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Group IB Secretory Phospholipase A2 Promotes Matrix Metalloproteinase-2-mediated Cell Migration via the Phosphatidylinositol 3-Kinase and Akt Pathway*

  • Young-Ae Choi
    Affiliations
    Department of Biochemistry and Molecular Biology, Yeungnam University, Daegu 705-717

    Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, South Korea
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  • Hyung-Kyu Lim
    Affiliations
    Department of Biochemistry and Molecular Biology, Yeungnam University, Daegu 705-717
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  • Jae-Ryong Kim
    Affiliations
    Department of Biochemistry and Molecular Biology, Yeungnam University, Daegu 705-717
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  • Chu-Hee Lee
    Affiliations
    Department of Biochemistry and Molecular Biology, Yeungnam University, Daegu 705-717
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  • Young-Jo Kim
    Affiliations
    Department of Internal Medicine, College of Medicine, Yeungnam University, Daegu 705-717
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  • Shin-Sung Kang
    Affiliations
    Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, South Korea
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  • Suk-Hwan Baek
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, 317-1 Daemyung 5-Dong, Nam-Gu, Daegu 705-717, South Korea. Tel.: 82-53-620-3981; Fax: 82-53-623-8032;
    Affiliations
    Department of Biochemistry and Molecular Biology, Yeungnam University, Daegu 705-717
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  • Author Footnotes
    * This work was supported by Korea Research Foundation Grant KRF-2002-041-C00232. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:June 25, 2004DOI:https://doi.org/10.1074/jbc.M314235200
      Secretory phospholipase A2 (sPLA2), abundantly expressed in various cells including fibroblasts, is able to promote proliferation and migration. Degradation of collagenous extracellular matrix by matrix metalloproteinase (MMP) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, tumor invasion, and metastasis. Here we show that group IB PLA2 increased pro-MMP-2 activation in NIH3T3 fibroblasts. MMP-2 activity was stimulated by group IB PLA2 in a dose- and time-dependent manner. Consistent with MMP-2 activation, sPLA2 decreased expression of type IV collagen. These effects are due to the reduction of tissue inhibitor of metalloproteinase-2 (TIMP-2) and the activation of the membrane type1-MMP (MT1-MMP). The decrease of TIMP-2 levels in conditioned media and the increase of MT1-MMP levels in plasma membrane were observed. In addition, treatment of cells with decanoyl Arg-Val-Lys-Arg-chloromethyl ketone, an inhibitor of pro-MT1-MMP, suppressed sPLA2-mediated MMP-2 activation, whereas treatment with bafilomycin A1, an inhibitor of H+-ATPase, sustained MMP-2 activation by sPLA2. The involvement of phosphatidylinositol 3-kinase (PI3K) and Akt in the regulation of MMP-2 activity was further suggested by the findings that PI3K and Akt were phosphorylated by sPLA2. Expression of p85α and Akt mutants, or pretreatment of cells with LY294002, a PI3K inhibitor, attenuated sPLA2-induced MMP-2 activation and migration. Taken together, these results suggest that sPLA2 increases the pro-MMP-2 activation and migration of fibroblasts via the PI3K and Akt-dependent pathway. Because MMP-2 is an important factor directly involved in the control of cell migration and the turnover of extracellular matrix, our study may provide a mechanism for sPLA2-promoted fibroblasts migration.
      Controlled degradation of extracellular matrix (ECM)
      The abbreviations used are: ECM, extracellular matrix; PLA2, phospholipase A2; sPLA2, secretory phospholipase A2; sPLA2R, secretory phospholipase A2 receptor; MMP, matrix metalloproteinase; PI3K, phosphatidylinositol 3-kinase; TIMP-2, tissue inhibitor of metalloproteinase-2; MT1, membrane type 1; Ab, antibody; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; dec, decanoyl; cmk, chloromethyl ketone; ERK, extracellular signal-regulated kinase; DN, dominant negative; MAPK, mitogen-activated protein kinase.
      1The abbreviations used are: ECM, extracellular matrix; PLA2, phospholipase A2; sPLA2, secretory phospholipase A2; sPLA2R, secretory phospholipase A2 receptor; MMP, matrix metalloproteinase; PI3K, phosphatidylinositol 3-kinase; TIMP-2, tissue inhibitor of metalloproteinase-2; MT1, membrane type 1; Ab, antibody; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; dec, decanoyl; cmk, chloromethyl ketone; ERK, extracellular signal-regulated kinase; DN, dominant negative; MAPK, mitogen-activated protein kinase.
      is essential in physiologic situations involving connective tissue remodeling. Nevertheless, excessive breakdown of connective tissue plays an important role in pathogenesis, e.g. chronic inflammatory processes and malignancy (
      • Birkedal-Hansen H.
      ,
      • Chang C.
      • Werb Z.
      ). The regulation of matrix metalloproteinases (MMPs) occurs on different levels, including gene expression, processing of the inactive proenzymes, and by inhibition of the active enzymes by their endogenous inhibitors delineated as tissue inhibitors of metalloproteinases (TIMPs). MMPs are important breakdown enzymes of ECM components such as collagen. These enzymes consist of gelatinases, collagenases, stromelysins, and membrane-type MMPs. MMPs are synthesized as preproenzymes, and most of them are secreted from the cells as proenzymes. Among them, MMP-2 is thought to be one of the key enzymes for degrading collagen, which is a major component of basement membranes (
      • McCawley L.J.
