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 Kato1,2, Shigeyuki Ozawa1,3, Mamoru Tsukuda2, Eiro Kubota3, Kaoru Miyazaki4, Yves St-Pierre5 and Ryu-Ichiro Hata1 Department of Biochemistry and Molecular Biology, Kanagawa Dental College, Yokosuka, Japan Department of Biology and Function in the Head and Neck, Yokohama City University Graduate School of Medicine, Japan Department of Oral and Maxillofacial Surgery, Kanagawa Dental College, Yokosuka, Japan Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Japan ´ ´ ´ INRS-Institut Armand-Frappier, Universite du Quebec, Laval, Quebec, Canada Keywords acidic sphingomylinase; Ca2+ 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: yasumasa@kdcnet.ac.jp (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 metalloproteinase-9 expression through phospholipase D, extracellular signal regulated kinase ⁄ 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 Ca2+ influx and acidic sphingomyelinase in mouse B16 melanoma cells We found that BAPTAAM [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 Ca2+ channel blockers, mibefradil (for T-type) and nimodipine (for L-type), dose-dependently inhibited acidic extracellular pH-induced matrix metalloproteinase-9 expression Intracellular free calcium concentration ([Ca2+]i) was transiently elevated by acidic extracellular pH, and this [Ca2+]i elevation was repressed by EGTA and the voltage dependent Ca2+ channel blockers but not by phospholipase C inhibitor, suggesting that acidic extracellular pH increased [Ca2+]i through voltage dependent Ca2+ channel In contrast, SR33557, an L-type voltage dependent Ca2+ channel blocker and acidic sphingomyelinase inhibitor, attenuated 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 expression BAPTA-AM reduced acidic extracellular pH-induced phospholipase 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; [Ca2+]i, intracellular Ca2+ concentration; DAG, diacylglycerol; ERK, extracellular signal regulated kinase; IL, interleukin; IP3, inositol 1,4,5-triphosphate; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; NF-jB, nuclear factor-jB; nSMase, neutral sphingomyelinase; PC, phosphatidylcholine; pHe, 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 Ca2+ channel; VEGF, vascular endothelial growth factor FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3171 aSMase and Ca2+ influx in acid induction of MMP-9 Y Kato et al sphingomyelinase inhibitors did not affect the phosphorylation of extracellular signal regulated kinase ⁄ 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 extracellular pH induced matrix metalloproteinase-9 expression, at least in part, through nuclear factor-jB activation Acidic extracellular pH (pHe) has been frequently observed in solid tumors, due to excess amounts of anaerobic glucose metabolites Acidic pHe has been reported to affect the efficacy of chemotherapy, including reducing the cytotoxicity of bleomycin, doxorubicin, daunorubicin, epirubicin, mitoxantrone, and vinca alkaloids, but potentiating 5-fluorouracil [1] A recent study demonstrated that acidic pHe is a predictor of metastasis-free survival in canine soft tissue sarcomas treated with thermoradiotherapy [2] The acidic microenvironment may also regulate tumor angiogenesis using a signal pathway different from that of hypoxia [3–7] Hypoxia was recently reported to affect expression of matrix metalloproteinases (MMPs) [8], which are important in inflammation, tumor invasion, and metastasis We have reported that acidic pHe induced the expression 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 pHe-induced MMP-9 expression was mediated via the phospholipase D (PLD)–mitogen-activated protein kinase (MAPK) [extracellular signal regulated kinase (ERK)1 ⁄ and p38] pathway, at least in part through acidic pHe signaling through nuclear factor-jB (NF-jB) [10] Acidic pHe has been shown to increase intracellular Ca2+ concentration ([Ca2+]i) in fibroblasts [11], endothelial cells [12], and smooth muscle cells [13–15] In addition, increased [Ca2+]i has been found to activate PLD [16,17], which is also involved in the acidic pHe induction of MMP-9 expression [10] [Ca2+]i elevation can be divided into major two pathways: Ca2+ influx through specific channels and release of Ca2+ from the endoplasmic reticulum by inositol 1,4,5-triphosphate (IP3), a product of phospholipase C (PLC) Voltage dependent Ca2+ 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 expression of acidic