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

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Acidic extracellular pH increases calcium influx-triggeredphospholipase D activity along with acidicsphingomyelinase activation to induce matrixmetalloproteinase-9 expression in mouse metastaticmelanomaYasumasa Kato1,2, Shigeyuki Ozawa1,3, Mamoru Tsukuda2, Eiro Kubota3, Kaoru Miyazaki4,Yves St-Pierre5and Ryu-Ichiro Hata11 Department of Biochemistry and Molecular Biology, Kanagawa Dental College, Yokosuka, Japan2 Department of Biology and Function in the Head and Neck, Yokohama City University Graduate School of Medicine, Japan3 Department of Oral and Maxillofacial Surgery, Kanagawa Dental College, Yokosuka, Japan4 Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Japan5 INRS-Institut Armand-Frappier, Universite´du Que´bec, Laval, Que´bec, CanadaKeywordsacidic sphingomylinase; Ca2+influx;extracellular acidic pH; MMP-9CorrespondenceY. Kato, Department of Biochemistry andMolecular Biology, Kanagawa DentalCollege, Yokosuka 238-8580, JapanFax: +81 46 822 8839Tel: +81 46 822 8840E-mail: yasumasa@kdcnet.ac.jp(Received 23 January 2007, revised 23 April2007, accepted 27 April 2007)doi:10.1111/j.1742-4658.2007.05848.xAcidic extracellular pH is a common feature of tumor tissues. We havereported 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 nuclearfactor-jB. Here, we show that acidic extracellular pH signaling involvesboth pathways of phospholipase D triggered by Ca2+influx and acidicsphingomyelinase 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 thevoltage dependent Ca2+channel blockers, mibefradil (for T-type) andnimodipine (for L-type), dose-dependently inhibited acidic extracellularpH-induced matrix metalloproteinase-9 expression. Intracellular free cal-cium concentration ([Ca2+]i) was transiently elevated by acidic extracellularpH, and this [Ca2+]ielevation was repressed by EGTA and the voltagedependent Ca2+channel blockers but not by phospholipase C inhibitor,suggesting that acidic extracellular pH increased [Ca2+]ithrough voltagedependent Ca2+channel. In contrast, SR33557, an L-type voltage depend-ent Ca2+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 acidicextracellular pH and that the specific acidic sphingomyelinase inhibitors(perhexiline and desipramine) and siRNA targeting aSMase ⁄ smpd1 couldinhibit 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 acidicAbbreviationsaSMase, 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 3171Acidic extracellular pH (pHe) has been frequentlyobserved in solid tumors, due to excess amounts ofanaerobic glucose metabolites. Acidic pHehas beenreported to affect the efficacy of chemotherapy, inclu-ding reducing the cytotoxicity of bleomycin, doxorubi-cin, daunorubicin, epirubicin, mitoxantrone, and vincaalkaloids, but potentiating 5-fluorouracil [1]. A recentstudy demonstrated that acidic pHeis a predictor ofmetastasis-free survival in canine soft tissue sarcomastreated with thermoradiotherapy [2]. The acidic micro-environment may also regulate tumor angiogenesisusing a signal pathway different from that of hypoxia[3–7]. Hypoxia was recently reported to affect expres-sion of matrix metalloproteinases (MMPs) [8], whichare important in inflammation, tumor invasion, andmetastasis.We have reported that acidic pHeinduced the expres-sion of MMP-9 ⁄ gelatinase B (EC in highlymetastatic mouse B16 melanoma cell lines, while notaffecting the expression of MMP-2 ⁄ gelatinase A [9].We have also reported that acidic pHe-induced MMP-9expression was mediated via the phospholipase D(PLD)–mitogen-activated protein kinase (MAPK)[extracellular signal regulated kinase (ERK)1 ⁄ 2 andp38] pathway, at least in part through acidic pHesigna-ling through nuclear factor-jB (NF-jB) [10].Acidic pHehas been shown to increase intracellularCa2+concentration ([Ca2+]i) in fibroblasts [11], endo-thelial cells [12], and smooth muscle cells [13–15]. Inaddition, increased [Ca2+]ihas been found to activatePLD [16,17], which is also involved in the acidic pHeinduction of MMP-9 expression [10]. [Ca2+]ielevationcan be divided into major two pathways: Ca2+influxthrough specific channels and release of Ca2+from theendoplasmic reticulum by inositol 1,4,5-triphosphate(IP3), a product of phospholipase C (PLC). Voltagedependent Ca2+channels (VDCC) have been classifiedinto low (T-type) and high (L-type) voltage types,which can be blocked by mibefradil and