradil
nimodipine and 0.001 for BAPTA-AM. Molecular markers are indica-
ted in kDa. Arrowheads indicate pro-MMP-9.
Y. Kato et al. aSMase and Ca
2+
influx in acid induction of MMP-9
FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3173
and 6.0 lm, respectively (Fig. 3A). Incubation of cells
at acidic pH
e
increased aSMase activity 2.0-fold but
had no effect on nSMase activity (Fig. 3B). When
aSMase blockers were added to the cultures, they sig-
nificantly inhibited acidic pH
e
-induced aSMase activity
(Fig. 3B), at concentrations sufficient to inhibit acidic

pH
e
-induced MMP-9 expression (Fig. 3A). To prove
the contribution of aSMase in this signaling cascade
to induce MMP-9, small interfering RNA (siRNA)
technology was used. Introduction of siRNA oligo-
nucleotide targeting smpd-1 ⁄ aSMase mRNA reduced
the acid induction of MMP-9 expression (Fig. 4A)
concomitantly with the decrease in smpd-1 ⁄ aSMase
mRNA (Fig. 4A) and its activity (Fig. 4B) and also
in vivo ceramide production that is a metabolite of
aSMase from SM (Fig. 4C). Interestingly, it was also
found that acid induction of smpd-1 ⁄ aSMase mRNA
expression, suggesting that the elevation of aSMase
activity by acidic pH
e
, as shown in Fig. 3B, is mainly
due to an increase in the mRNA level rather than its
activation. These data showed a significant contribu-
tion of aSMase in acidic pH
e
signaling to induce
MMP-9 expression.
Chelation of [Ca
2+
]
i
inhibits acidic pH
e
-induced

PLD activation and MMP-9 expression but not
aSMase activation
To determined the effect of [Ca
2+
]
i
elevation on PLD
activity, cells were cultured with the [Ca
2+
]
i
chelater
BAPTA-AM. We found that this reagent dose-depend-
ently reduced acidic pH
e
-induced PLD activity, but
had no effect on aSMase activity (Fig. 5), suggesting
that acidic pH
e
triggers Ca
2+
influx, which is followed
by PLD activation independent of the aSMase path-
way.
[Ca
2+
]
i
elevation by thapsigargin at neutral pH
e

mimics acidic pH
e
-induced PLD activation and
MMP-9 expression
If Ca
2+
influx triggers PLD activation and this is fol-
lowed by MMP-9 expression, we expected that we
could mimic this effect at neutral pH
e
by increasing
[Ca
2+
]
i
pharmacologically. When the cells were cul-
tured at neutral pH
e
with thapsigargin, a releaser of
intracellular free Ca
2+
from the endoplasmic reticu-
lum, PLD activity was increased and MMP-9 was
expressed [36,37] (Fig. 6). 12-O-tetradecanoylphorbol
13-acetate (TPA) did not induce MMP-9 expression in
B16 melanoma cells [9,10,38], but did so, through PLD
activation, in HT1080 cells [39]. Here, we found that
TPA could not increase PLD activity (Fig. 6), suggest-
ing a reason that TPA could not induce MMP-9
expression in this model. Besides, acid induction of

MMP-9 expression was found without activation of
AP-1 [10], generally known as the responsible factor
for MMP-9 transcription which could be activated by
TPA.
In contrast, we found that exogenous addition of
SMase dose-dependently stimulated the level observed
in the presence of thapsigargin (Fig. 7A). Similarly,
C
2
-ceramide, a cell permeable ceramide analogue,
increased MMP-9 expression in the presence, but not
in the absence, of thapsigargin at neutral pH
e
(Fig. 7B), suggesting that both SM and PC (phosphat-
idylcholine) metabolites are important in acidic pH
e
induction of MMP-9 expression.
A
B
Fig. 3. aSMase mediates acidic pH
e
induction of MMP-9 expres-
sion. Nearly confluent cells in a 24-well culture plate were serum-
starved overnight and cultured for 2 days in acidic medium (pH 5.9)
in the presence of the indicated concentrations of aSMase inhibi-
tors perhexiline maleate (perhexiline) and desipramine hydrochloride
(desipramine). (A) Proteins in CM were concentrated and analyzed
by gelatin zymography. The arrowhead indicates MMP-9 activity.
(B) Membrane fractions (50 lg), prepared using a 0.2% Triton X-
100 buffer, were incubated for 60 min at 37 °C in 250 m