sphingomyelinase (aSMase) ⁄ acid lysosomal sphingomyelin phosphodiesterase (smpd1) in the signal transduction pathways of interleukin (IL)-1 and tumor necrosis factor a (TNF-a) [18,19] 3172 MMP-9 can be up-regulated by various stimuli, including IL-1 and TNF-a, which trigger the ceramidesignaling 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 kinase (JNK), and p38, in various cell types [22–25] Moreover, ceramide can induce MMP expression [26,27] Here, we report that PLD, which is activated by Ca2+ influx and aSMase, mediates the acidic pHe induction of MMP-9, at least in part through NF-jB activation Results Acidic pHe increases Ca2+ influx through VDCC Increased [Ca2+]i has been shown to activate PLD [16,17] and acidic pHe has been shown to elevate [Ca2+]i in fibroblasts [11], endothelial cells [12], and smooth muscle cells [13–15] To determine the involvement of [Ca2+]i, in acidic pHe signaling, we treated cells with the calcium chelator BAPTA-AM [1,2bis(2-aminophenoxy)-ethane-N,N,N1,N1-tetraacetic acid tetrakis (acetoxymethyl) ester] We found that BAPTA-AM dose-dependently attenuated the acidic pHe-induced MMP-9 expression with an IC50 of 5.1 lm (Fig 1A) When we tested the effects of VDCC blockers on acidic pHe-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 pHe-induced MMP-9 expression, with an IC50 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 measure [Ca2+]i, we observed a transient increase in [Ca2+]i in the presence, but not in the absence, of extracellular Ca2+ (Fig 2A) The calcium chelator, EGTA, but not the broad PLC inhibitor U73122, attenuated the acidic pHe-induced transient increase in [Ca2+]i, suggesting that [Ca2+]i is increased by Ca2+ entry not by inositol 1,4,5-triphosphate (IP3)-induced Ca2+ release from the endoplasmic reticulum (Fig 2B) Mibefradil and FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS aSMase and Ca2+ influx in acid induction of MMP-9 Y Kato et al A A B B C Fig Intracellular Ca2+ chelator and VDCC blockers reduce acidic pHe-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 nimodipine 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 reduction was seen with P-values less than 0.01 for SR33557, mibefradil nimodipine and 0.001 for BAPTA-AM Molecular markers are indicated in kDa Arrowheads indicate pro-MMP-9 nimodipine prevented acidic pHe-induced Ca2+ influx (Fig 2B), suggesting that Ca2+ influx, which occurred through T-type and L-type VDCCs, triggered acidic pHe-induced MMP-9 expression SR33557 (25 lm) did not affect acidic pHe-induced Ca2+ influx (Fig 2B) but suppressed MMP-9 expression (Fig 1B), suggesting that aSMase may be involved in acidic pHe signaling Fig [Ca2+]i is increased through VDCC but not from the endoplasmic reticulum Cells (40 000) cells were incubated overnight with serum-containing growing medium and with serum-free medium (pH 7.3) for h and loaded with Fluo-4-AM (0.9 lM) in NaCl ⁄ Pi containing 0.495 mM MgCl2 for 30 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 mM Hepes, mM phosphoric acid, and 0.495 mM MgCl2] in the presence (open circle) or absence (closed circle) of 0.901 mM CaCl2 [Ca2+]i was measured at 490 nm excitation and 535 nm emission wavelengths, at 0.26 s intervals (B) Cells were treated with EGTA (5 mM), mibefradil (2.5 lM), nimodipine (5 lM), SR33557 (25 lM), and U73122 (50 lM) on [Ca2+]i for 15 and stimulated as above Bars indicate SD aSMase mediates acidic pHe-induced MMP-9 expression To investigate the involvement of aSMase in acidic pHe signaling, we tested the effects of the aSMase specific inhibitors perhexiline [30–32] and desipramine [32–35] Both dose-dependently inhibited acidic pHeinduced MMP-9 expression, with an IC50 of 0.5 lm FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3173 aSMase and Ca2+ influx in acid induction of MMP-9 Y Kato et al 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 pHe, as shown in Fig 3B, is mainly due to an increase in the mRNA level rather than its activation These data showed a significant contribution of aSMase in acidic pHe signaling to induce MMP-9 expression A B Chelation of [Ca2+]i inhibits acidic pHe-induced PLD activation and MMP-9 expression but not aSMase activation To determined the effect of [Ca2+]i elevation on PLD activity, cells were cultured with the [Ca2+]i chelater BAPTA-AM We found that this reagent dose-dependently reduced acidic pHe-induced PLD activity, but had no effect on aSMase activity (Fig 5), suggesting that