nimodipine,respectively. SR33557, which is another type of theL-type VDCC blocker, can also inhibit mRNA expres-sion of acidic sphingomyelinase (aSMase) ⁄ acid lyso-somal sphingomyelin phosphodiesterase 1 (smpd1)inthe signal transduction pathways of interleukin (IL)-1and 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 generatedby the hydrolysis of sphingomyelin (SM) acts as a lipidsecond messenger for apoptotic signaling [21]. BothaSMase and neutral sphingomyelinase (nSMase) canactivate 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 Ca2+influx and aSMase, mediates the aci dic pHeinduction ofMMP-9, at least in part through NF-jB activation.ResultsAcidic pHeincreases Ca2+influx through VDCCIncreased [Ca2+]ihas been shown to activate PLD[16,17] and acidic pHehas been shown to elevate[Ca2+]iin fibroblasts [11], endothelial cells [12], andsmooth muscle cells [13–15]. To determine the involve-ment of [Ca2+]i, in acidic pHesignaling, we treatedcells with the calcium chelator BAPTA-AM [1,2-bis(2-aminophenoxy)-ethane-N,N,N1,N1-tetraacetic acidtetrakis (acetoxymethyl) ester]. We found thatBAPTA-AM dose-dependently attenuated the acidicpHe-induced MMP-9 expression with an IC50of5.1 lm (Fig. 1A). When we tested the effects of VDCCblockers on acidic pHe-induced MMP-9 expression, wefound that the L-type VDCC blockers SR33557 [28,29]and nimodipine and the T-type blocker mibefradildose-dependently inhibited acidic pHe-induced MMP-9expression, with an IC50of 13.7 lm, 3.0 lm, and1.0 lm, respectively (Fig. 1B,C). These agents at thesame concentrations showed neither cellular toxicitynor any other gelatinolytic activity.Using Fluo4-AM, a fluorescent probe used to meas-ure [Ca2+]i, we observed a transient increase in [Ca2+]iin the presence, but not in the absence, of extracellularCa2+(Fig. 2A). The calcium chelator, EGTA, but notthe broad PLC inhibitor U73122, attenuated the acidicpHe-induced transient increase in [Ca2+]i, suggestingthat [Ca2+]iis increased by Ca2+entry not by inositol1,4,5-triphosphate (IP3)-induced Ca2+release fromthe endoplasmic reticulum (Fig. 2B). Mibefradil andsphingomyelinase inhibitors did not affect the phosphorylation of extracel-lular signal regulated kinase 1 ⁄ 2 and p38, but they suppressed nuclearfactor-jB activity. These data suggest that the calcium influx-triggeredphospholipase 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 Ca2+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 FEBSnimodipine prevented acidic pHe-induced Ca2+influx(Fig. 2B), suggesting that Ca2+influx, which occurredthrough T-type and L-type VDCCs, triggered acidicpHe-induced MMP-9 expression. SR33557 (25 lm) didnot affect acidic pHe-induced Ca2+influx (Fig. 2B) butsuppressed MMP-9 expression (Fig. 1B), suggestingthat aSMase may be involved in acidic pHesignaling.aSMase mediates acidic pHe-induced MMP-9expressionTo investigate the involvement of aSMase in acidicpHesignaling, we tested the effects of the aSMasespecific inhibitors perhexiline [30–32] and desipramine[32–35]. Both dose-dependently inhibited acidic pHe-induced MMP-9 expression, with an IC50of 0.5 lmABFig. 2. [Ca2+]iis increased through VDCC but not from the endo-plasmic reticulum. Cells (40 000) cells were incubated overnightwith serum-containing growing medium and with serum-free med-ium (pH 7.3) for 4 h and loaded with Fluo-4-AM (0.9 lM) in NaCl ⁄ Picontaining 0.495 mM MgCl2for 30 min at room temperature. (A)After washing, the cells were simulated by overlaying an acidic pHbuffer [horizontal gray bar; NaCl ⁄ Pi (pH 5.9) supplemented with15 mM Hepes, 4 mM phosphoric acid, and 0.495 mM MgCl2] in thepresence (open circle) or absence (closed circle) of 0.901 mMCaCl2. [Ca2+]iwas measured at 490 nm excitation and 535 nmemission wavelengths, at 0.26 s intervals. (B) Cells were treatedwith EGTA (5 mM), mibefradil (2.5 lM), nimodipine (5 lM), SR33557(25 lM), and U73122 (50 lM) on [Ca2+]ifor 15 min and stimulatedas above. Bars indicate SD.ABCFig. 