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Bone sialoprotein does not interact with pro-gelatinase A (MMP-2) or mediate MMP-2 activation Hwang, Queena; Cheifetz, Sela; Overall, Christopher M; McCulloch, Christopher A; Sodek, Jaro Apr 22, 2009

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ralssBioMed CentBMC CancerOpen AcceResearch articleBone sialoprotein does not interact with pro-gelatinase A (MMP-2) or mediate MMP-2 activationQueena Hwang1, Sela Cheifetz†1, Christopher M Overall2, Christopher A McCulloch*1 and Jaro Sodek†1Address: 1CIHR Group in Matrix Dynamics, University of Toronto, Toronto, Canada and 2CIHR Group in Matrix Dynamics, University of British Columbia, Vancouver, CanadaEmail: Queena Hwang - queena.hwang@utoronto.ca; Sela Cheifetz - christopher.mcculloch@utoronto.ca; Christopher M Overall - chris.overall@ubc.ca; Christopher A McCulloch* - christopher.mcculloch@utoronto.ca; Jaro Sodek - christopher.mcculloch@utoronto.ca* Corresponding author    †Equal contributorsAbstractBackground: A recent model for activation of the zymogen form of matrix metalloproteinase 2(MMP-2, also known as gelatinase A) has suggested that interactions between the SIBLING proteinbone sialoprotein (BSP) and MMP-2 leads to conformational change in MMP-2 that initiates theconversion of the pro-enzyme into a catalytically active form. This model is particularly relevant tocancer cell metastasis to bone since BSP, bound to the αvβ3 integrin through its arginine-glycine-aspartic acid motif, could recruit MMP-2 to the cell surface.Methods: We critically assessed the relationship between BSP and proMMP-2 and its activationusing various forms of recombinant and purified BSP and MMP-2. Gelatinase and collagenase assays,fluorescence binding assays, real-time PCR, cell culture and pull-down assays were employed totest the model.Results: Studies with a fluorogenic substrate for MMP-2 showed no activation of proMMP-2 byBSP. Binding and pull-down assays demonstrated no interaction between MMP-2 and BSP. WhileBSP-mediated invasiveness has been shown to depend on its integrin-binding RGD sequence,analysis of proMMP-2 activation and the level of membrane type 1 (MT1)-MMP in cells grown on aBSP substratum showed that the BSP-αvβ3 integrin interaction does not induce the expression ofMT1-MMP.Conclusion: These studies do not support a role for BSP in promoting metastasis throughinteractions with pro-MMP-2.BackgroundBone sialoprotein (BSP) is a highly glycosylated and sul-fated phosphoprotein that is expressed largely in mineral-tumors and serum from patients with breast, lung, pros-tate, or thyroid cancer [2]. Expression of BSP in cancer hasbeen associated with metastasis of tumor cells to bone [3]Published: 22 April 2009BMC Cancer 2009, 9:121 doi:10.1186/1471-2407-9-121Received: 23 September 2008Accepted: 22 April 2009This article is available from: http://www.biomedcentral.com/1471-2407/9/121© 2009 Hwang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 11(page number not for citation purposes)izing tissues [1] but is also associated with cancermetastasis. Elevated levels of BSP have been reported inas well as hydroxyapatite crystal formation in tumor tis-sues and breast cancer cell lines [4].BMC Cancer 2009, 9:121 http://www.biomedcentral.com/1471-2407/9/121Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that cooperate to modulatehomeostasis of the extracellular environment by regulat-ing oncogenic signaling networks and degrading extracel-lular matrix components, thereby contributing to tumorcell progression [5-7]. MMP-2 (also known as gelatinaseA) is made up of five structural domains including aninhibitory pro-domain [8-10]. Functional activity is regu-lated by enzymatic removal of the inhibitory