acidic pHe triggers Ca2+ influx, which is followed by PLD activation independent of the aSMase pathway Fig aSMase mediates acidic pHe induction of MMP-9 expression Nearly confluent cells in a 24-well culture plate were serumstarved overnight and cultured for days in acidic medium (pH 5.9) in the presence of the indicated concentrations of aSMase inhibitors 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 X100 buffer, were incubated for 60 at 37 °C in 250 mM sodium acetate, mM EDTA (pH 5.0) for aSMase or 250 mM Tris ⁄ HCl (pH 7.4) for nSMase, each containing 0.05 lCi [choline methyl-14C]SM Radioactive phosphorylcholine was extracted with chloroform ⁄ 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 and 6.0 lm, respectively (Fig 3A) Incubation of cells at acidic pHe increased aSMase activity 2.0-fold but had no effect on nSMase activity (Fig 3B) When aSMase blockers were added to the cultures, they significantly inhibited acidic pHe-induced aSMase activity (Fig 3B), at concentrations sufficient to inhibit acidic pHe-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 oligonucleotide 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 3174 [Ca2+]i elevation by thapsigargin at neutral pHe mimics acidic pHe-induced PLD activation and MMP-9 expression If Ca2+ influx triggers PLD activation and this is followed by MMP-9 expression, we expected that we could mimic this effect at neutral pHe by increasing [Ca2+]i pharmacologically When the cells were cultured at neutral pHe with thapsigargin, a releaser of intracellular free Ca2+ from the endoplasmic reticulum, 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), suggesting 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, C2-ceramide, a cell permeable ceramide analogue, increased MMP-9 expression in the presence, but not in the absence, of thapsigargin at neutral pHe (Fig 7B), suggesting that both SM and PC (phosphatidylcholine) metabolites are important in acidic pHe induction of MMP-9 expression FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS aSMase and Ca2+ influx in acid induction of MMP-9 Y Kato et al A B Fig Knockdown of aSMase ⁄ smpd1 expression reduces acidic pHe-induced MMP-9 expression Cells, which have been transfected with siRNA oligonucleotide targeting aSMase ⁄ smpd1, were treated with acidic or neutral pHe (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 specific primer sets (B) Membrane fractions (50 lg), prepared using a 0.2% Triton X-100 buffer, were incubated for 60 at 37 °C in 250 mM sodium acetate, mM EDTA (pH 5.0) containing 0.05 lCi [choline methyl-14C]-SM Radioactive phosphorylcholine was extracted 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 lCiỈmL)1 [9,10-3H]-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 [3H]-ceramide formed was identified by comigration of N-palmitoyl-D-erythro-sphingosine The spots, which were identified as [3H]-ceramide, were scrapped off and the radioactivities were counted by liquid scintillation counting Bars indicate SD *P < 0.05; ***P < 0.001 (Student’s t-test) C Inhibition of aSMase activity has no effect on ERK1 ⁄ and p38 phosphorylations To assess the contribution of MAPKs to the downstream signaling of aSMase at acidic pHe, we measured the levels of the phosphorylated (active) forms of MAPKs in these cultures We previously showed that phosphorylation of ERK1 ⁄ and p38 MAPKs was significantly decreased by the PLD inhibitor (1-butanol), whereas the total amounts of ERK1 ⁄ and p38 MAPKs were not affected [10] We found that perhexiline and desipramine inhibition of aSMase did not affect the activation of ERK1 ⁄ and p38 MAPKs Fig [Ca2+]i chelation reduces acidic pHe-induced PLD but not aSMase activity Nearly confluent cells in a 60 mm culture dish were serum-starved overnight and cultured for days in acidic medium (pH 5.9), in the presence or absence of the indicated concentrations of BAPTA-AM The membrane fractions (50 lg) were prepared, and aSMase activity (closed column) was measured by incubation for 60 at 37 °C in 250 mM sodium acetate, mM EDTA (pH 5.0) containing 0.05 lCi [choline methyl-14C]-SM, followed by scintillation counting of the aqueous phase PLD activity (open column) of the membrane fractions was measured using an AmplexTM Red PLD assay kit and a fluorescence microplate reader, with an excitation wavelength of 535 nm and a detection wavelength of 590 nm Bars indicate SD (Fig 8) Similar findings were observed with the other aSMase inhibitor, SR33557 (data not shown) The JNK phosphorylation level was not affected by acidic pHe [10] and aSMase inhibitors had no effect on its basal phosphorylation level (data not shown) These data suggested that ERK1 ⁄ and p38 MAPKs were FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3175 aSMase and Ca2+ influx in acid induction of MMP-9 Y Kato et al Fig Thapsigargin increased [Ca2+]i induces PLD activity and MMP-9 expression at neutral pHe Nearly confluent cells were serum-starved and incubated with thapsigargin (Thap, 2.5 lM), TPA (80 nM) or vehicle at pHe 7.3 Gelatinolytic activity in CM was analyzed by zymography (inset) The cells were lysed with 0.2% Triton X-100, and the lysates were subjected to AmplexTM Red PLD assay Bars indicate SD *P < 0.05; ***P < 0.001 (Student’s t-test) NS, not significant Arrowhead indicates pro-MMP-9 Fig aSMase inhibitors not affect acidic pHe-induced phosphorylation of ERK1 ⁄ and p38 Nearly confluent cells were serum-starved and incubated with or without 10 lM desipramine hydrochloride (desipramine) or 10 lM perhexiline maleate (perhexiline) or at pHe 5.9 for 48 h The cells were lysed and MAPK phosphorylation was analyzed by western blotting using phosphospecific ERK1 ⁄ 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 pHe 7.3 p-ERK1 ⁄ 2, phosphorylated ERK1 ⁄ 2; p-p38, phosphorylated p38 A B Fig 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 aureus) (A) or 25 lM C2-ceramide (B) in the presence or absence of 2.5 lM thapsigargin at pHe 7.3 CM was collected, concentrated, and MMP-9 activity was assayed by zymography Arrowheads indicate pro-MMP-9 not downstream targets of aSMase in acidic pHe signaling Inhibition of aSMase activity attenuates acidic pHe-induced NF-jB and MMP-9 promoter activities The MAPK kinase inhibitor PD098059 and the p38 inhibitor SB203580 have been shown to inhibit acidic 3176 pHe-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 inhibition partially reduced acidic pHe-induced NF-jB activity (Fig 9B), suggesting that acidic pHe-induced NF-jB is coregulated by the Ca2+ ⁄ PLD ⁄ MAPK and aSMase pathways These cascades proposed were schematically 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 pHe and that this induction was attenuated by the aSMase inhibitors Although acidic pHe-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 Among the transcription factors known within the minimal MMP-9 promoter region, Ets1 and SP1 were potentially involved in the acidic pHe 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) The upstream signaling cascade leading to their activations (e.g MAPKs and aSMase) is currently under investigation FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS aSMase and Ca2+ influx in acid induction of MMP-9 Y Kato et al Discussion Fig aSMase inhibitors inhibit acidic pHe-induced NF-jB activity and MMP-9 promoter activity Cells cultured overnight with 10% fetal bovine serum in six-well plates were transfected with lg of mouse MMP-9 promoter-luciferase reporter construct (A) or PathoDetectÒ NF-jB-luciferase reporter construct (B) using TransfectinTM in serum-free DMEM ⁄ F12 at pHe 7.3 After 18 h, the cells were washed twice and cultured for 24 h with or without perhexiline maleate (perhexiline) or desipramine hydrochloride (desipramine) at pHe 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 construct); DNF-jB, pGL3MMP9DNF-jB (MMP-9 promoter construct mutated at the NF-jB binding site) **P < 0.05; ***P < 0.01 (Student’s t-test) Fig 10 Schematic representation of a proposed acidic pHe signaling to induce MMP-9 expression Acidic pHe, a common feature of solid tumors, is thought to decrease the efficacy of chemotherapy regimens [40–44] Angiogenesis-related gene expression was found to be induced by acidic pHe 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 glioblastoma [7] cells and IL-8 expression in human pancreatic 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 pHe (pHe 6.5–5.4) and that, among B16 clones, the rate of induction was correlated with metastatic potential [9] Most recently, acidic pHe 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 pHe induction of MMP-9 expression was mediated through the PLD–MAPK pathway [10] Here, we further examined whether increased [Ca2+]i and SM metabolism contributed to the acidic pHe 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 pHe-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 Ca2+ influx is essential for acidic pHe signaling but