M sodium
acetate, 1 m
M EDTA (pH 5.0) for aSMase or 250 mM Tris ⁄ HCl
(pH 7.4) for nSMase, each containing 0.05 lCi [choline methyl-
14
C]-
SM. Radioactive phosphorylcholine was extracted with chloro-
form ⁄ methanol (2 : 1, v ⁄ v) and the radioactivities in the aqueous
phase were determined by liquid scintillation counting. Closed and
open columns indicate aSMase and nSMase activities, respectively.
Bars indicate SD.
aSMase and Ca
2+
influx in acid induction of MMP-9 Y. Kato et al.
3174 FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS
Inhibition of aSMase activity has no effect on
ERK1
⁄
2 and p38 phosphorylations
To assess the contribution of MAPKs to the down-
stream signaling of aSMase at acidic pH
e
, we meas-
ured the levels of the phosphorylated (active) forms of
MAPKs in these cultures. We previously showed that
phosphorylation of ERK1 ⁄ 2 and p38 MAPKs was
significantly decreased by the PLD inhibitor (1-buta-
nol), whereas the total amounts of ERK1 ⁄ 2 and
p38 MAPKs were not affected [10]. We found that
perhexiline and desipramine inhibition of aSMase did

not affect the activation of ERK1⁄ 2 and p38 MAPKs
(Fig. 8). Similar findings were observed with the other
aSMase inhibitor, SR33557 (data not shown). The
JNK phosphorylation level was not affected by acidic
pH
e
[10] and aSMase inhibitors had no effect on its
basal phosphorylation level (data not shown). These
data suggested that ERK1 ⁄ 2 and p38 MAPKs were
A
B
C
Fig. 4. Knockdown of aSMase ⁄ smpd1 expression reduces acidic
pH
e
-induced MMP-9 expression. Cells, which have been transfect-
ed with siRNA oligonucleotide targeting aSMase ⁄ smpd1, were
treated with acidic or neutral pH
e
. (A) Proteins in CM were ethanol
concentrated, and gelatinolytic activity was detected by gelatin
zymography. Total RNA was extracted and mmp-9, aSMase ⁄ smpd1
and b-actin gene expressions were analyzed by RT-PCR using spe-
cific primer sets. (B) Membrane fractions (50 lg), prepared using a
0.2% Triton X-100 buffer, were incubated for 60 min at 37 °Cin
250 m
M sodium acetate, 1 mM EDTA (pH 5.0) containing 0.05 lCi
[choline methyl-
14
C]-SM. Radioactive phosphorylcholine was extrac-

ted with chloroform ⁄ methanol (2 : 1, v ⁄ v) and the radioactivities in
the aqueous phase were determined by liquid scintillation counting.
(C) The aSMase siRNA-transfected cells were labelled with
0.5 lCiÆmL
)1
[9,10-
3
H]-palmitic acid and then stimulated with acidic
pH medium for 24 h. Lipids were extracted from the cells with
chloroform ⁄ methanol and analyzed by thin layer chromatography.
The [
3
H]-ceramide formed was identified by comigration of N-palmi-
toyl-
D-erythro-sphingosine. The spots, which were identified as
[
3
H]-ceramide, were scrapped off and the radioactivities were coun-
ted by liquid scintillation counting. Bars indicate SD. *P < 0.05;
***P < 0.001 (Student’s t-test).
Fig. 5. [Ca
2+
]
i
chelation reduces acidic pH
e
-induced PLD but not
aSMase activity. Nearly confluent cells in a 60 mm culture dish
were serum-starved overnight and cultured for 2 days in acidic
medium (pH 5.9), in the presence or absence of the indicated con-