pro-domain.A primary mechanism of proMMP-2 activation involvesformation of a tri-molecular complex on cell surfacesinvolving tissue inhibitor of metalloproteinase-2 andmembrane type 1-MMP (MT1-MMP) [11,12]. An alterna-tive mechanism for controlling MMP-2 activity hasinvoked apparent structural changes that arise from spe-cific interactions between BSP and proMMP-2 [13,14]. Itwas reported that upon binding to BSP the proteolyticactivity of proMMP-2 increased significantly, but paradox-ically without removal of the inhibitory pro-peptide [13].It was suggested that BSP-mediated conformationalchanges upon partnering with proMMP-2 may facilitateremoval of the inhibitory pro-peptide by another pro-tease, which is similar to the binding and activation ofproMMP-2 by MT1-MMP. A 26 amino acid domain of BSPappears to be involved in the displacement of MMP-2'spropeptide from the active site of MMP2, thereby enhanc-ing protease activity [14].Since BSP and MMP-2 are associated with tumor progres-sion [2,15-17,7], the potential modulation of proMMP-2activity by BSP is particularly relevant to tumor metastasis.We critically assessed potential interactions between BSPand proMMP-2 that mediate proMMP-2 activation.MethodsReagentsRecombinant proMMP-2 was produced as described [18].Bacterial recombinant rat BSP, rat native BSP, and BSPfragments were produced by Harvey Goldberg (Universityof Western Ontario). The BSP fragments contained aminoacids 1–100, 99–200, 200–301, 51–150, and 99–250) ofBSP. Recombinant human BSP expressed in human bonemarrow stromal cells was from N.S. Fedarko (Johns Hop-kins). Porcine BSP, G2 BSP, human BSP, and pig OPNwere purified from 0.5 M EDTA and 4 M guanidine-HCL(G2) extracts of bone tissues. OPN was purified frombovine milk.Cell CultureHuman breast cancer cell lines MDA-MB231, MCF7,T47D, and fibrosarcoma HT1080 cells were obtainedfrom ATCC. Rat bone marrow stromal cells were from S.Pitaru (Tel Aviv, Israel). Human gingival fibroblasts weremaintained in α-minimum essential medium (MEM)containing 10% fetal bovine serum. T47D cells weremaintained in a monolayer culture in RPM1 containing1% Glutamax 1 and10% FBS.Cell Culture on BSP substratumCells were seeded on to 24-well plates coated with 30 nMrat recombinant or rat native BSP. For analysis ofproMMP-2 activation, conditioned medium was collectedafter 24 hours in serum-free medium, concentrated, andanalyzed for gelatinase activity by zymography. To ana-lyze MT1-MMP mRNA, cells were seeded at 1.0 × 106 cells/mL on a non-tissue culture 96-well ELISA plates coatedwith rat native BSP (0.15 μM) or poly-L-Lysine (0.1%).Tryptophan fluorescence binding assaySince BSP contains no tryptophans, the binding of BSP toproMMP-2 was measured from the shift in tryptophan flu-orescence of proMMP-2 (15 tryptophan residues) (excita-tion = 295 nm; emission = 300–400 nm) after addition ofBSP, BSP peptides, or control proteins (from 17 nM-1165nM) to proMMP-2 (333 nM). All spectra were correctedfor buffer and dilution effects. Under these conditions,fluorescence observed was attributed exclusively to tryp-tophans from proMMP-2 as described previously [13]. Toestimate dissociation constants (Kd), a saturation curve forBSP-proMMP-2 complex formation was obtained. Kd val-ues were calculated using the Scatchard equation r/[freeBSP] = n/Kd - r/Kd where n represents the number of bind-ing sites and r = [bound BSP]/[total proMMP-2]. Eachexperiment was carried out in triplicate.Analysis of proMMP-2 auto-activationGelatinase activities were determined by gelatin zymogra-phy [19]. BSP-mediated proMMP-2 activation was moni-tored by incubating 0.2 ng/2 μL of BSP with proMMP-2(0.05, 0.2, 0.5, or 2 ng) and adding collagenase assaybuffer[20] (total volume of 8 μL). For