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SIKVAV, a Laminin α1-Derived Peptide, Interacts with Integrins and Increases Protease Activity of a Human Salivary Gland Adenoid Cystic Carcinoma Cell Line through the ERK 1/2 Signaling Pathway
2007; Elsevier BV; Volume: 171; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.051264
ISSN1525-2191
AutoresVanessa M. Freitas, Vanessa F. Vilas-Boas, Daniel C. Pimenta, Vânia Loureiro, María A. Juliano, Márcia Regina Dias de CARVALHO, João de Jesus Viana Pinheiro, Antônio Carlos Martins de Camargo, Anselmo Sigari Moriscot, Matthew P. Hoffman, Ruy Gastaldoni Jaeger,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoAdenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm. We studied the induction of protease activity by the laminin-derived peptide, SIKVAV, in cells (CAC2) derived from this neoplasm. Laminin α1 and matrix metalloproteinases (MMPs) 2 and 9 were immunolocalized in adenoid cystic carcinoma cells in vivo and in vitro. CAC2 cells cultured on SIKVAV showed a dose-dependent increase of MMP9 as detected by zymography and colocalization of α3 and α6 integrins. Small interfering RNA (siRNA) knockdown of integrin expression in CAC2 cells resulted in decreased adhesion to the peptide. SIKVAV affinity chromatography and immunoblot analysis showed that α3, α6, and β1 integrins were eluted from the SIKVAV column, which was confirmed by mass spectrometry and a solid-phase binding assay. Small interfering RNA experiments also showed that these integrins, through extracellular signal-regulated kinase (ERK) 1/2 signaling, regulate MMP secretion induced by SIKVAV in CAC2 cells. We propose that SIKVAV increases protease activity of a human salivary gland adenoid cystic carcinoma cell line through α3β1 and α6β1 integrins and the ERK 1/2 signaling pathway. Adenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm. We studied the induction of protease activity by the laminin-derived peptide, SIKVAV, in cells (CAC2) derived from this neoplasm. Laminin α1 and matrix metalloproteinases (MMPs) 2 and 9 were immunolocalized in adenoid cystic carcinoma cells in vivo and in vitro. CAC2 cells cultured on SIKVAV showed a dose-dependent increase of MMP9 as detected by zymography and colocalization of α3 and α6 integrins. Small interfering RNA (siRNA) knockdown of integrin expression in CAC2 cells resulted in decreased adhesion to the peptide. SIKVAV affinity chromatography and immunoblot analysis showed that α3, α6, and β1 integrins were eluted from the SIKVAV column, which was confirmed by mass spectrometry and a solid-phase binding assay. Small interfering RNA experiments also showed that these integrins, through extracellular signal-regulated kinase (ERK) 1/2 signaling, regulate MMP secretion induced by SIKVAV in CAC2 cells. We propose that SIKVAV increases protease activity of a human salivary gland adenoid cystic carcinoma cell line through α3β1 and α6β1 integrins and the ERK 1/2 signaling pathway. 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162: 821-829Crossref PubMed Scopus (106) Google Scholar This peptide, located at the carboxy-terminal portion of the laminin α1 chain is liberated during tumor invasion through basement membranes.18Faisal Khan KM Laurie GW McCaffrey TA Falcone DJ Exposure of cryptic domains in the alpha 1-chain of laminin-1 by elastase stimulates macrophages urokinase and matrix metalloproteinase-9 expression.J Biol Chem. 2002; 277: 13778-13786Crossref PubMed Scopus (48) Google Scholar, 28Shintani S Alcalde RE Matsumura T Terakado N Extracellular matrices expression in invasion area of adenoid cystic carcinoma of salivary glands.Cancer Lett. 1997; 116: 9-14Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar SIKVAV is involved in malignant transformation, cell proliferation, angiogenesis, and protease activity in a number of cell types. Because of its biological significance, this peptide has been proposed as a therapeutic agent.29Kasai S Ohga Y Mochizuki M Nishi N Kadoya Y Nomizu M Multifunctional peptide fibrils for biomedical materials.Biopolymers. 