      • Matrisian L.M.
      ,
      • Sternlicht M.D.
      • Werb Z.
      ). MMP-2 is abundantly expressed in various tissues and cell types including fibroblasts (
      • Brooks P.C.
      • Stromblad S.
      • Sanders L.C.
      • von Schalscha T.L.
      • Aimes R.T.
      • Stetler-Stevenson W.G.
      • Quigley J.P.
      • Cheresh D.A.
      ). Transient fibroblast activation is probably regulated by a variety of mediators produced by infiltrating platelets, monocytes, and other inflammatory cells (
      • Ihn H.
      • Tamaki K.J.
      ,
      • Ihn H.
      • Yamane K.
      • Asano Y.
      • Kubo M.
      • Tamaki K.
      ). Numerous in vitro and in vivo studies have suggested that some cytokines and growth factors regulate fibroblast proliferation and ECM deposition (
      • Ihn H.
      • Tamaki K.J.
      ,
      • Ihn H.
      • Yamane K.
      • Asano Y.
      • Kubo M.
      • Tamaki K.
      ,
      • Medrano E.E.
      ).
      Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond in phospholipids as they generate free fatty acids, such as arachidonic acid (
      • Balsinde J.
      • Winstead M.V.
      • Dennis E.A.
      ,
      • Murakami M.
      • Nakatani Y.
      • Atsumi G.
      • Inoue K.
      • Kudo I.
      ). Arachidonic acid is the key substrate for the synthesis of potent lipid mediators of inflammation. Several mammalian secretory PLA2s (sPLA2s) have been characterized and classified into different families (
      • Hurt-Camejo E.
      • Camejo G.
      • Peilot H.
      • Oorni K.
      • Kovanen P.
      ). At present, 12 distinct sPLA2s have been identified in mammals and classified into different groups, depending on their primary structures as characterized by the number and position of cysteine residues. Initially, sPLA2s were thought to carry out digestive functions. However, the recent reports of a class of high affinity cell surface PLA2 receptors and the delineation of novel receptor-mediated biological effects have changed this notion (
      • Lambeau G.
      • Lazdunski M.
      ,
      • Cupillard L.
      • Mulherkar R.
      • Gomez N.
      • Kadam S.
      • Valentin E.
      • Lazdunski M.
      • Lambeau G.
      ). PLA2 has been implicated in a wide range of cellular responses, including cell proliferation, signal transduction, host defense, chemokinesis, and membrane remodeling (
      • Kundu G.C.
      • Mukherjee A.B.
      ,
      • Yuan C.J.
      • Mandal A.K.
      • Zhang Z.
      • Mukherjee A.B.
      ,
      • Attiga F.A.
      • Fernandez P.M.
      • Weeraratna A.T.
      • Manyak M.J.
      • Patierno S.R.
      ). In addition to an extensive understanding of its action in phospholipid hydrolysis, a role for sPLA2 has been suggested in the remodeling of ECM, although little evidence for such involvement has yet been published (
      • Kundu G.C.
      • Mukherjee A.B.
      ). A previous study reported that porcine pancreatic PLA2 promotes cell migration and ECM invasion via its high affinity receptor (
      • Kundu G.C.
      • Mukherjee A.B.
      ). Attiga et al. (
      • Attiga F.A.
      • Fernandez P.M.
      • Weeraratna A.T.
      • Manyak M.J.
      • Patierno S.R.
      ) showed that the sPLA2 inhibitor p-bromophenacyl bromide could reduce the pro-MMP-2 and active MMP-2 from prostate cancer cells. All these results support the possible role of sPLA2 as a factor in MMP activation. However, the mechanism for this MMP-2 activation, including related TIMP in fibroblast, is not clear. Moreover, the intracellular signaling pathway that regulates MMP-2 expression has not been characterized.
      To investigate the role of group IB PLA2 on MMP-2 activation in fibroblasts, we established an MMP-2 activation profile by using the mouse fibroblast cell line NIH3T3. In this study, we found that group IB PLA2 increases the activity of MMP-2 in cell conditioned medium and that this increase is the result of a decreased level of TIMP-2 protein and an increased level of MT1-MMP activity. Moreover, the sPLA2 regulation of MMP-2 seems to be mediated by PI3K and Akt. Because group IB PLA2 is involved in promoting NIH3T3 migration, our results suggest that sPLA2-promoted cell migration is mediated by MMP-2.

      EXPERIMENTAL PROCEDURES

      Materials—Group IB PLA2, gelatin, and bafilomycin A1 were purchased from Sigma; the ECL reagent was from PerkinElmer Life Sciences; RPMI 1640, LipofectAMINE 2000, and Opti-MEM were from Invitrogen; the MMP-2 ELISA kit was from R & D Systems (Minneapolis, MN); FCS was from Hyclone (Logan, UT); mouse polyclonal MMP-2, TIMP-2, type IV collagen, p85α subunit antibodies, and the horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); the MT1-MMP antibody was from Chemicon (Temecula, CA); the phospho-Akt antibody was from New England Biolabs (Beverly, MA); the furin inhibitor decanoyl Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk) was from Alexis Biochemicals (San Diego, CA); and LY294002 was from Biomol (Plymouth Meeting, PA). The latter was dissolved in Me2SO prior to adding to the cell cultures. The final Me2SO concentrations were 0.1% or less, and these concentrations had no effect on cell viability.