centrations of BAPTA-AM. The membrane fractions (50 lg) were
prepared, and aSMase activity (closed column) was measured by
incubation for 60 min at 37 °C in 250 m
M sodium acetate, 1 mM
EDTA (pH 5.0) containing 0.05 lCi [choline methyl-
14
C]-SM, fol-
lowed by scintillation counting of the aqueous phase. PLD activity
(open column) of the membrane fractions was measured using an
Amplex
TM
Red PLD assay kit and a fluorescence microplate reader,
with an excitation wavelength of 535 nm and a detection wave-
length of 590 nm. Bars indicate SD.
Y. Kato et al. aSMase and Ca
2+
influx in acid induction of MMP-9
FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3175
not downstream targets of aSMase in acidic pH
e
signaling.
Inhibition of aSMase activity attenuates acidic
pH
e
-induced NF-jB and MMP-9 promoter
activities
The MAPK kinase inhibitor PD098059 and the p38
inhibitor SB203580 have been shown to inhibit acidic
pH
e

-induced NF-jB activity [10]. We found that the
aSMase inhibitors reduced wild-type MMP-9 promoter
activity, as well as altering NF-jB-mutant MMP-9
promoter activity (Fig. 9A). Moreover, aSMase inhibi-
tion partially reduced acidic pH
e
-induced NF-jB activ-
ity (Fig. 9B), suggesting that acidic pH
e
-induced
NF-jB is coregulated by the Ca
2+
⁄ PLD ⁄ MAPK and
aSMase pathways. These cascades proposed were sche-
matically summarized in Fig. 10. We found, however,
that a mutant MMP-9 promoter lacking the NF-jB
binding site (DNF-jB) still showed inducibility at
acidic pH
e
and that this induction was attenuated by
the aSMase inhibitors. Although acidic pH
e
-induced
NF-jB activity was down-regulated by these inhibitors
at the same concentrations, this inhibition was only
partial, suggesting that other transcription factor(s)
may be the downstream target(s) of aSMase. Some
candidates were considered
1
. Among the transcription

factors known within the minimal MMP-9 promoter
region, Ets1 and SP1 were potentially involved in the aci-
dic pH
e
signaling. Indeed, using transcription factor-decoy
and siRNA technologies, we found that Ets1 and SP1
were responsible for acid induction of MMP-9 expression
(Y. Kato, S. Ozawa and R. I. Hata, unpublished data)
2
.
The upstream signaling cascade leading t o their activa-
tions (e.g. MAPKs and aSMase) is currently under inves-
tigation.
Fig. 6. Thapsigargin increased [Ca
2+
]
i
induces PLD activity and
MMP-9 expression at neutral pH
e
. Nearly confluent cells were
serum-starved and incubated with thapsigargin (Thap, 2.5 l
M), TPA
(80 n
M) or vehicle at pH
e
7.3. Gelatinolytic activity in CM was ana-
lyzed by zymography (inset). The cells were lysed with 0.2% Triton
X-100, and the lysates were subjected to Amplex
TM

Red PLD
assay. Bars indicate SD. *P < 0.05; ***P < 0.001 (Student’s t-test).
NS, not significant. Arrowhead indicates pro-MMP-9.
A
B
Fig. 7. SM hydrolysis contributes to MMP-9 expression. Nearly
confluent cells were serum-starved and incubated for 48 h with the
indicated concentrations of bacterial SMase (Staphylococcus aure-
us) (A) or 25 l
M C
2
-ceramide (B) in the presence or absence of
2.5 l
M thapsigargin at pH
e
7.3. CM was collected, concentrated,
and MMP-9 activity was assayed by zymography. Arrowheads indi-
cate pro-MMP-9.
Fig. 8. aSMase inhibitors do not affect acidic pH
e
-induced
phosphorylation of ERK1 ⁄ 2 and p38. Nearly confluent cells were
serum-starved and incubated with or without 10 l
M desipramine
hydrochloride (desipramine) or 10 l
M perhexiline maleate (perhexi-
line) or at pH
e
5.9 for 48 h. The cells were lysed and MAPK phos-
phorylation was analyzed by western blotting using phospho-