1. Intracellular Ca2+chelator and VDCC blockers reduce acidicpHe-induced MMP-9 expression. Nearly confluent cells in a 24-wellculture plate were serum-starved overnight and cultured with acidicmedium (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 representativeexperiment is shown accompanied with the induction rate, whichwas estimated by densitometry. Concentration dependent reduc-tion was seen with P-values less than 0.01 for SR33557, mibefradilnimodipine and 0.001 for BAPTA-AM. Molecular markers are indica-ted in kDa. Arrowheads indicate pro-MMP-9.Y. Kato et al. aSMase and Ca2+influx in acid induction of MMP-9FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3173and 6.0 lm, respectively (Fig. 3A). Incubation of cellsat acidic pHeincreased aSMase activity 2.0-fold buthad no effect on nSMase activity (Fig. 3B). WhenaSMase blockers were added to the cultures, they sig-nificantly inhibited acidic pHe-induced aSMase activity(Fig. 3B), at concentrations sufficient to inhibit acidicpHe-induced MMP-9 expression (Fig. 3A). To provethe contribution of aSMase in this signaling cascadeto induce MMP-9, small interfering RNA (siRNA)technology was used. Introduction of siRNA oligo-nucleotide targeting smpd-1 ⁄ aSMase mRNA reducedthe acid induction of MMP-9 expression (Fig. 4A)concomitantly with the decrease in smpd-1 ⁄ aSMasemRNA (Fig. 4A) and its activity (Fig. 4B) and alsoin vivo ceramide production that is a metabolite ofaSMase from SM (Fig. 4C). Interestingly, it was alsofound that acid induction of smpd-1 ⁄ aSMase mRNAexpression, suggesting that the elevation of aSMaseactivity by acidic pHe, as shown in Fig. 3B, is mainlydue to an increase in the mRNA level rather than itsactivation. These data showed a significant contribu-tion of aSMase in acidic pHesignaling to induceMMP-9 expression.Chelation of [Ca2+]iinhibits acidic pHe-inducedPLD activation and MMP-9 expression but notaSMase activationTo determined the effect of [Ca2+]ielevation on PLDactivity, cells were cultured with the [Ca2+]ichelaterBAPTA-AM. We found that this reagent dose-depend-ently reduced acidic pHe-induced PLD activity, buthad no effect on aSMase activity (Fig. 5), suggestingthat acidic pHetriggers Ca2+influx, which is followedby PLD activation independent of the aSMase path-way.[Ca2+]ielevation by thapsigargin at neutral pHemimics acidic pHe-induced PLD activation andMMP-9 expressionIf Ca2+influx triggers PLD activation and this is fol-lowed by MMP-9 expression, we expected that wecould mimic this effect at neutral pHeby increasing[Ca2+]ipharmacologically. When the cells were cul-tured at neutral pHewith thapsigargin, a releaser ofintracellular free Ca2+from the endoplasmic reticu-lum, PLD activity was increased and MMP-9 wasexpressed [36,37] (Fig. 6). 12-O-tetradecanoylphorbol13-acetate (TPA) did not induce MMP-9 expression inB16 melanoma cells [9,10,38], but did so, through PLDactivation, in HT1080 cells [39]. Here, we found thatTPA could not increase PLD activity (Fig. 6), suggest-ing a reason that TPA could not induce MMP-9expression in this model. Besides, acid induction ofMMP-9 expression was found without activation ofAP-1 [10], generally known as the responsible factorfor MMP-9 transcription which could be activated byTPA.In contrast, we found that exogenous addition ofSMase dose-dependently stimulated the level observedin the presence of thapsigargin (Fig. 7A). Similarly,C2-ceramide, a cell permeable ceramide analogue,increased MMP-9 expression in the presence, but notin the absence, of thapsigargin at neutral pHe(Fig. 7B), suggesting that both SM and PC (phosphat-idylcholine) metabolites are important in acidic pHeinduction of MMP-9 expression.ABFig. 3. aSMase mediates acidic pHeinduction 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 analyzedby 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 mM sodiumacetate, 1 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 chloro-form ⁄ methanol (2 : 1, v ⁄ v) and the radioactivities in the aqueousphase were determined by liquid scintillation counting. Closed andopen columns indicate aSMase and nSMase activities, respectively.Bars indicate SD.aSMase and Ca2+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 FEBSInhibition of aSMase activity has no effect onERK1⁄2 and p38 phosphorylationsTo assess the contribution of MAPKs to the down-stream signaling of aSMase at acidic pHe, we meas-ured the levels of the phosphorylated (active) forms ofMAPKs in these cultures. We previously showed thatphosphorylation of ERK1 ⁄ 2 and p38 MAPKs wassignificantly decreased by the PLD inhibitor (1-buta-nol), whereas the total amounts of ERK1 ⁄ 2 andp38 MAPKs were not affected [10]. We found thatperhexiline and desipramine inhibition of aSMase didnot affect the activation of ERK1⁄ 2 and p38 MAPKs(Fig. 8). Similar findings were observed with the otheraSMase inhibitor, SR33557 (data not shown). TheJNK phosphorylation level was not affected by acidicpHe[10] and aSMase inhibitors had no effect on itsbasal phosphorylation level (data not shown). Thesedata suggested that ERK1 ⁄ 2 and p38 MAPKs wereABCFig. 