      Cell Culture—The NIH3T3 mouse fibroblast cell line was obtained from the American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 2 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% FCS. The cells were grown at 37 °C in 5% CO2 fully humidified air and were subcultured twice weekly. They were then seeded in a 6-well plate at 3 × 105 cells/well and stimulated in the presence of group IB PLA2, with or without inhibitors, for various times ranging from a few minutes to 24 h.
      Zymogram Analysis—MMP-2 activity was determined by gelatin zymography using 0.1% gelatin as a substrate. The conditioned medium was mixed with SDS-PAGE sample buffer in the absence of reducing agent and electrophoresed in 10% polyacrylamide gel. After electrophoresis, the gels were washed three times with 2.5% Triton X-100 in water and then incubated overnight in a closed container at 37 °C in 0.2% Brij 35, 5 mm CaCl2, 1 mm NaCl, and 50 mm Tris, pH 7.4. The gels were stained for 30 min with 0.25% Coomassie Blue R-250 in 10% acetic acid and 45% methanol and then destained for 30 min using an aqueous mix of 20% acetic acid, 20% methanol, and 17% ethanol. Areas of protease activity appeared as clear bands.
      PI3K and Akt Activity Assay—NIH3T3 cells were seeded in 35-mm dishes and cultured overnight before they were serum-starved for 24 h and treated with or without group IB PLA2 for the indicated times. Whole-cell lysates were prepared in ice-cold lysis buffer containing 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 150 mm NaCl, 1% Triton X-100, 50 mm NaF, 1 μm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture. For PI3K activity, 0.5 mg of total protein was incubated with phosphotyrosine Ab (4G10) for 4 h and then with protein A-agarose for 2 h at 4 °C. After washing seven times in lysis buffer, the immunocomplexes were resolved by SDS-PAGE and transferred to nitrocellulose for subsequent immunoblot analysis by using p85 PI3K Ab. For Akt activity, immunoblotting was performed using Ab specific for the phosphorylated, activated forms of Akt.
      Transfection of Akt cDNA—Akt and K179M-Akt (DN-Akt) were subcloned into the mammalian expression vector pEGFP. Plasmid DNAs (pSRα) encoding wild type and deletion mutant of p85α (Δp85 α, deletion of 479–513) were generously provided by Dr. Wataru Ogawa at Kobe University, Japan. LipofectAMINE 2000 reagent was used to transfect PI3K or Akt cDNA into NIH3T3 cells, according to the manufacturer's instructions. One microgram of the plasmid was mixed with 3 μl of LipofectAMINE 2000 in 0.2 ml of Opti-MEM medium for 20 min and then added to the cells, which had grown to 40–50% confluency in the 6-well plate. After incubation for 6–18 h, the medium was replaced with fresh culture medium. To obtain stable transfectants, the cells transfected with the cDNA were cloned by serial dilution in a culture medium containing 750 μg/ml G418 in a 96-well plate. The subculturing was continued for 4 weeks, and then wells representing a single colony were selected, and the expression was confirmed by using its protein as determined by Western blot analysis.
      Preparation of Plasma Membranes—For preparation of crude plasma membranes, the cells were harvested and suspended in ice-cold phosphate-buffered saline containing protease inhibitor mixture, washed twice, and subjected to three cycles of freeze-thaw in dry ice/ethanol/37 °C baths. Lysates were sonicated for 3 s, and the nuclei and debris were removed from the homogenate by centrifugation at 1,000 × g at 4 °C. The resulting supernatant was centrifuged at 100,000 × g for 70 min at 4 °C. The membrane pellet was solubilized in buffer (10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 0.5% Triton X-100, and protease inhibitor mixture (Roche Diagnostics)) for 1 h at 4 °C. Insoluble materials were removed by centrifugation at 14,000 × g for 10 min and subjected to SDS-PAGE for MT1-MMP expression.
      Western Blot Analysis—NIH3T3 cells were treated with group IB PLA2 for the indicated times and doses. The cells were washed with cold phosphate-buffered saline, trypsinized, and pelleted at 700 × g at 4 °C. Cell pellets were resuspended in lysis buffer (50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 150 mm NaCl, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture), and the preparation was cleared by centrifugation, and the supernatant was retained as a whole-cell lysate. The protein was separated by 8% reducing SDS-PAGE and immunoblotted in 20% methanol, 25 mm Tris, and 192 mm glycine to a nitrocellulose membrane, which was then blocked with 5% nonfat dry milk in TTBS (25 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 0.2% Tween 20) and subsequently incubated with the indicated antibodies for 4 h. The membrane was then washed and incubated for 1 h with secondary antibodies conjugated to horseradish peroxidase, rewashed, and developed using an ECL system.
      Cell Migration—The effect of sPLA2 on cell migration was assessed by using a modified cell dispersion assay (
      • Gilles C.
      • Polette M.
      • Coraux C.
      • Tournier J.-M.
      • Meneguzzi G.
      • Munaut C.
      • Volders L.
      • Rousselle P.
      • Birembaut P.
      • Foidart J.-M.