specific ERK1 ⁄ 2 or p38 polyclonal antibodies. The induction rate of
phosphorylated ratio was estimated by the densitometry and
expressed as the relative values for the ratio of vehicle control at
pH
e
7.3. p-ERK1 ⁄ 2, phosphorylated ERK1 ⁄ 2; p-p38, phosphorylated
p38.
aSMase and Ca
2+
influx in acid induction of MMP-9 Y. Kato et al.
3176 FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS
Discussion
Acidic pH
e
, a common feature of solid tumors, is
thought to decrease the efficacy of chemotherapy regi-
mens [40–44]. Angiogenesis-related gene expression
was found to be induced by aci dic pH
e
through hypoxia
independent pathways involving platelet-derived
endothelial cell growth factor ⁄ thymidine phosphorylase
in human breast tumor cells [45], the inducible isoform
of nitric oxide synthase in macrophages [46], vascular
endothelial cell growth factor in glioma [6] and gliobla-
stoma [7] cells and IL-8 expression in human pancre-
atic adenocarcinoma [3,47,48] and ovarian carcinoma
cells [4]. In addition, we have reported that expression
of MMP-9 in mouse metastatic B16 melanoma cells
was induced by acidic pH

e
(pH
e
6.5–5.4) and that,
among B16 clones, the rate of induction was correlated
with metastatic potential [9]. Most recently, acidic pH
e
was reported to enhance the metastatic potential of
human melanoma cells, accompanied by elevation of
proteinases and proangiogenic factors such as MMP-9,
MMP-2, cathepsin B, cathepsin L, vascular endothelial
growth factor (VEGF)-A, and IL-8 [49]. We also
reported that acidic pH
e
induction of MMP-9 expres-
sion was mediated through the PLD–MAPK pathway
[10]. Here, we further examined whether increased
[Ca
2+
]
i
and SM metabolism contributed to the acidic
pH
e
signaling induction of MMP-9 expression. These
contributions were also investigated in human lung
adenocarcinoma cell line A549. Perhexiline (aSMase
inhibitor) and nimodipine (L-type VDCC blocker)
reduced acidic pH
e

-induced MMP-9 expression in
A549 but mibefradile (T-type VDCC blocker) had no
effect on this induction (data not shown), suggesting
that the contribution of aSMase and Ca
2+
influx is
essential for acidic pH
e
signaling but the majority of
the VDCC type
3
involved in this signaling is cell type
specific.
NF-jB is a transcription factor responsible for
MMP-9 expression [50] and can mediate acidic pH
e
signaling [10]. Acidic pH
e
-induced activity of PLD, but
not aSMase, was suppressed by chelating [Ca
2+
]
i
, sug-
gesting that Ca
2+
influx activated PLD, but not
aSMase. It has been reported that aSMase activity
could be induced by PC-derived diacylglycerol (DAG)
through PC-PLC but not by phosphatidylinositol 4,5-

biphosphate-derived DAG through PLD followed by
phosphatidate phosphatase. Because U73122 had little
effect on [Ca
2+
]
i
,IP
3
is not likely to be involved in aci-
dic pH
e
induced [Ca
2+
]
i
elevation. PC, a metabolite of
PC-PLC, decreased after pH
e
dropped and D609, an
inhibitor of PC-PLC, did not dose-dependently inhibit
acidic pH
e
-induced MMP-9 expression [10]. Thus,
Fig. 10. Schematic representation of a proposed acidic pH
e
signa-
ling to induce MMP-9 expression.
Fig. 9. aSMase inhibitors inhibit acidic pH
e
-induced NF-jB activity

and MMP-9 promoter activity. Cells cultured overnight with 10%
fetal bovine serum in six-well plates were transfected with 1 lg
of mouse MMP-9 promoter-luciferase reporter construct (A) or
PathoDetectÒ NF-jB-luciferase reporter construct (B) using
Transfectin
TM
in serum-free DMEM ⁄ F12 at pH
e
7.3. After 18 h,
the cells were washed twice and cultured for 24 h with or
without perhexiline maleate (perhexiline) or desipramine hydro-
chloride (desipramine) at pH
e
7.3 or 5.9. The cells were lysed
and subjected to dual luciferase assay; and transfection efficiency
was normalized by cotransfecting a Renilla luciferase reporter
construct. WT, pGL3MMP9 (wild-type MMP-9 promoter con-
struct); DNF-jB, pGL3MMP9DNF-jB (MMP-9 promoter con-
struct mutated at the NF-jB binding site). **P < 0.05;
***P < 0.01 (Student’s t-test).
Y. Kato et al. aSMase and Ca
2+
influx in acid induction of MMP-9
FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3177
although the pathway involving PC-PLC may be ruled
out, DAG derived from PC through PLD and
phosphatidate phosphatase, but not from phosphati-
dylinositol 4,5-biphosphate through phosphatidyl-
inositol specific PLC, may be involved in acidic pH
e