2004; 76: 27-33Crossref PubMed Scopus (69) Google Scholar, 30Almiñana N Grau-Oliete MR Reig F Rivera-Fillat MP In vitro effects of SIKVAV retro and retro-enantio analogues on tumor metastatic events.Peptides. 2004; 25: 251-259Crossref PubMed Scopus (8) Google Scholar, 31Almiñana N Alsina MA Espina M Reig F Synthesis and physicochemical study of the laminin active sequence: SIKVAV.J Colloid Interface Sci. 2003; 263: 432-440Crossref PubMed Scopus (12) Google Scholar, 32Hashimoto T Suzuki Y Tanihara M Kakimaru Y Suzuki K Development of alginate wound dressings linked with hybrid peptides derived from laminin and elastin.Biomaterials. 2004; 25: 1407-1414Crossref PubMed Scopus (148) Google Scholar, 33Tong YW Shoichet MS Enhancing the neuronal interaction on fluoropolymer surfaces with mixed peptides or spacer group linkers.Biomaterials. 2001; 22: 1029-1034Crossref PubMed Scopus (85) Google Scholar, 34Grant DS Zukowska Z Revascularization of ischemic tissues with SIKVAV and neuropeptide Y (NPY).Adv Exp Med Biol. 2000; 476: 139-154Crossref PubMed Scopus (16) Google Scholar In salivary gland neoplasms, we have demonstrated that SIKVAV regulates the morphology and protease activity of a cell line (CAC2) derived from human adenoid cystic carcinoma.11Freitas VM Jaeger RG The effect of laminin and its peptide SIKVAV on a human salivary gland adenoid cystic carcinoma cell line.Virchows Arch. 2002; 441: 569-576Crossref PubMed Scopus (19) Google Scholar, 12Freitas VM Scheremeta B Hoffman MP Jaeger RG Laminin-1 and SIKVAV a laminin-1-derived peptide, regulate the morphology and protease activity of a human salivary gland adenoid cystic carcinoma cell line.Oral Oncol. 2004; 40: 483-489Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Despite its biological relevance, the mechanisms regulating the bioactivity of SIKVAV are not fully understood.Here, we studied the regulatory mechanisms resulting in increased protease activity induced by SIKVAV in CAC2 cells. Cells were cultured on SIKVAV, and the secretion of matrix metalloproteinases was measured by zymography. We show that α3β1 and α6β1 integrins elute from SIKVAV affinity columns. Our experiments suggest that integrin signaling, through the extracellular signal-regulated kinase (ERK) pathway, regulates the increased protease activity in CAC2 cells cultured on SIKVAV.Materials and MethodsImmunolocalization of Laminin α1 and Matrix Metalloproteinases in Adenoid Cystic Carcinoma in VivoTen cases of adenoid cystic carcinoma were retrieved from the files of the Department of Oral Pathology, UNIMES, and the Department of Oral Pathology, School of Dentistry, University of Pará. Growth patterns were cribriform (six cases), tubular (two cases), and solid (two cases). Formalin-fixed paraffin-embedded tissues were studied by immunohistochemistry. Sections (3 μm) were stained by the EnVision method (Dako Corp., Carpinteria, CA). Sections mounted on 3-aminopropyltriethoxy-silane-coated slides (Sigma Chemical Corp., St. Louis, MO) were dewaxed in xylene and hydrated in graded ethanol. Endogenous peroxidase activity was inhibited with 3% H2O2 in methanol for 20 minutes. Antigen retrieval involved treating sections with 1% pepsin in 10 mmol/L HCl for 1 hour at 37°C. Sections were blocked with 1% bovine serum albumin (BSA; Sigma) in Tris-HCl. A goat polyclonal antibody to laminin α1 chain [C-20 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), kindly