4. Knockdown of aSMase ⁄ smpd1 expression reduces acidicpHe-induced MMP-9 expression. Cells, which have been transfect-ed with siRNA oligonucleotide targeting aSMase ⁄ smpd1, weretreated with acidic or neutral pHe. (A) Proteins in CM were ethanolconcentrated, and gelatinolytic activity was detected by gelatinzymography. Total RNA was extracted and mmp-9, aSMase ⁄ smpd1and b-actin gene expressions were analyzed by RT-PCR using spe-cific primer sets. (B) Membrane fractions (50 lg), prepared using a0.2% Triton X-100 buffer, were incubated for 60 min at 37 °Cin250 mM sodium acetate, 1 mM EDTA (pH 5.0) containing 0.05 lCi[choline methyl-14C]-SM. Radioactive phosphorylcholine was extrac-ted with chloroform ⁄ methanol (2 : 1, v ⁄ v) and the radioactivities inthe aqueous phase were determined by liquid scintillation counting.(C) The aSMase siRNA-transfected cells were labelled with0.5 lCiÆmL)1[9,10-3H]-palmitic acid and then stimulated with acidicpH medium for 24 h. Lipids were extracted from the cells withchloroform ⁄ methanol and analyzed by thin layer chromatography.The [3H]-ceramide formed was identified by comigration of N-palmi-toyl-D-erythro-sphingosine. The spots, which were identified as[3H]-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. [Ca2+]ichelation reduces acidic pHe-induced PLD but notaSMase activity. Nearly confluent cells in a 60 mm culture dishwere serum-starved overnight and cultured for 2 days in acidicmedium (pH 5.9), in the presence or absence of the indicated con-centrations of BAPTA-AM. The membrane fractions (50 lg) wereprepared, and aSMase activity (closed column) was measured byincubation for 60 min at 37 °C in 250 mM sodium acetate, 1 mMEDTA (pH 5.0) containing 0.05 lCi [choline methyl-14C]-SM, fol-lowed by scintillation counting of the aqueous phase. PLD activity(open column) of the membrane fractions was measured using anAmplexTMRed 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 Ca2+influx in acid induction of MMP-9FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3175not downstream targets of aSMase in acidic pHesignaling.Inhibition of aSMase activity attenuates acidicpHe-induced NF-jB and MMP-9 promoteractivitiesThe MAPK kinase inhibitor PD098059 and the p38inhibitor SB203580 have been shown to inhibit acidicpHe-induced NF-jB activity [10]. We found that theaSMase inhibitors reduced wild-type MMP-9 promoteractivity, as well as altering NF-jB-mutant MMP-9promoter activity (Fig. 9A). Moreover, aSMase inhibi-tion partially reduced acidic pHe-induced NF-jB activ-ity (Fig. 9B), suggesting that acidic pHe-inducedNF-jB is coregulated by the Ca2+⁄ PLD ⁄ MAPK andaSMase pathways. These cascades proposed were sche-matically summarized in Fig. 10. We found, however,that a mutant MMP-9 promoter lacking the NF-jBbinding site (DNF-jB) still showed inducibility atacidic pHeand that this induction was attenuated bythe aSMase inhibitors. Although acidic pHe-inducedNF-jB activity was down-regulated by these inhibitorsat the same concentrations, this inhibition was onlypartial, suggesting that other transcription factor(s)may be the downstream target(s) of aSMase. Somecandidates were considered1. Among the transcriptionfactors known within the minimal MMP-9 promoterregion, Ets1 and SP1 were potentially involved in the aci-dic pHesignaling. Indeed, using transcription factor-decoyand siRNA technologies, we found that Ets1 and SP1were 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 [Ca2+]iinduces PLD activity andMMP-9 expression at neutral pHe. Nearly confluent cells wereserum-starved and incubated with thapsigargin (Thap, 2.5 lM), TPA(80 nM) or vehicle at pHe7.3. Gelatinolytic activity in CM was ana-lyzed by zymography (inset). The cells were lysed with 0.2% TritonX-100, and the lysates were subjected to AmplexTMRed PLDassay. Bars indicate SD. *P < 0.05; ***P < 0.001 (Student’s t-test).NS, not significant. Arrowhead indicates pro-MMP-9.ABFig. 7. SM hydrolysis contributes to MMP-9 expression. Nearlyconfluent cells were serum-starved and incubated for 48 h with theindicated concentrations of bacterial SMase (Staphylococcus aure-us) (A) or 25 lM C2-ceramide (B) in the presence or absence of2.5 lM thapsigargin at pHe7.3. 