signaling.
Because thapsigargin induced MMP-9 expression
along with a 1.8-fold increase in PLD activity [16,36],
the basal activity of aSMase may be sufficient, but that
of PLD may be defective, for induction of MMP-9
expression at neutral pH
e
. We have reported that aci-
dic pH
e
increased ERK1 ⁄ 2 and p38, but not JNK,
phosphorylation and that the former was attenuated
by 1-butanol, a PLD inhibitor [10]. ERK1 ⁄ 2, JNK and
p38 are activated as downstream targets of nSMase
and induce MMP-1 expression in fibroblasts [26]. In
B16-BL6 cells, however, aSMase inhibitors had little
effect on the phosphorylation of ERK1 ⁄ 2 and p38.
Because ceramide can be metabolized from SM by
both SMases, this difference may be cell type specific.
Further studies are needed to clarify the role of
aSMase in each cell type.
We have shown here that, although NF-jBisa
downstream target of aSMase, the signaling pathway
connecting the two is still unclear. One candidate
mediator is protein kinase Cf (PKCf), because cera-
mide is an activator of PKCf [51,52] and because
PKCf can directly phosphorylate the p65 (Ser311)
subunit of NF-jB [53]. We found that a PKCf pseudo-
substrate can inhibit acidic pH
e

-induced MMP-9
expression (Y. Kato, S. Ozawa and R. I. Hata, unpub-
lished data)
4
.
Because aSMase not only contributes to apoptosis,
but also to metastatic ability, its ability to adapt and
be selected for resistance to microenvironmental stress
such as acidic pH
e
may be indicative of its more
aggressive phenotype, using an ‘apoptotic signal’. This
concept is supported by results showing that hypoxia
inducible factor 1a, a key transcription factor for
VEGF during angiogenesis, induces apoptosis in nor-
mal pancreatic islets [54] but prevents cell death and
even stimulates growth of pancreatic cancer cells [55].
Although the pH
e
of tumor tissues is acidic and
anaerobic glucose metabolites are the major source of
acidity, tumor acidity was shown to be caused by
excess amounts of CO
2
, regardless of pO
2
, through the
pentose phosphate pathway, in glycolysis-impaired
(phosphoglucose isomerase-deficient) cells [56]. This
pathway provides cells with ribose 5-phosphate, which

is used to synthesize nucleic acids. Thus, highly prolif-
erating cells need more ribose 5-phosphate for DNA
replication and RNA synthesis, thereby producing
excess amounts of CO
2
. These observations suggest
that extracellular acidity in tumors is partly regulated
by an hypoxia-independent pathway. Because tumor
acidity affects the response radiation therapy and che-
motherapy, pharmacological blockade of VDCC
5
may
prevent tumor invasion and metastasis.
In conclusion, we found that two independent path-
ways; Ca
2+
–PLD–MAPKs (ERK1 ⁄ 2 and p38) and
aSMase, leading to NF-jB activation, are essential in
acidic pH
e
induction of MMP-9 expression.
Experimental procedures
Reagents
SR33557 [([2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxy-
phenethyl) amino] propyloxy) benzenesulfonyl]) indolizine],
an aSMase specific inhibitor, was kindly provided by San-
ofi-Aventis (Paris, France). BAPTA-AM and N-palmitoyl-
d-erythro-sphingosine [C16:0 (palmitoyl) ceramide] were
purchased from Calbiochem (La Jolla, CA, USA), and fluo
4-AM was obtained from Dojindo (Kumamoto, Japan).