      ). NIH3T3 cells were seeded and cultured in a 6-well plate containing a 6-mm glass ring. The cells were washed twice with serum-free media and serum-starved for an additional 12 h. After serum starvation, glass ring was removed, and the media were then switched to Dulbecco's modified Eagle's medium containing group IB PLA2. In selected experiments, the sPLA2 dependence of migration was determined by adding the PI3K inhibitor LY294002. To quantify the relative motility, the migratory front was photographed every 12 h for the indicated times, and the percentage of cells crossing a line designated “migration (%)” was enumerated.

      RESULTS

      Activation of MMP-2 by Group IB PLA2 in NIH3T3 Fibroblast—Initially, we wanted to determine the effect of group IB PLA2 on MMP induction and activation in fibroblasts. MMP activity was measured in the conditioned medium by using standard zymography. To investigate the effects of sPLA2 on the induction of MMP-2 release and activation, time kinetic analysis was performed with mouse fibroblast NIH3T3 cells. The results indicated a significant response to the treatment at 6, 12, and 24 h (Fig. 1A). sPLA2 stimulated MMP-2 activity in a dose-dependent manner by zymography and Western blot analysis (Fig. 1B). Therefore, further analysis was performed with supernatants at 24 h of culture. We further examined the total protein levels of MMP-2 and found that there was no dramatic change of MMP-2 protein by ELISA (Fig. 1C). An induction of the unknown band (85 kDa) was also detected by zymography in these experiments. However, MMP-9 was not detected under these experimental conditions, and therefore, no further studies on MMP-9 were conducted. To determine whether the induced MMP-2 activity was associated with collagen expression, Western blots were performed by using an antibody against type IV collagen. The collagen expression was carried out by treating the NIH3T3 cells with sPLA2. Fig. 1B shows the dose response of collagen expression as a function of cell treatment with varying concentrations of sPLA2. The decrease of collagen expression by sPLA2 is clearly dose-dependent and almost completely disappeared at an sPLA2 concentration of 50 nm.
      Figure thumbnail gr1
      Fig. 1Group IB PLA2 stimulates NIH3T3 fibroblast MMP-2 activity. A, NIH3T3 cells were plated on 6-well plates in medium containing 10% FCS. Following overnight serum starvation, cells were treated with 100 nm sPLA2. The conditioned media were collected at the indicated times and analyzed for MMP activity and type IV collagen by gelatin zymography (zymo) and Western blot (WB), respectively. B and C, the cells were starved overnight and treated with sPLA2 at the indicated concentrations. The conditioned media were collected at 24 h and analyzed for MMP activity by gelatin zymography, for MMP-2 (2 μg/ml antibody) and collagen expression by Western blot, and for total MMP-2 amounts by ELISA. The values for the MMP-2 production are represented as average ± S.D. The Western blot and zymography data shown are representative of four independent experiments.
      Involvement of TIMP-2 and MT1-MMP in the sPLA2-induced MMP-2 Activity—We speculated that the sPLA2-mediated MMP-2 activation in cells may be through the regulation of TIMP and MT-MMP. The latter was initially considered, because it has been demonstrated that TIMP-2 can form a complex with pro-MMP-2 through their carboxyl terminus (
      • Toth M.
      • Bernardo M.M.
      • Gervasi D.C.
      • Soloway P.D.
      • Wang Z.
      • Bigg H.F.
      • Overall C.M.
      • DeClerck Y.A.
      • Tschesche H.
      • Cher M.L.
      • Brown S.
      • Mobashery S.
      • Fridman R.
      ). To test this hypothesis, we measured sPLA2-stimulated TIMP-2 expression. The conditioned medium from NIH3T3 cells stimulated by sPLA2 was resolved by Western blot with monoclonal antibody against TIMP-2. As expected, TIMP-2 protein was strongly expressed at basal states, but this protein level was dramatically decreased by sPLA2 in a dose-dependent manner (Fig. 2A). We confirmed the effect of TIMP-2 on the activation of pro-MMP-2 by sPLA2. Cells were incubated with sPLA2 and recombinant TIMP-2 protein. Activation of pro-MMP-2 by sPLA2 was not enhanced in the presence of TIMP-2 at concentrations greater than 5 nm (Fig. 2B). MT1-MMP is a transmembrane MMP known to bind and activate pro-MMP-2 at the cell surface (
      • Seiki M.
      ). To determine the change of MT1-MMP activation in sPLA2 stimulation, we prepared plasma membrane fractions after treatment of sPLA2, and we performed Western blot analysis. The active form of MT1-MMP protein was increased by sPLA2 treatment in a dose-dependent manner (Fig. 3A). To investigate the involvement of MT1-MMP in sPLA2-dependent pro-MMP-2 activation, cells were treated with a furin inhibitor dec-RVKR-cmk, which has been shown to block activation of pro-MT1-MMP (
      • Seiki M.
      ,
      • Sato T.
      • Kondo T.
      • Seiki M.
      • Ito A.
      ,
      • Hess A.R.
      • Seftor E.A.
      • Seftor R.E.
      • Hendrix M.J.
      ). Treatment of cells with dec-RVKR-cmk inhibited sPLA2-mediated MMP-2 activation, implicating MT1-MMP in the sPLA2-induced pro-MMP-2 activation (Fig. 3B). A previous report (
      • Maquoi E.
      • Peyrollier K.
      • Noel A.
      • Foidart J.M.
      • Frankenne F.