provided by Dr. Vilma R. Martins, Ludwig Institute for Cancer Research, São Paulo Branch, Brazil] was diluted 1:50 in Tris-HCl. Anti-matrix metalloproteinase (MMP) 2 monoclonal antibody (mAb; clone 42-5D11) was diluted 1:50, and anti-MMP9 mAb (clone 56-2A4) was diluted 1:25 (both from Calbiochem-Novabiochem Co., La Jolla, CA). Diaminobenzidine (Sigma) was used as the chromogen, and sections were counterstained with Mayer's hematoxylin (Sigma). A nonspecific serum served as negative control.Cell CultureThe CAC2 cell line was derived from a human adenoid cystic carcinoma and has been characterized previously.11Freitas VM Jaeger RG The effect of laminin and its peptide SIKVAV on a human salivary gland adenoid cystic carcinoma cell line.Virchows Arch. 2002; 441: 569-576Crossref PubMed Scopus (19) Google Scholar, 12Freitas VM Scheremeta B Hoffman MP Jaeger RG Laminin-1 and SIKVAV a laminin-1-derived peptide, regulate the morphology and protease activity of a human salivary gland adenoid cystic carcinoma cell line.Oral Oncol. 2004; 40: 483-489Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Cells were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Cultilab; Campinas, São Paulo, Brazil) and 1% antibiotic-antimycotic solution (Sigma). The cells were maintained in 25-cm2 flasks in a humidified atmosphere of 5% CO2 at 37°C.ImmunofluorescenceCells were fixed in 1% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes, blocked with 1% BSA in PBS, and stained with goat antibody against laminin α1 chain (1:50 in PBS; Chemicon, Temecula, CA) and an anti-goat Cy3-conjugated secondary antibody (Zymed-Invitrogen Co., Carlsbad, CA). To stain for MMPs, cells were fixed and permeabilized with 0.5% Triton X-100 (Sigma) in PBS for 15 minutes and incubated with mAbs to MMP2 and MMP9 diluted in PBS 1:50 revealed by an anti-mouse Cy3-conjugated secondary antibody (Zymed-Invitrogen). All incubations were performed for 60 minutes at room temperature, and Pro Long (Invitrogen-Molecular Probes, Eugene, OR) was used as a mounting medium. Nuclei were counterstained with either 4,6-diamidino-2-phenylindole or Sytox Green (Invitrogen-Molecular Probes). A nonspecific serum served as negative control.For immunofluorescence analysis of α3 and α6 integrins, CAC2 cells were cultured on SIKVAV and IVSKVA for 4 hours and subjected to the immunofluorescence protocol described by Hogervorst et al.35Hogervorst F Admiraal LG Niessen C Kuikman I Janssen H Daams H Sonnenberg A Biochemical characterization and tissue distribution of the A and B variants of the integrin α6 subunit.J Cell Biol. 1993; 121: 179-191Crossref PubMed Scopus (125) Google Scholar Cells were fixed in 1% paraformaldehyde in PBS for 10 minutes, rinsed, permeabilized with 0.5% Triton X-100 (Sigma) in PBS for 5 minutes, and blocked with 1% BSA for 30 minutes. Samples were then double-labeled with antibodies against α3 (mAb, clone P1B5; Chemicon) and α6 (rat mAb, clone GoH3; Chemicon) integrins diluted 1:100 in PBS for 1 hour. Secondary antibodies were anti-mouse Cy3 (Zymed-Invitrogen) and anti-rat FITC (Zymed-Invitrogen). Nuclei were counterstained with 4,6-diamidino-2-phenylindole and mounted in Pro Long.Zymography of SIKVAV-Treated CAC2 CellsCAC2 cells were cultured in six-multiwell plates coated with 100 μl of either SIKVAV or the scrambled control IVSKVA (50 μg/ml, 200 μg/ml, 500 μg/ml, and 1 mg/ml) diluted in Milli-Q water and allowed to evaporate overnight in a hood. CAC2 cells (1 × 104) were plated and allowed to adhere and spread for at least 8 hours. The adherent cells were washed three times with PBS, and the culture medium was replaced by serum-free medium for 24 hours. The presence of MMPs in the conditioned medium was assessed by