the majority of the VDCC type involved in this signaling is cell type specific NF-jB is a transcription factor responsible for MMP-9 expression [50] and can mediate acidic pHe signaling [10] Acidic pHe-induced activity of PLD, but not aSMase, was suppressed by chelating [Ca2+]i, suggesting that Ca2+ 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 4,5biphosphate-derived DAG through PLD followed by phosphatidate phosphatase Because U73122 had little effect on [Ca2+]i, IP3 is not likely to be involved in acidic pHe induced [Ca2+]i elevation PC, a metabolite of PC-PLC, decreased after pHe dropped and D609, an inhibitor of PC-PLC, did not dose-dependently inhibit acidic pHe-induced MMP-9 expression [10] Thus, FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3177 aSMase and Ca2+ influx in acid induction of MMP-9 Y Kato et al although the pathway involving PC-PLC may be ruled out, DAG derived from PC through PLD and phosphatidate phosphatase, but not from phosphatidylinositol 4,5-biphosphate through phosphatidylinositol specific PLC, may be involved in acidic pHe 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 pHe We have reported that acidic pHe increased ERK1 ⁄ 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 ⁄ 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-jB is a downstream target of aSMase, the signaling pathway connecting the two is still unclear One candidate mediator is protein kinase Cf (PKCf), because ceramide 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 pseudosubstrate can inhibit acidic pHe-induced MMP-9 expression (Y Kato, S Ozawa and R I Hata, unpub4 lished data) 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 pHe 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 normal pancreatic islets [54] but prevents cell death and even stimulates growth of pancreatic cancer cells [55] Although the pHe 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 CO2, regardless of pO2, 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 proliferating cells need more ribose 5-phosphate for DNA replication and RNA synthesis, thereby producing excess amounts of CO2 These observations suggest 3178 that extracellular acidity in tumors is partly regulated by an hypoxia-independent pathway Because tumor acidity affects the response radiation therapy and che5 motherapy, pharmacological blockade of VDCC may prevent tumor invasion and metastasis In conclusion, we found that two independent pathways; Ca2+–PLD–MAPKs (ERK1 ⁄ and p38) and aSMase, leading to NF-jB activation, are essential in acidic pHe induction of MMP-9 expression Experimental procedures Reagents SR33557 [([2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxyphenethyl) amino] propyloxy) benzenesulfonyl]) indolizine], an aSMase specific inhibitor, was kindly provided by Sanofi-Aventis (Paris, France) BAPTA-AM and N-palmitoyld-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); TransfectinTM and siLentFectTM 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-14C]-SM was obtained from Amersham Biosciences (Piscataway, NJ, USA); [9,10-3H]palmitic acid (50.0 CiỈmmol)1) was obtained from Moravec Biochemicals (Brea, CA, USA); Immobilon-P [poly(vinylidene 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 ImmunostarTM Western blotting detection kits, which included a chemiluminescent reagent and peroxidase-conjugated swine anti-rabbit IgG or goat antimouse 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 antibodies 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 F254 plate was obtained from Merck KGaA (Darmstadt, Germany) Vectors The PathoDetectÒ NF-jB cis-reporting system (pNF-jBLuc) was obtained from Stratagene (La Jolla, CA, USA) FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS aSMase and Ca2+ influx in acid induction of MMP-9 Y Kato et al 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 expression occurred from pHe 6.5–5.4 [9], we fixed the pH of the assay media at 5.9 for acidic pHe and at 7.3 for neutral pHe To prepare serum-free assay media (DMEM ⁄ F-12), a : mixture of DMEM and F-12 was supplemented with 15 mm Hepes and 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 siLentFectTM 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 medium (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 SDS7.5% polyacrylamide gels containing 0.1% gelatin The gels were washed in 2.5% Triton X-100 with gentle shaking for 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 CaCl2, and 