      ) showed that the catalytic activity of MT1-MMP on the cell surface is regulated through a vacuolar H+-ATPase-dependent degradation process. To confirm whether or not the sPLA2-induced MMP-2 activation reflected enhanced MT1-MMP activation, the vacuolar H+-ATPase inhibitor bafilomycin A1 was utilized. Cells were cultured in sPLA2 for 24 h in various concentrations of bafilomycin A1, and the conditioned media were collected, concentrated, and evaluated for MMP-2 activity by zymography and Western blot. As shown in Fig. 3C, bafilomycin A1 potentiated the sPLA2-induced pro-MMP-2 activation.
      Figure thumbnail gr2
      Fig. 2sPLA2 decreases soluble TIMP-2 level. A, cells were cultured for 24 h in medium at the indicated sPLA2 concentrations. The conditioned media were collected, concentrated using Centricon-10, and Western blotted with anti-TIMP-2 antibody (5 μg/ml). B, the cells were cultured for 12 h with 50 nm sPLA2 in the presence or absence of recombinant (r) TIMP-2 protein. The conditioned media were collected and analyzed for MMP activity by gelatin zymography. The data shown are representative of three independent experiments.
      Figure thumbnail gr3
      Fig. 3sPLA2-induced pro-MMP-2 activation involves MT1-MMP. A, the cells were treated with the indicated concentrations of sPLA2 for 24 h. The crude plasma membranes were isolated from each sample and subjected to Western blot with MT1-MMP antibody (10 μg/ml). The data shown are representative of two independent experiments. B, the cells were plated on 6-well plates, subjected to overnight serum starvation, and treated with 100 nm sPLA2 with the indicated concentrations of dec-RVKR-cmk. C, the cells were serum-starved overnight and incubated for an additional 24 h with 100 nm sPLA2 with the indicated concentrations of bafilomycin A1. The conditioned media were collected at 24 h and analyzed for MMP activity by gelatin zymography (zymo). Western blot (WB) was performed with anti-MMP-2 antibody. The data shown are representative of three independent experiments.
      MMP-2 Activation by sPLA2 Is Mediated through the PI3K and Akt Pathway but Not the MAPK Pathway—It has been suggested that PI3K plays a role in pro-MMP activation and cell migration in various cells (
      • Okkenhaug K.
      • Vanhaesebroeck B.
      ). To investigate the role of PI3K/Akt in MMP-2 regulation, sPLA2-treated cells were analyzed for activation of PI3K and Akt. Quiescent cells were exposed to 100 nm sPLA2 for different periods (0–30 min), and protein extracts were analyzed either by immunoprecipitation with phosphotyrosine Ab for PI3K activity or by Western blot with phosphospecific Ab for Akt activity. Phosphorylation of PI3K by sPLA2 peaked at 5 min, whereas that of Akt began to occur within 3 min, peaked between 5 and 10 min, and then progressively declined to the basal level (Fig. 4, A and B). To gain insight into the mechanism of PI3K/Akt leading to pro-MMP-2 activation, cells were exposed to sPLA2 alone or in combination with the specific PI3K inhibitor LY294002. As shown in Fig. 5A, LY294002 dose-dependently suppressed the sPLA2-induced pro-MMP-2 activation. To confirm whether the sPLA2 signal was dependent on PI3K, cells were transfected with wild type p85α or the deletion mutant of p85α (Δp85α). NIH3T3 cells transfected with wild type p85α or Δp85α cDNA were exposed to sPLA2, and conditioned medium from these cells was assayed in order to measure MMP-2 activity. Overexpression of wild type p85α and Δp85α was checked by Western blot analysis. Overexpression of Δp85α resulted in significant attenuation of sPLA2 response. Moreover, the overexpression of wild type p85α resulted in an increase of MMP-2 activity by sPLA2 (Fig. 5B). Furthermore, we evaluated the effect of exogenous expression of wild type or DN-Akt on the MMP-2 activation by sPLA2. The overexpression of the DN-Akt was also shown to inhibit sPLA2-induced MMP-2 activation, whereas the overexpression of wild type Akt potentiated sPLA2-induced MMP-2 activation (Fig. 5C).
      Figure thumbnail gr4
      Fig. 4sPLA2 induced phosphorylation of PI3K and Akt. A, cells were treated with 100 nm sPLA2 for the indicated times. Total protein (0.5 mg) was immunoprecipitated (IP) with 4G10 antibody (10 μg/ml), followed by immunoblotting (IB) with anti-p85 antibody (1 μg/ml). Whole-cell extracts (30 μg) were subjected to Western blot analysis to determine total p85 protein levels. B, cells were treated with 100 nm sPLA2 for the indicated times. The lysates were analyzed by Western blot with antibodies to phospho-Akt and Akt. The two protein levels shown are representative of three independent experiments.
      Figure thumbnail gr5
      Fig. 5PI3K and Akt are linked to sPLA2-induced pro-MMP-2 activation. A, cells were serum-starved overnight and incubated for an additional 24 h with 100 nm sPLA2 with the indicated concentrations of LY294002. The conditioned media were collected at 24 h and analyzed for MMP activity by gelatin zymography. B and C, cells were transfected with a control vector (pSRα), wild type p85α, or Δp85α (B). Cells were transfected with a control vector (pEGFP), wild type Akt, or DN-Akt (C). Cells were stimulated with 100 nm sPLA2 for 24 h, and whole-cell lysates were obtained. Western blot analysis was performed by using p85 or Akt antibody. The conditioned media were collected at 24 h and analyzed for MMP activity by gelatin zymography. The data shown are representative of two independent experiments.