zymography. The conditioned medium was collected, concentrated (Microcon 30K; Millipore Co., Bedford, MA), and resuspended in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis sample buffer (without β-mercaptoethanol). The remaining CAC2 cells were lysed, and the amount of protein was estimated by bicinchoninic acid assay (Pierce Biotechnology Inc., Rockford, IL). In zymograms, the volume of conditioned medium loaded per lane was standardized on the basis of protein content in the cell lysate. Samples were separated on 10% polyacrylamide gels containing 0.2% gelatin (Novex-Invitrogen). After electrophoresis, the gels were washed in 2.5% Triton X-100 for 30 minutes, equilibrated in 10 mmol/L Tris, pH 8.0, and incubated at 37°C in a development buffer containing 50 mmol/L Tris, pH 8.0, 5 mmol/L CaCl2, and 0.02% NaN3 for 16 to 24 hours. The gels were stained with 0.2% Coomassie blue R250 (Amersham Co., Arlington Heights, IL) and destained with acetic acid/methanol.The MMP activity was also inhibited with either 10 mmol/L ethylenediamine tetraacetic acid (EDTA) (calcium chelator; Sigma) or 5 mmol/L 1,10-phenanthroline (heavy metal chelator; Sigma) in the zymogram development buffer to confirm that the bands were MMPs. We also ran parallel polyacrylamide gels with CAC2 cell lysates and did Western blots for β-actin to confirm equal loading conditions.CAC2 cells treated with small interfering RNA (siRNA) to integrins and syndecan-1 were cultured in six-multiwell plates on SIKVAV or IVSKVA (scrambled control) diluted in serum-free medium (50 μg/ml). The MMP activity in the conditioned medium was analyzed by zymography as described above.CAC2 Cell Adhesion AssaysCell adhesion to SIKVAV was compared with laminin-111 and with IVSKVA. Adhesion assays were performed in 96-well round-bottomed plates. Wells were coated overnight at 4°C with 100 μl of SIKVAV (1 μg/well), laminin-111 (1 μg/well), or IVSKVA (1 μg/well). Wells were blocked with 3% BSA for 30 minutes at 37°C and rinsed in PBS with 0.1% BSA, and cells were incubated for 20 minutes at 37°C. Attached cells were fixed/stained for 10 minutes with 0.2% (w/v) crystal violet in 20% (v/v) methanol. After three washes with H2O, cells were dissolved in 10% (w/v) SDS, and the absorbance at 600 nm was measured. We analyzed the effects of EDTA (calcium chelator) on the adhesion of CAC2 cells to SIKVAV. Cells were preincubated with 1 mmol/L EDTA in PBS for 10 minutes before being added to cell adhesion assays.CAC2 cells treated with siRNA to α3, α6, β1 integrin subunits and syndecan-1 were used in adhesion assays to SIKVAV, laminin-111, type I collagen, and fibronectin. Adhesion assays were performed in 96-well round-bottomed plates. Wells were coated overnight at 4°C with 100 μl of SIKVAV (1 μg/well), laminin-111 (1 μg/well), type I collagen (1 μg/well), and fibronectin (1 μg/well). Adhesion assays were performed as described above. To assess the specificity of the α3 and α6 integrin knockdown on adhesion, we performed assays to substrates where adhesion is not dependent on these integrins, such as fibronectin and type I collagen. Furthermore, we also silenced a nonintegrin receptor, syndecan-1. CAC2 cells with reduced syndecan expression were submitted to the same adhesion assays described above.RNA InterferenceCAC2 cells were transfected with commercially available siRNA targeting α3 and β1 integrins and syndecan-1 (Santa Cruz Biotechnology) and α6 integrin (Invitrogen) following the manufacturer's instructions. One day before transfection, subconfluent CAC2 cells were cultured in Dulbecco's modified Eagle's medium, supplemented by 10% fetal bovine serum without antibiotic-antimycotic solution. Cells were incubated with a complex formed by the siRNA, transfection