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 pHe-inducedphosphorylation of ERK1 ⁄ 2 and p38. Nearly confluent cells wereserum-starved and incubated with or without 10 lM desipraminehydrochloride (desipramine) or 10 lM perhexiline maleate (perhexi-line) or at pHe5.9 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 ofphosphorylated ratio was estimated by the densitometry andexpressed as the relative values for the ratio of vehicle control atpHe7.3. p-ERK1 ⁄ 2, phosphorylated ERK1 ⁄ 2; p-p38, phosphorylatedp38.aSMase and Ca2+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 FEBSDiscussionAcidic pHe, a common feature of solid tumors, isthought to decrease the efficacy of chemotherapy regi-mens [40–44]. Angiogenesis-related gene expressionwas found to be induced by aci dic pHethrough hypoxiaindependent pathways involving platelet-derivedendothelial cell growth factor ⁄ thymidine phosphorylasein human breast tumor cells [45], the inducible isoformof nitric oxide synthase in macrophages [46], vascularendothelial 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 carcinomacells [4]. In addition, we have reported that expressionof MMP-9 in mouse metastatic B16 melanoma cellswas induced by acidic pHe(pHe6.5–5.4) and that,among B16 clones, the rate of induction was correlatedwith metastatic potential [9]. Most recently, acidic pHewas reported to enhance the metastatic potential ofhuman melanoma cells, accompanied by elevation ofproteinases and proangiogenic factors such as MMP-9,MMP-2, cathepsin B, cathepsin L, vascular endothelialgrowth factor (VEGF)-A, and IL-8 [49]. We alsoreported that acidic pHeinduction of MMP-9 expres-sion was mediated through the PLD–MAPK pathway[10]. Here, we further examined whether increased[Ca2+]iand SM metabolism contributed to the acidicpHesignaling induction of MMP-9 expression. Thesecontributions were also investigated in human lungadenocarcinoma cell line A549. Perhexiline (aSMaseinhibitor) and nimodipine (L-type VDCC blocker)reduced acidic pHe-induced MMP-9 expression inA549 but mibefradile (T-type VDCC blocker) had noeffect on this induction (data not shown), suggestingthat the contribution of aSMase and Ca2+influx isessential for acidic pHesignaling but the majority ofthe VDCC type3involved in this signaling is cell typespecific.NF-jB is a transcription factor responsible forMMP-9 expression [50] and can mediate acidic pHesignaling [10]. Acidic pHe-induced activity of PLD, butnot aSMase, was suppressed by chelating [Ca2+]i, sug-gesting that Ca2+influx activated PLD, but notaSMase. It has been reported that aSMase activitycould be induced by PC-derived diacylglycerol (DAG)through PC-PLC but not by phosphatidylinositol 4,5-biphosphate-derived DAG through PLD followed byphosphatidate phosphatase. Because U73122 had littleeffect on [Ca2+]i,IP3is not likely to be involved in aci-dic pHeinduced [Ca2+]ielevation. PC, a metabolite ofPC-PLC, decreased after pHedropped and D609, aninhibitor of PC-PLC, did not dose-dependently inhibitacidic pHe-induced MMP-9 expression [10]. Thus,Fig. 10. Schematic representation of a proposed acidic pHesigna-ling to induce MMP-9 expression.Fig. 9. aSMase inhibitors inhibit acidic pHe-induced NF-jB activityand MMP-9 promoter activity. Cells cultured overnight with 10%fetal bovine serum in six-well plates were transfected with 1 lgof mouse MMP-9 promoter-luciferase reporter construct (A) orPathoDetectÒ NF-jB-luciferase reporter construct (B) usingTransfectinTMin serum-free DMEM ⁄ F12 at pHe7.3. After 18 h,the cells were washed twice and cultured for 24 h with orwithout perhexiline maleate (perhexiline) or desipramine hydro-chloride (desipramine) at pHe7.3 or 5.9. The cells were lysedand subjected to dual luciferase assay; and transfection efficiencywas normalized by cotransfecting a Renilla luciferase reporterconstruct. 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 Ca2+influx in acid induction of MMP-9FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3177although the pathway involving PC-PLC may be ruledout, DAG derived from PC through PLD andphosphatidate phosphatase, but not from phosphati-dylinositol 4,5-biphosphate through phosphatidyl-inositol specific PLC, may be involved in acidic pHesignaling.Because thapsigargin induced MMP-9 expressionalong with a 1.8-fold increase in PLD activity [16,36],the basal activity of aSMase may be sufficient, but thatof PLD may be defective, for induction of MMP-9expression at neutral pHe. We have reported that aci-dic pHeincreased ERK1 ⁄ 