DMEM and Ham’s F-12 (F-12), and TRIzolÒ Regent were
obtained from Invitrogen (Carlsbad, CA, USA); Trans-
fectin
TM
and siLentFect
TM
Lipid Reagents were obtained
from Bio-Rad (Hercules, CA, USA); the Dual Luciferase
Reporter Assay kit was obtained from Toyo Ink (Tokyo,
Japan); Staphylococcus aureus SMase, perhexiline maleate
salt, and desipramine hydrochloride were obtained from
Sigma (St. Louis, MO, USA); fetal bovine serum was
obtained from Cell Culture Technologies GmbH (Zurich,
Switzerland); [choline methyl-
14
C]-SM was obtained from
Amersham Biosciences (Piscataway, NJ, USA); [9,10-
3
H]-
palmitic acid (50.0 CiÆmmol
)1
) was obtained from Moravec
Biochemicals (Brea, CA, USA); Immobilon-P [poly(vinylid-
ene difluoride)] membrane was obtained from Millipore
(Bedford, MA, USA); and the Nuclear Extract kit was
obtained from Active Motif (Carlsbad, CA, USA). EGTA,
TPA and the Immunostar
TM
Western blotting detection
kits, which included a chemiluminescent reagent and

peroxidase-conjugated swine anti-rabbit IgG or goat anti-
mouse IgG, were obtained from Wako (Tokyo, Japan). The
blocking reagent N102 was obtained from NOF Corp.
(Tokyo, Japan); siRNA oligonucleotide targeting aSMase ⁄
smpd1 and a control oligonucleotide (scramble), and anti-
bodies directed against total or phosphorylated MAPKs
(sc-7976-R, sc-154, sc-7149, sc-7975-R, sc-571, sc-6254)
were obtained from Santa Cruz (Santa Cruz, CA, USA).
Silica Gel60 F
254
plate was obtained from Merck KGaA
(Darmstadt, Germany).
Vectors
The PathoDetectÒ NF-jB cis-reporting system (pNF-jB-
Luc) was obtained from Stratagene (La Jolla, CA, USA).
aSMase and Ca
2+
influx in acid induction of MMP-9 Y. Kato et al.
3178 FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS
The MMP-9 promoter luciferase reporter construct and its
mutant construct of the NF-jB binding site have been
described previously [10,57]. The cytomegalovirus-driven
Renilla luciferase reporter vector (pRL-CMV, Promega,
Madison, WI, USA) was used to monitor transfection
efficiency.
Cells and cell culture
B16-BL6 cells were cultured in DMEM containing 15 mm
Hepes (pH 7.3) supplemented with heat-inactivated 10%
fetal bovine serum. Because induction of MMP-9 expres-
sion occurred from pH

e
6.5–5.4 [9], we fixed the pH of
the assay media at 5.9 for acidic pH
e
and at 7.3 for neut-
ral pH
e
. To prepare serum-free assay media (DMEM ⁄
F-12), a 1 : 1 mixture of DMEM and F-12 was supple-
mented with 15 mm Hepes and 4 mm phosphoric acid and
adjusted to pH 5.9 with HCl or to pH 7.3 with NaOH
[9,10,38].
SiRNA-mediated gene silencing
To suppress aSMase mRNA expression, siRNA technology
was used. Oligonucleotide (2 nm) targeting aSMase⁄ smpd1
was transfected into cells with siLentFect
TM
Lipid Reagent
in a serum-free DMEM ⁄ F-12 at pH 7.3 and cultured for
48 h. The transfectants were stimulated with acidic medium
for 48 h. The scrambled siRNA were used for a control. At
the end of incubation period, proteins in conditioned med-
ium (CM) and total RNA were obtained for zymography
to detect MMP-9 activity and RT-PCR was used to detect
mmp-9 gene expression.
Preparation of concentrated CM for zymography
Proteins in CM were concentrated by adding three volumes
of ice-cold ethanol as described previously [10,58]. The
quantity of samples was normalized for zymography assay
based on the DNA contents of the cultures (1.5 lg DNA ⁄