reagent (Lipofectamine 2000; Invitrogen), and transfection medium (Opti-MEM I; Invitrogen) for 30 hours at 37°C. A siRNA-scrambled sequence (Invitrogen and Santa Cruz proprietary target sequences) was used as negative control. The following oligonucleotide sequences for siRNA were used: α3 integrin (a pool of three mRNA strands), 5′-UUCACUGUGAUGUUCAUCCTTGGAUGAACAUCACAGUGAATT-3′, 5′-UUCAUGAAGACAUAGAUGGTTCCAUCUAUGUCUUCAUGAATT-3′, and 5′-UGAUAGAUGUACACUUUGCTTGCAAAGUGUACAUCUAUCATT-3′; α6 integrin, 5′-UAACCUGGAGGCAUAUCCCACUAGGTT-3′; β1 integrin (a pool of three mRNA strands), 5′-GAGAUGAGGUUCAAUUUGA-3′, 5′-GAUGAGGUUCAAUUUGAAA-3′, and 5′-GUACAGAUCCGAAGUUUCA-3′; and syndecan-1 (a pool of three mRNA strands), 5′-CGCAAAUUGUGGCUACU-AA-3′, 5′-AGCAGGACUUCACCUUUGA-3′, and 5′-CGUGGGGCUCAUCUUUGCU-3′. Transfection efficiency was confirmed by immunoblot and real-time quantitative polymerase chain reaction (PCR).Western BlotsImmunoblots for α3, α6, or β1 integrins and syndecan-1 were used to test the efficiency of RNA silencing in CAC2 cells. Samples were lysed in radioimmunoprecipitation assay buffer (150 mmol/L NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 50 mmol/L Tris, pH 8.0) with protease inhibitor cocktail (Sigma) and centrifuged (10,000 × g) for 10 minutes at 4°C. The supernatants were recovered, quantified (bicinchoninic acid kit; Pierce), and resuspended in Laemmli sample buffer containing 62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% mercaptoethanol, and 0.001% bromphenol blue. Equal amounts (20 μg) of cell lysates were electrophoresed in 4 to 12% polyacrylamide gradient gels. Proteins were transferred to a Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham Co.) and blocked in Tris-buffered saline with 2.5% nonfat milk overnight at 4°C. After one wash in Tris-buffered saline with 0.05% Tween 20, the membrane was probed with anti-α3 (rabbit polyclonal, 1:1000; Chemicon), anti-α6A (mouse monoclonal, clone 1A10, 1:1000; Chemicon), anti-β1 (rabbit polyclonal, 1:1000; Chemicon) integrin antibodies, and anti-syndecan-1 (mouse mAb, clone B-B4, 1:100; Chemicon). Primary antibodies were detected by horseradish peroxidase-conjugated secondary anti-bodies (1:10,000) and developed using an enhanced chemiluminescence (ECL) substrate (Amersham Co.).Real-Time PCRRNA was collected after siRNA treatment using the TRIzol reagent (Invitrogen). Reverse transcription of total RNA (1 μg) with oligo dT (500 μg/ml), 10 mmol/L of each dNTP, 5× first-strand buffer, 0.1 mol/L dithiothreitol, and 200 U of reverse transcriptase (Moloney murine leukemia virus; Promega, Madison, WI) was performed at 70°C for 10 minutes followed by 42°C for 60 minutes and 10 minutes at 95°C. Quantitative reverse transcriptase-PCR was quantified with the ABI Prism 5700 sequence detector (Applied Biosystems, Foster City, CA). The PCR reactions contained 40 to 160 ng/μl cDNA in 25 μl of SYBR Green PCR master mix (Invitrogen) and 50 to 900 nmol/L primers (forward and reverse). Cycling conditions were as follows: 50°C for 2 minutes and 95°C for 10 minutes, followed by 50 cycles of 15 seconds at 95°C and 60 seconds at 60°C. The primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA), with the following sequences: human α3 integrin,36Nagata M Fujita H Ida H Hoshina H Inoue T Seki Y Ohnishi M Ohyama T Shingaki S Kaji M Saku T Takagi R Identification of potential biomarkers of lymph node metastasis in oral squamous cell carcinoma by cDNA microarray analysis.Int J Cancer. 2003; 106: 683-689Crossref PubMed Scopus (136) Google Scholar 5′-TAAGAGGCAGAAGGCGGAGATG-3′ (forward) and 5′-CCTGTGGACTGTCGAGGCATAAC-3′ (reverse); human α6 integrin,37Stossi F Barnett DH Frasor J Komm B Lyt
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