      Because the activation of extracellular signal-regulated kinase (ERK) is also central to sPLA2-induced cell proliferation, we were interested in defining the role of ERK in the stimulation of pro-MMP-2 activity. Quiescent cells were exposed to 100 nm sPLA2 for different times (3–30 min), and protein extracts were analyzed by Western blotting with phosphospecific antibody. sPLA2-stimulated phosphorylation of ERK1/2 occurred within 5 min, peaked between 10 and 20 min, and then progressively declined. In addition, sPLA2 stimulated phosphorylation of p38 over a similar period to the time kinetics of the ERK activation (Fig. 6A). To gain insight into the mechanism of sPLA2 signaling leading to pro-MMP-2 activation, cells were exposed to sPLA2 alone or in combination with each specific inhibitor. The results of zymography demonstrated that none of them had any effect on the sPLA2-induced MMP activation (Fig. 6B). These results suggested that sPLA2 acts through the PI3K/Akt pathway, rather than the MAPK pathway, to activate pro-MMP-2.
      Figure thumbnail gr6
      Fig. 6sPLA2-induced MMP-2 activation mediated independently of the MAPKs pathway. A, cells were treated with 100 nm sPLA2 for the indicated times. Cell lysates were analyzed by immunoblotting with anti-phospho-ERK1/2, anti-phospho-p38, or anti-phosphoc-Jun NH2-terminal kinase (pJNK) Ab to recognize specifically the phosphorylated of ERK1/2, p38, and c-Jun NH2-terminal kinase, respectively. B, the cells were serum-starved overnight and incubated for an additional 24 h with sPLA2 in the presence of PD98059 (25 μm), SB203580 (5 μm), or SP600125 (2.5 μm). The conditioned media were collected at 24 h and analyzed for MMP activity by gelatin zymography. The data shown are representative of three independent experiments.
      sPLA2 Stimulates Cell Migration via a PI3K- and Akt-dependent Pathway—A critical response of skin fibroblasts during wound healing is their migration into regions of repair and remodeling. For migration, fibroblasts must degrade the surrounding basement membrane, rich in type IV collagen (
      • Ellerbroek S.M.
      • Halbleib J.M.
      • Benavidez M.
      • Warmka J.K.
      • Wattenberg E.V.
      • Stack M.S.
      • Hudson L.G.
      ,
      • Mackay A.R.
      • Corbitt R.H.
      • Hartzler J.L.
      • Thorgeirsson U.P.
      ), which is the principal substrate for MMP-2. To evaluate the functional consequences of sPLA2-induced MMP-2 activation, the effect of sPLA2 on NIH3T3 cell migration was examined. Migration was quantified by using an in vitro cell dispersion assay, in which cells are plated at high density in a hole made in the middle of a 6-well plate and allowed to migrate after sPLA2 treatment. Cell migration was greatly increased in a dose- and time-dependent manner (Fig. 7A). Previous results have suggested that active MMP-2 mediates cell migration (
      • Kundu G.C.
      • Mukherjee A.B.
      ). Thus far, our results have led us to speculate that PI3K and Akt are able to regulate the stimulation of migration through MMP-2, and we therefore sought to investigate the possibility that PI3K could mediate the stimulation of sPLA2-associated cell migration. To determine whether migration was PI3K and Akt-dependent, the PI3K-specific inhibitor LY294002 was added, which greatly reduced cell migration (Fig, 7B). To confirm whether the sPLA2-stimulated cell migration was dependent on PI3K and Akt, cells were transfected with wild type or dominant negative mutants of p85α and Akt. Overexpression of the Δp85α or DN-Akt was also shown to inhibit sPLA2-induced cell migration, whereas overexpression of wild type p85α or Akt potentiated sPLA2-induced cell migration (Fig. 7C). These data suggest that the PI3K and Akt pathway is able to regulate the cleavage of collagen through MMP-2 and ultimately affects fibroblast migration.
      Figure thumbnail gr7
      Fig. 7PI3K Akt mediates sPLA2-induced cell migration. A, cells were plated in medium containing 10% FCS, serum-starved for 24 h, and then placed inside a hole made in the middle of a 6-well plate. Cells were treated with the indicated concentrations of sPLA2 for the indicated times and then allowed to migrate. To quantify the relative motility, the migratory hole was photographed, and the percentage of cells crossing a line designated migration (%) was enumerated. B, the cells were serum-starved overnight, treated with 100 nm sPLA2 with the indicated concentrations of LY294002 (LY), and then allowed to migrate for 24 h. The relative motility was quantified in the same manner as in A. C, cells were transfected with wild type p85α and Δp85α or wild type Akt and DN-Akt. Cells were treated with 100 nm sPLA2 and then allowed to migrate. The data shown are representative of five independent experiments.

      DISCUSSION

      Group IB PLA2 has been identified as a mitogen for fibroblasts, smooth muscle cells, chondrocytes, and synovial cells (
      • Ortega N.
      • Werb Z.