2 and p38, but not JNK,phosphorylation and that the former was attenuatedby 1-butanol, a PLD inhibitor [10]. ERK1 ⁄ 2, JNK andp38 are activated as downstream targets of nSMaseand induce MMP-1 expression in fibroblasts [26]. InB16-BL6 cells, however, aSMase inhibitors had littleeffect on the phosphorylation of ERK1 ⁄ 2 and p38.Because ceramide can be metabolized from SM byboth SMases, this difference may be cell type specific.Further studies are needed to clarify the role ofaSMase in each cell type.We have shown here that, although NF-jBisadownstream target of aSMase, the signaling pathwayconnecting the two is still unclear. One candidatemediator is protein kinase Cf (PKCf), because cera-mide is an activator of PKCf [51,52] and becausePKCf can directly phosphorylate the p65 (Ser311)subunit of NF-jB [53]. We found that a PKCf pseudo-substrate can inhibit acidic pHe-induced MMP-9expression (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 andbe selected for resistance to microenvironmental stresssuch as acidic pHemay be indicative of its moreaggressive phenotype, using an ‘apoptotic signal’. Thisconcept is supported by results showing that hypoxiainducible factor 1a, a key transcription factor forVEGF during angiogenesis, induces apoptosis in nor-mal pancreatic islets [54] but prevents cell death andeven stimulates growth of pancreatic cancer cells [55].Although the pHeof tumor tissues is acidic andanaerobic glucose metabolites are the major source ofacidity, tumor acidity was shown to be caused byexcess amounts of CO2, regardless of pO2, through thepentose phosphate pathway, in glycolysis-impaired(phosphoglucose isomerase-deficient) cells [56]. Thispathway provides cells with ribose 5-phosphate, whichis used to synthesize nucleic acids. Thus, highly prolif-erating cells need more ribose 5-phosphate for DNAreplication and RNA synthesis, thereby producingexcess amounts of CO2. These observations suggestthat extracellular acidity in tumors is partly regulatedby an hypoxia-independent pathway. Because tumoracidity affects the response radiation therapy and che-motherapy, pharmacological blockade of VDCC5mayprevent tumor invasion and metastasis.In conclusion, we found that two independent path-ways; Ca2+–PLD–MAPKs (ERK1 ⁄ 2 and p38) andaSMase, leading to NF-jB activation, are essential inacidic pHeinduction of MMP-9 expression.Experimental proceduresReagentsSR33557 [([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] werepurchased from Calbiochem (La Jolla, CA, USA), and fluo4-AM was obtained from Dojindo (Kumamoto, Japan).DMEM and Ham’s F-12 (F-12), and TRIzolÒ Regent wereobtained from Invitrogen (Carlsbad, CA, USA); Trans-fectinTMand siLentFectTMLipid Reagents were obtainedfrom Bio-Rad (Hercules, CA, USA); the Dual LuciferaseReporter Assay kit was obtained from Toyo Ink (Tokyo,Japan); Staphylococcus aureus SMase, perhexiline maleatesalt, and desipramine hydrochloride were obtained fromSigma (St. Louis, MO, USA); fetal bovine serum wasobtained from Cell Culture Technologies GmbH (Zurich,Switzerland); [choline methyl-14C]-SM was obtained fromAmersham Biosciences (Piscataway, NJ, USA); [9,10-3H]-palmitic acid (50.0 CiÆmmol)1) was obtained from MoravecBiochemicals (Brea, CA, USA); Immobilon-P [poly(vinylid-ene difluoride)] membrane was obtained from Millipore(Bedford, MA, USA); and the Nuclear Extract kit wasobtained from Active Motif (Carlsbad, CA, USA). EGTA,TPA and the ImmunostarTMWestern blotting detectionkits, which included a chemiluminescent reagent andperoxidase-conjugated swine anti-rabbit IgG or goat anti-mouse IgG, were obtained from Wako (Tokyo, Japan). Theblocking 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 F254plate was obtained from Merck KGaA(Darmstadt, Germany).VectorsThe PathoDetectÒ NF-jB cis-reporting system (pNF-jB-Luc) was obtained from Stratagene (La Jolla, CA, USA).aSMase and Ca2+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 FEBSThe MMP-9 promoter luciferase reporter construct and itsmutant construct of the NF-jB binding site have beendescribed previously [10,57]. The cytomegalovirus-drivenRenilla luciferase reporter vector (pRL-CMV, Promega,Madison, WI, USA) was used to monitor transfectionefficiency.Cells and cell cultureB16-BL6 cells were cultured in DMEM containing 15 mmHepes (pH 7.3) supplemented with heat-inactivated 10%fetal bovine serum. Because induction of MMP-9 expres-sion occurred from pHe6.5–5.4 [9], we fixed the pH ofthe assay media at 5.9 for acidic pHeand at 7.3 for neut-ral pHe. 