lane), as measured using bisbenzimide [59].
Gelatin zymography
Gelatinolytic activities in the CM were analyzed by gelatin
zymography, as described previously [9,10,60,61]. Briefly,
ethanol-precipitated proteins were electrophoresed in SDS-
7.5% polyacrylamide gels containing 0.1% gelatin. The gels
were washed in 2.5% Triton X-100 with gentle shaking for
1 h at room temperature to remove SDS and incubated for
20 h in reaction buffer [50 mm Tris ⁄ HCl (pH 7.5), 100 mm
NaCl, 10 mm CaCl
2
, and 0.002% NaN
3
]at37°C. Gela-
tinolytic activity was visualized as a clear zone on a
blue background following Coomassie Brilliant Blue R250
staining.
[Ca
2+
]
i
measurements
Cells were inoculated at a density of 40 000 cells ⁄ well in
96-well culture p lates. Following overnight incubation, the cells
were washed twice with Ca
2+
- and Mg
2+
-free Dulbecco’s
phosphate-balanced saline (NaCl ⁄ Pi) and incubated in

serum-free DMEM for 4 h. The cells were incubated with
Fluo 4-AM (final concentration, 0.9 lm) in NaCl ⁄ Pi
(pH 7.3) containing 0.901 mm CaCl
2
and 0.495 mm MgCl
2
for 30 min at room temperature and washed four times with
NaCl ⁄ Pi (pH 7.3) containing 0.495 mm MgCl
2
. The cells
were overlain with NaCl ⁄ Pi (pH 5.9) supplemented with
15 mm Hepes, 4 mm phosphoric acid, and 0.495 mm MgCl
2
in the presence or absence of 0.901 mm CaCl
2
. The [Ca
2+
]
i
was measured at 490 nm excitation and 535 nm emission
wavelengths, at 0.26 s intervals using Tecan GENiosPro
TM
fluorescence plate reader (Gro
¨
dig, Salzburg, Austria).
Where indicated, cells were incubated for 5 min with cal-
cium channel blockers dissolved in the NaCl ⁄ Pi (pH 7.3)
containing 0.901 mm CaCl
2
and 0.495 mm MgCl

2
, and
the cells were overlain with channel blocker-containing
NaCl ⁄ Pi (pH 5.9) supplemented with 15 mm Hepes, 4 mm
phosphoric acid, 0.495 mm MgCl
2
, and 0.901 mm CaCl
2
.
PLD activiy
Membrane fractions of the cells were prepared using 0.2%
Triton X-100. PLD activity was detected using the
Amplex
TM
Red PLD assay kit (Molecular Probes, Eugene,
OR, USA) [10]. Whole cell lysates were incubated with
250 lm PC, 100 mU Æ mL
)1
Alcaligenes sp. choline oxidase,
1UÆmL
)1
horseradish peroxidase, and 50 lm 10-acetyl-3,7-
dihydrophenoxazine (Amplex
TM
Red reagent) in reaction
buffer consisting of 50 m m Tris ⁄ HCl (pH 8.0), 5 mm
CaCl
2
, and 0.2% Triton X-100. PLD activity was measured
with a fluorescence microplate reader using an excitation

wavelength of 535 nm and detection wavelength of 590 nm.
SMase activities
SMase activities were measured as described previously [18].
Briefly, membrane fractions (50 lg), prepared using 0.2%
Triton X-100, were incubated for 60 min at 37 °Cin200lL
250 mm sodium acetate, 1 mm EDTA (pH 5.0) for aSMase
or 200 lL 250 mm Tris ⁄ HCl (pH 7.4) for nSMase, each con-
taining 0.05 lCi [choline methyl-
14
C]-SM. Radioactive phos-
phorylcholine was extracted with 750 lL of chloroform ⁄
methanol (2 : 1, v ⁄ v), and the radioactivity in the aqueous
phase was determined by liquid scintillation counting.
In vivo ceramide production
In vivo ceramide production
6
was measured as described
previously [62,63]. Cells were inoculated into six-well
Y. Kato et al. aSMase and Ca
2+
influx in acid induction of MMP-9
FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3179
culture plate at a density of 2.5 · 10
5
cells ⁄ well. Follow-
ing overnight incubation, the cells were washed twice
with NaCl ⁄ Pi and labelled with 1.5 lCi ⁄ well [9,10-
3
H]-
palmitic acid in serum-free DMEM for 18 h. The cells