      ). The growth-promoting effects of sPLA2 are observed at relatively low concentrations (1–20 nm) in a variety cells, including NIH3T3 cells (
      • Hanasaki K.
      • Arita H.
      ). In addition, a previous study showed that fibroblast cell proliferation and migration were stimulated by group IB PLA2 treatment (
      • Kundu G.C.
      • Mukherjee A.B.
      ,
      • Fuentes L.
      • Hernandez M.
      • Fernandez-Aviles F.J.
      • Crespo M.S.
      • Nieto M.L.
      ), and the authors suggested the role of sPLA2 in MMP regulation. However, the detailed biological significance of sPLA2 is not clear. In the present study, we demonstrated first that the enhanced production of the active form of MMP-2 after group IB PLA2 treatment is in agreement with the continued reduction of TIMP-2 protein, and second, we demonstrated that selective elevation of only the MMP-2 activity elevated zymogen secretion but not the increased pro-form of the enzyme. The observed lack of elevation in the MMP-2 total protein suggests that these molecules did not contribute to the synthetic processing of pro-MMP-2 during sPLA2 stimulation.
      PLA2-induced pro-MMP-2 activation was associated with an MT1-MMP and TIMP-2 combination. Our data showed that sPLA2 induced a decline of TIMP-2 levels in culture media. A similar phenomenon has been reported recently in other model systems. For example, phorbol 12-myristate 13-acetate or calcium-induced MMP-2 activation in HT1080 or SCC25 cells is coupled with TIMP-2 expression (
      • Munshi H.G.
      • Wu Y.I.
      • Ariztia E.V.
      • Stack M.S.
      ). We also found that MT1-MMP is required to trigger the activation of pro-MMP-2 by showing direct MT1-MMP activation or dec-RVKR-cmk and bafilomycin A1 effects. MT1-MMP activity is regulated by several mechanisms. Pro-MT1-MMP is activated by furin (
      • Yana I.
      • Weiss S.J.
      ,
      • Maquoi E.
      • Frankenne F.
      • Baramova E.
      • Munaut C.
      • Sounni N.E.
      • Remacle A.
      • Noel A.
      • Murphy G.
      • Foidart J.M.
      ). Mature MT1-MMP can be inhibited by all the TIMPs except TIMP-1. Although TIMP-2 is the major inhibitor of MT1-MMP (
      • Yana I.
      • Weiss S.J.
      ), it also participates in the activation of MMP-2 by mediating the binding of pro-MMP-2 to MT1-MMP through the formation of a ternary complex (
      • Hernandez-Barrantes S.
      • Toth M.
      • Bernardo M.M.
      • Yurkova M.
      • Gervasi D.C.
      • Raz Y.
      • Sang Q.A.
      • Fridman R.
      ,
      • Bernardo M.M.
      • Fridman R.
      ). It is well established that acidification of intracellular vacuolar compartments plays an important role in membrane trafficking, protein sorting, and degradation (
      • Forgac M.
      ). This acidification is caused by vacuolar H+-ATPase, which can be selectively inhibited by bafilomycin A1 (
      • Bowman E.J.
      • Siebers A.
      • Altendorf K.
      ). Bafilomycin A1 effectively promotes the sPLA2-induced pro-MMP-2 activation, suggesting that inhibition of vacuolar H+-ATPase prevents the degradation of MT1-MMP and increases its activity at the cell surface.
      Fibroblasts play an essential role in regulating the MMP-TIMP balance, and their primary function, to maintain tissue ECM integrity, is significantly altered in inflammation due to changed composition of growth factors and cytokines (
      • McKaig B.C.
      • McWilliams D.
      • Watson S.A.
      • Mahida Y.R.
      ,
      • Leonardi A.
      • Cortivo R.
      • Fregona I.
      • Plebani M.
      • Secchi A.G.
      • Abatangelo G.
      ,
      • Saad S.
      • Gottlieb D.J.
      • Bradstock K.F.
      • Overall C.M.
      • Bendall L.J.
      ). The sPLA2-stimulated increase of the MMP-2 activity, which was implicated in connective tissue degradation in some specific cells, suggests common signaling mechanisms and supports a catabolic effect for sPLA2. We found that sPLA2 increases the activation of pro-MMP-2 in NIH3T3 as well as in human fibroblasts. The present study also investigated whether the sPLA2-induced modulation of MMP-2 and TIMP-2 was the result of regulation of signaling molecules. In investigating a number of possible signaling transduction molecules that might mediate the activation of pro-MMP-2, it was found that sPLA2 activated the PI3K and Akt pathway in NIH3T3 cells. Moreover, specific blockage of PI3K and Akt activation resulted in the down-regulation of the MMP-2 activity induced by sPLA2. These results strongly suggest that the PI3K and Akt pathway is essential for sPLA2 signaling responses, including pro-MMP-2 activation. In contrast, MAPKs activities were not required for sPLA2-stimulated pro-MMP-2 activation.
      The sPLA2s are members of a large family of enzymes that are capable of producing biologically active compounds and signaling molecules from membrane lipids. Such phospholipase activity has been delineated in the production of eicosanoids and in the generation of other intracellular signaling molecules (
      • Balsinde J.
      • Winstead M.V.
      • Dennis E.A.
      ,
      • Murakami M.
      • Nakatani Y.
      • Atsumi G.
      • Inoue K.
      • Kudo I.
      ,
      • Balsinde J.