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 andadjusted to pH 5.9 with HCl or to pH 7.3 with NaOH[9,10,38].SiRNA-mediated gene silencingTo suppress aSMase mRNA expression, siRNA technologywas used. Oligonucleotide (2 nm) targeting aSMase⁄ smpd1was transfected into cells with siLentFectTMLipid Reagentin a serum-free DMEM ⁄ F-12 at pH 7.3 and cultured for48 h. The transfectants were stimulated with acidic mediumfor 48 h. The scrambled siRNA were used for a control. Atthe end of incubation period, proteins in conditioned med-ium (CM) and total RNA were obtained for zymographyto detect MMP-9 activity and RT-PCR was used to detectmmp-9 gene expression.Preparation of concentrated CM for zymographyProteins in CM were concentrated by adding three volumesof ice-cold ethanol as described previously [10,58]. Thequantity of samples was normalized for zymography assaybased on the DNA contents of the cultures (1.5 lg DNA ⁄lane), as measured using bisbenzimide [59].Gelatin zymographyGelatinolytic activities in the CM were analyzed by gelatinzymography, as described previously [9,10,60,61]. Briefly,ethanol-precipitated proteins were electrophoresed in SDS-7.5% polyacrylamide gels containing 0.1% gelatin. The gelswere washed in 2.5% Triton X-100 with gentle shaking for1 h at room temperature to remove SDS and incubated for20 h in reaction buffer [50 mm Tris ⁄ HCl (pH 7.5), 100 mmNaCl, 10 mm CaCl2, and 0.002% NaN3]at37°C. Gela-tinolytic activity was visualized as a clear zone on ablue background following Coomassie Brilliant Blue R250staining.[Ca2+]imeasurementsCells were inoculated at a density of 40 000 cells ⁄ well in96-well culture p lates. Following overnight incubation, the cellswere washed twice with Ca2+- and Mg2+-free Dulbecco’sphosphate-balanced saline (NaCl ⁄ Pi) and incubated inserum-free DMEM for 4 h. The cells were incubated withFluo 4-AM (final concentration, 0.9 lm) in NaCl ⁄ Pi(pH 7.3) containing 0.901 mm CaCl2and 0.495 mm MgCl2for 30 min at room temperature and washed four times withNaCl ⁄ Pi (pH 7.3) containing 0.495 mm MgCl2. The cellswere overlain with NaCl ⁄ Pi (pH 5.9) supplemented with15 mm Hepes, 4 mm phosphoric acid, and 0.495 mm MgCl2in the presence or absence of 0.901 mm CaCl2. The [Ca2+]iwas measured at 490 nm excitation and 535 nm emissionwavelengths, at 0.26 s intervals using Tecan GENiosProTMfluorescence 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 CaCl2and 0.495 mm MgCl2, andthe cells were overlain with channel blocker-containingNaCl ⁄ Pi (pH 5.9) supplemented with 15 mm Hepes, 4 mmphosphoric acid, 0.495 mm MgCl2, and 0.901 mm CaCl2.PLD activiyMembrane fractions of the cells were prepared using 0.2%Triton X-100. PLD activity was detected using theAmplexTMRed PLD assay kit (Molecular Probes, Eugene,OR, USA) [10]. Whole cell lysates were incubated with250 lm PC, 100 mU Æ mL)1Alcaligenes sp. choline oxidase,1UÆmL)1horseradish peroxidase, and 50 lm 10-acetyl-3,7-dihydrophenoxazine (AmplexTMRed reagent) in reactionbuffer consisting of 50 m m Tris ⁄ HCl (pH 8.0), 5 mmCaCl2, and 0.2% Triton X-100. PLD activity was measuredwith a fluorescence microplate reader using an excitationwavelength of 535 nm and detection wavelength of 590 nm.SMase activitiesSMase 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 °Cin200lL250 mm sodium acetate, 1 mm EDTA (pH 5.0) for aSMaseor 200 lL 250 mm Tris ⁄ HCl (pH 7.4) for nSMase, each con-taining 0.05 lCi [choline methyl-14C]-SM. Radioactive phos-phorylcholine was extracted with 750 lL of chloroform ⁄methanol (2 : 1, v ⁄ v), and the radioactivity in the aqueousphase was determined by liquid scintillation counting.In vivo ceramide productionIn vivo ceramide production6was measured as describedpreviously [62,63]. Cells were inoculated into six-wellY. Kato et al. aSMase and Ca2+influx in acid induction of MMP-9FEBS Journal 274 (2007) 3171–3183 ª 2007 The Authors Journal compilation ª 2007 FEBS 3179culture plate at a density of 2.5 · 105cells ⁄ well. Follow-ing overnight incubation, the cells were washed twicewith NaCl ⁄ Pi and labelled with 1.5 lCi ⁄ well [9,10-3H]-palmitic acid in serum-free DMEM for 18 h. The cellswere 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 chloroformphase were collected and analyzed by thin-layer chroma-tography using a Silica Gel60 F254plate (20 · 20 cm) andethyl acetate ⁄ acetic acid ⁄ 2,2,4-trimethypentane (9 : 2 : 5)as a