were then stimulated with acidic assay medium for 24 h.
Lipids were extracted from the cells with chloro-
form ⁄ methanol (2 : 1, v ⁄ v). Lipids in the chloroform
phase were collected and analyzed by thin-layer chroma-
tography using a Silica Gel60 F
254
plate (20 · 20 cm) and
ethyl acetate ⁄ acetic acid ⁄ 2,2,4-trimethypentane (9 : 2 : 5)
as a solvent. The spots, which were identified as [
3
H]-cer-
amide by comigration of N-palmitoyl-d-erythro-sphingo-
sine [C16:0 (palmitoyl) ceramide], were scrapped off and
their radioactivities were counted by liquid scintillation
counter.
RT-PCR
Total RNA was extracted by using TRIsol Ò Reagent,
reverse-transcribed by MMLV super transcriptase, and
amplified by Taq polymerase with specific primer sets:
aSMase ⁄ smpd1 (26 cycles, 258 bp), 5¢-TTC CTG CCA
GAG CTT ATC-3¢ (forward) and 5¢-TCC TCA AAG
AGA TGG ACG-3¢ (Reverse); mmp-9 (28 cycles, 471 bp),
5¢-GTA TGG TCG TGG CTC TAA GC-3¢ (forward)
and 5¢-AAA ACC CTC TTG GTC TGC GG-3¢ (reverse);
b-actin (18 cycles, 555 bp) 5¢-CAT CGT GGG CCG
CTC TAG GCA CCA AG-3¢ (forward) and 5¢-GCA
CAG CTT CTC TTT GAT GTC ACG CAC-3¢ (reverse).
PCR thermal conditions used were: aSMase ⁄ smpd1 and
mmp-9,94°C for 30 s; annealing, 56 °C for 30 s;
extention, 72 °C for 30 s; b-actin, denature, 94 °C for

30 s; annealing, 62 °C for 30 s; extention, 72 °C for
30 s.
Western blot analysis
The active forms of MAPKs were detected by western
blotting as described previously [10,64]. Cells were lysed
with the Nuclear Extract kit according to the manufac-
turer’s protocol. Proteins in the cell lysate (20 lg) were
separated on SDS-containing 10% polyacrylamide gels
and transferred to Immobilon-P membranes using the
Bio-Rad western blot apparatus. After blocking with
20% blocking reagent N102 in Tris-buffered saline solu-
tion [20 mm Tris ⁄ HCl (pH 7.6), 137 mm NaCl] containing
0.05% Tween-20, the membrane was incubated with
primary antibody in the same buffer containing 10%
Blocking Regent N102. After sequential incubations
with biotin-conjugated secondary antibody and horserad-
ish peroxidase-conjugated avidin, the blots were incubated
with a chemiluminescent substrate using an Immuno-
star
TM
detection kit, and the signals were detected
with the LAS3000 imaging system (Fuji Film, Tokyo,
Japan).
Luciferase reporter assay
The PathoDetectÒ NF-jB cis-reporting system, an inducible
reporter vector containing the luciferase reporter gene
driven by a basic promoter element (TATA box) and the
cis-enhancer NF-jB, was used to measure NF-jB activity
[10]. An MMP-9 promoter luciferase reporter construct and
its mutant construct were used to measure MMP-9 promo-

ter activity [10,57]. These reporter vectors (1 lg ⁄ 35 mm
dish) were transfected into B16-BL6 cells with Trans-
fectin
TM
in six-well culture plates according to the manufac-
turer’s protocol, and transfection efficiency was monitored
by cotransfection of the Renilla luciferase reporter vector
(pRL-CMV) and a dual luciferase reporter assay kit.
Protein concentrations
Protein concentration was determined according to the
Bradford method, using the Bio-Rad protein assay kit and
bovine serum albumin as the standard.
Statistical analysis
The two-tailed Student’s t-test was used for statistical com-
parisons. A value of P < 0.05 was considered statistically
significant.
Acknowledgements
We thank Drs Charles A. Lambert, Pierre Mineur,
Agne
´
s Noe
¨
l, Francis Frankenne, and Jean-Michel
Foidart of the Universite
´
de Lie
`
ge, Belgium, for their
critical discussions. This work was supported in part
by the Grants-in-Aid for ‘High-Tech Research Center

Project’ from the Ministry of Education, Culture,
Sports, Science and Technology of Japan and for
Scientific Research (B) and (C)
7
from the Japan Society
for the Promotion of Science, Japan.
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