      • Balboa M.A.
      • Dennis E.A.
      ). However, several papers (
      • Lambeau G.
      • Lazdunski M.
      ,
      • Cupillard L.
      • Mulherkar R.
      • Gomez N.
      • Kadam S.
      • Valentin E.
      • Lazdunski M.
      • Lambeau G.
      ) have suggested that sPLA2 has a diverse range of functions unrelated to phospholipase activity, including cell proliferation, migration, and hormone release in certain cells. sPLA2 was responsible for the activation of pro-MMP-2 in fibroblasts. Similar evidence was detected in culture media from human fibroblasts and chondrocytes (data not shown). High levels of sPLA2 subtype, the so-called pancreatic-type sPLA2 IB, are also found in the non-digestive tissues, including during acute pancreatitis (
      • Murakami M.
      • Kudo I.
      ,
      • Beck S.
      • Lambeau G.
      • Scholz-Pedretti K.
      • Gelb M.H.
      • Janssen M.J.
      • Edwards S.H.
      • Wilton D.C.
      • Pfeilschifter J.
      • Kaszkin M.
      ), suggesting that group IB PLA2 may also contribute to the pathophysiological effects in such conditions. In rat mesangial cells, exogenously added group IB PLA2 can stimulate prostaglandin synthesis (
      • Kishino J.
      • Kawamoto K.
      • Ishizaki J.
      • Verheij H.M.
      • Ohara O.
      • Arita H.
      ,
      • Kishino J.
      • Ohara O.
      • Nomura K.
      • Kramer R.M.
      • Arita H.
      ). When exogenously added to other cell types, group IB PLA2 was also found to activate the expression of a number of pro-inflammatory genes, including COX-2 (
      • Yuan C.J.
      • Mandal A.K.
      • Zhang Z.
      • Mukherjee A.B.
      ,
      • Tohkin M.
      • Kishino J.
      • Ishizaki J.
      • Arita H.
      ), sphingomyelinase, and ceramidase (
      • Mandal A.K.
      • Zhang Z.
      • Chou J.Y.
      • Mukherjee A.B.
      ). This effect is thought to involve binding of sPLA2 to the M-type sPLA2R (
      • Kishino J.
      • Ohara O.
      • Nomura K.
      • Kramer R.M.
      • Arita H.
      ). The nature of the group IB PLA2 cellular target involved in these biological effects remains, however, to be clearly identified. NIH3T3 cells expressed M-type sPLA2R, suggesting that these cells could also be targets of sPLA2 (
      • Cupillard L.
      • Mulherkar R.
      • Gomez N.
      • Kadam S.
      • Valentin E.
      • Lazdunski M.
      • Lambeau G.
      ). To determine whether MMP-2 activity is required to induce a signal through sPLA2R, we examined the effect of M-type sPLA2R overexpression on sPLA2-induced activation of pro-MMP-2. Cells were transfected either by vector (pCIneo) alone as a control or by the full length of the mouse M-type sPLA2R construct. Reverse transcriptase-PCR detection by using specific primers against the M-type sPLA2R increased the specific band of ∼300 bp in the sPLA2R-transfected cells. However, gelatin zymography of conditioned medium showed that overexpression of M-type sPLA2R did not affect pro-MMP-2 activation by sPLA2 (data not shown). These results suggest that this receptor may not be involved and that other surface molecules may therefore play a major role.
      The purpose of this study was also to analyze the role of cell migration in the activation of pro-MMP-2 by exogenous sPLA2 in NIH3T3 cells. Our data demonstrated that migration is enhanced in sPLA2-stimulated cells and under conditions that down-regulate TIMP-2 expression. The effect of cell migration, which we observed, was both dose- and time-dependent. Secreted PLA2 from Indian cobra (Naja naja) venom induced migration of a gastric epithelial cell line (
      • Minami T.
      • Tojo H.
      • Zushi S.
      • Shinomura Y.
      • Matsuzawa Y.
      ), whereas pancreatic PLA2 induced migration of rat embryonic thoracic aorta smooth muscle cells (
      • Kanemasa T.
      • Hanasaki K.
      • Arita H.
      ). Moreover, N. naja and pancreatic PLA2 induced migration of vascular endothelial cells (
      • Rizzo M.T.
      • Nguyen E.
      • Aldo-Benson M.
      • Lambeau G.
      ). Although previous studies have demonstrated a role for group IB PLA2 as a positive regulator of cell motility, there is no direct evidence regarding the effect of sPLA2-induced MMP-2 activation on cell migration. Our data additionally link PI3K and Akt activities with MMP-2 activity and cell migration by demonstrating that inhibition of PI3K results in decreased cell migration. We hypothesize that the decrease in cell migration is a result of the diminution of MMP-2 activation.
      In summary, our findings implicate a physiologic role for sPLA2 as a mediator of fibroblast migration. The mechanisms by which sPLA2 influences cell movement appear to depend on MMP-2 activity. In addition, we identified the PI3K and Akt pathway as a key regulator of pro-MMP-2 activation, collagen cleavage, and ultimately cell migration. These results provide important clues into the regulatory mechanisms underlying fibroblast migration and specifically identify sPLA2 as a potential new target in a novel signaling cascade.

      Acknowledgments

      We thank Dr. Wataru Ogawa for providing plasmids p85α and mutant.

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