solvent. The spots, which were identified as [3H]-cer-amide by comigration of N-palmitoyl-d-erythro-sphingo-sine [C16:0 (palmitoyl) ceramide], were scrapped off andtheir radioactivities were counted by liquid scintillationcounter.RT-PCRTotal RNA was extracted by using TRIsol Ò Reagent,reverse-transcribed by MMLV super transcriptase, andamplified by Taq polymerase with specific primer sets:aSMase ⁄ smpd1 (26 cycles, 258 bp), 5¢-TTC CTG CCAGAG CTT ATC-3¢ (forward) and 5¢-TCC TCA AAGAGA 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 CCGCTC TAG GCA CCA AG-3¢ (forward) and 5¢-GCACAG CTT CTC TTT GAT GTC ACG CAC-3¢ (reverse).PCR thermal conditions used were: aSMase ⁄ smpd1 andmmp-9,94°C for 30 s; annealing, 56 °C for 30 s;extention, 72 °C for 30 s; b-actin, denature, 94 °C for30 s; annealing, 62 °C for 30 s; extention, 72 °C for30 s.Western blot analysisThe active forms of MAPKs were detected by westernblotting as described previously [10,64]. Cells were lysedwith the Nuclear Extract kit according to the manufac-turer’s protocol. Proteins in the cell lysate (20 lg) wereseparated on SDS-containing 10% polyacrylamide gelsand transferred to Immobilon-P membranes using theBio-Rad western blot apparatus. After blocking with20% blocking reagent N102 in Tris-buffered saline solu-tion [20 mm Tris ⁄ HCl (pH 7.6), 137 mm NaCl] containing0.05% Tween-20, the membrane was incubated withprimary antibody in the same buffer containing 10%Blocking Regent N102. After sequential incubationswith biotin-conjugated secondary antibody and horserad-ish peroxidase-conjugated avidin, the blots were incubatedwith a chemiluminescent substrate using an Immuno-starTMdetection kit, and the signals were detectedwith the LAS3000 imaging system (Fuji Film, Tokyo,Japan).Luciferase reporter assayThe PathoDetectÒ NF-jB cis-reporting system, an induciblereporter vector containing the luciferase reporter genedriven by a basic promoter element (TATA box) and thecis-enhancer NF-jB, was used to measure NF-jB activity[10]. An MMP-9 promoter luciferase reporter construct andits mutant construct were used to measure MMP-9 promo-ter activity [10,57]. These reporter vectors (1 lg ⁄ 35 mmdish) were transfected into B16-BL6 cells with Trans-fectinTMin six-well culture plates according to the manufac-turer’s protocol, and transfection efficiency was monitoredby cotransfection of the Renilla luciferase reporter vector(pRL-CMV) and a dual luciferase reporter assay kit.Protein concentrationsProtein concentration was determined according to theBradford method, using the Bio-Rad protein assay kit andbovine serum albumin as the standard.Statistical analysisThe two-tailed Student’s t-test was used for statistical com-parisons. A value of P < 0.05 was considered statisticallysignificant.AcknowledgementsWe thank Drs Charles A. Lambert, Pierre Mineur,Agne´s Noe¨l, Francis Frankenne, and Jean-MichelFoidart of the Universite´de Lie`ge, Belgium, for theircritical discussions. This work was supported in partby the Grants-in-Aid for ‘High-Tech Research CenterProject’ from the Ministry of Education, Culture,Sports, Science and Technology of Japan and forScientific Research (B) and (C)7from the Japan Societyfor the Promotion of Science, Japan.References1 Raghunand N & Gillies RJ (2001) pH and chemother-apy. Novartis Found Symp 240, 199–211; discussion265–198.2 Lora-Michiels M, Yu D, Sanders L, Poulson JM,Azuma C, Case B, Vujaskovic Z, Thrall DE, CharlesHC & Dewhirst MW (2006) Extracellular pH and P-31magnetic resonance spectroscopic variables are relatedto outcome in canine soft tissue sarcomas treatedwith thermoradiotherapy. Clin Cancer Res 12, 5733–5740.3 Shi Q, Abbruzzese JL, Huang S, Fidler IJ, Xiong Q &Xie K (1999) Constitutive and inducible interleukin 8expression by hypoxia and acidosis renders humanaSMase and Ca2+influx in acid induction of MMP-9 Y. 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Acidic extracellular pH increases calcium in ux-triggered phospholipase D activity along with acidic sphingomyelinase activation to induce matrix metalloproteinase-9. found that acidic sphingomyelinase activity was induced by acidic extracellular pH and that the specific acidic sphingomyelinase inhibitors(perhexiline and
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Xem thêm: 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, 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, 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