0.002% NaN3] at 37 °C Gelatinolytic activity was visualized as a clear zone on a blue background following Coomassie Brilliant Blue R250 staining [Ca2+]i measurements Cells were inoculated at a density of 40 000 cells ⁄ well in 96-well culture plates Following overnight incubation, the cells were washed twice with Ca2+- and Mg2+-free Dulbecco’s phosphate-balanced saline (NaCl ⁄ Pi) and incubated in serum-free DMEM for h The cells were incubated with Fluo 4-AM (final concentration, 0.9 lm) in NaCl ⁄ Pi (pH 7.3) containing 0.901 mm CaCl2 and 0.495 mm MgCl2 for 30 at room temperature and washed four times with NaCl ⁄ Pi (pH 7.3) containing 0.495 mm MgCl2 The cells were overlain with NaCl ⁄ Pi (pH 5.9) supplemented with 15 mm Hepes, mm phosphoric acid, and 0.495 mm MgCl2 in the presence or absence of 0.901 mm CaCl2 The [Ca2+]i was measured at 490 nm excitation and 535 nm emission wavelengths, at 0.26 s intervals using Tecan GENiosProTM fluorescence plate reader (Grodig, Salzburg, Austria) ă Where indicated, cells were incubated for with calcium channel blockers dissolved in the NaCl ⁄ Pi (pH 7.3) containing 0.901 mm CaCl2 and 0.495 mm MgCl2, and the cells were overlain with channel blocker-containing NaCl ⁄ Pi (pH 5.9) supplemented with 15 mm Hepes, mm phosphoric acid, 0.495 mm MgCl2, and 0.901 mm CaCl2 PLD activiy Membrane fractions of the cells were prepared using 0.2% Triton X-100 PLD activity was detected using the AmplexTM Red PLD assay kit (Molecular Probes, Eugene, OR, USA) [10] Whole cell lysates were incubated with 250 lm PC, 100 mmL)1 Alcaligenes sp choline oxidase, mL)1 horseradish peroxidase, and 50 lm 10-acetyl-3,7dihydrophenoxazine (AmplexTM Red reagent) in reaction buffer consisting of 50 mm Tris ⁄ HCl (pH 8.0), mm CaCl2, 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 at 37 °C in 200 lL 250 mm sodium acetate, mm EDTA (pH 5.0) for aSMase or 200 lL 250 mm Tris ⁄ HCl (pH 7.4) for nSMase, each containing 0.05 lCi [choline methyl-14C]-SM Radioactive phosphorylcholine 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 was measured as described previously [62,63] Cells were inoculated into six-well FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3179 aSMase and Ca2+ influx in acid induction of MMP-9 Y Kato et al culture plate at a density of 2.5 · 105 cells ⁄ well Following overnight incubation, the cells were washed twice with NaCl ⁄ Pi and labelled with 1.5 lCi ⁄ well [9,10-3H]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 chloroform ⁄ methanol (2 : 1, v ⁄ v) Lipids in the chloroform phase were collected and analyzed by thin-layer chromatography using a Silica Gel60 F254 plate (20 · 20 cm) and ethyl acetate ⁄ acetic acid ⁄ 2,2,4-trimethypentane (9 : : 5) as a solvent The spots, which were identified as [3H]-ceramide by comigration of N-palmitoyl-d-erythro-sphingosine [C16:0 (palmitoyl) ceramide], were scrapped off and their radioactivities were counted by liquid scintillation counter 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 promoter activity [10,57] These reporter vectors (1 lg ⁄ 35 mm dish) were transfected into B16-BL6 cells with TransfectinTM in six-well culture plates according to the manufacturer’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 RT-PCR Total RNA was extracted by using TRIsolÒ 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 manufacturer’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 solution [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 horseradish peroxidase-conjugated avidin, the blots were incubated with a chemiluminescent substrate using an ImmunostarTM detection kit, and the signals were detected with the LAS3000 imaging system (Fuji Film, Tokyo, Japan) 3180 Protein 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Exp Metastasis 21, 419–425 FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3183 ... likely to be involved in acidic pHe induced [Ca2+]i elevation PC, a metabolite of PC-PLC, decreased after pHe dropped and D6 09, an inhibitor of PC-PLC, did not dose-dependently inhibit acidic pHe-induced... nuclear factor-jB activity These data suggest that the calcium in? ??ux-triggered phospholipase D and acidic sphingomyelinase pathways of acidic extracellular pH induced matrix metalloproteinase-9 expression, ... activity attenuates acidic pHe-induced NF-jB and MMP-9 promoter activities The MAPK kinase inhibitor PD098059 and the p38 inhibitor SB203580 have been shown to inhibit acidic 3176 pHe-induced