positive controls,activated MMP-2 was obtained from concanavalin A-treated fibroblast-conditioned medium. After 4 hours ofincubation at 21°C, samples extracted in SDS-PAGE sam-ple buffer (without DTT) were analyzed by gelatin zymog-raphy.Gelatinase substrate assayProMMP-2 activity was measured with a highly quenched,fluorescein-labeled (DQ) gelatin substrate at 21°C. Uponproteolytic digestion, its fluorescence is revealaed and canbe used to measure enzymatic activity. Each assay wasconducted at 21°C in collagenase assay buffer. ProMMP-2 (1.4 nM or 2.8 nM) was added to 12.5 μg/mL substratein the presence or absence of BSP (4.9 nM or 9.8 nM).Cleavage of the substrate was monitored using a micro-Page 2 of 11(page number not for citation purposes)grown in primary culture and for production of activatedMMP2, cells were treated with concanavalin A. Cells wereplate based multi-detection reader (485 nm excitation,520 nm emission filters; FLUOstar OPTIMA, BMGBMC Cancer 2009, 9:121 http://www.biomedcentral.com/1471-2407/9/121Labtech, Offenburg, Germany). Changes in fluorescenceintensity were monitored in relation to controls: substrate+ BSP, substrate or proMMP-2 as negative controls, andsubstrate + proMMP-2 activated with aminophenylmercu-ric acetate (APMA) as a positive control.Binding assaysFor analysis of bound and unbound proMMP-2, 25 ng/50μL of the pro-enzyme was added to ELISA plates coatedwith various concentrations of pBSPE (porcine BSP-extract), pBSPG2 (G2-extract), human bone proteins(hBP), pig bone OPN (OPN), and incubated for 1 hour at21°C. Supernatants and bound proteins were analyzed bygelatin zymography. For analysis of potential adaptormolecules, conditioned medium from cells was added toELISA plates coated with 40 nM rat recombinant BSP,BSA, or gelatin and incubated for 1 hour at 21°C. Super-natant and bound proteins extracted with sample bufferwere analyzed for gelatinase activity by zymography. Foranalysis of potential adaptor molecules, 200 μL of condi-tioned medium collected from MDA-MB231, rat bonemarrow stromal cells, HT1080 cells, and human gingivalfibroblasts at 60 hours after seeding was added to a 96-well ELISA plated coated with 40 nM rat recombinantBSP, BSA or gelatin. Each mixture was incubated for 1hour at 21°C. Supernatant and bound proteins extractedwith sample buffer were analyzed for gelatinase activityusing zymography.Biotinylation of bone proteinsFor biotinylation, 22 moles of biotin were used per moleof bone proteins. Correspondingly, appropriate amount sof biotin (1 mg of biotin dissolved in mL DMSO) wereadded to each protein preparation. The mixtures werestirred for 2 hr at 4°C. To remove free biotin, the mixtureswere desalted on a 10 mL desalting column equilibratedin 50 mM ammonium bicarbonate buffer, pH 8.5. Bioti-nylation of the eluate fractions was assessed using dot blotanalysis, where 2 μL of each fraction was taken andprobed with streptavidin horseradish peroxidase. Finally,the highly biotinylated fractions were pooled, speed vacu-umed and reconstituted in water.Solution phase binding assayBiotinylated BSP was utilized to examine the potentialinteraction between BSP and proMMP-2 in solution andin these experiments 25 ng proMMP-2 was incubated with5 μg biotinylated protein in 50 μL Tris-Tween (0.05%; pH~7.6) for 1 hour at 21°C. To isolate BSP along with boundproteins, streptavidin beads were added, incubated for 30minute at 21°C, centrifuged, and supernatants were col-lected. Beads were rinsed and supernatants and bead elu-ates were analyzed by gelatin zymography. ControlsReal-time PCRRNA was extracted from cells using a Stratagene RNA min-iprep kit. Total RNA (1 μg) was reverse transcribed andreal-time PCR for MT1 was performed using the TaqMan®Gene Expression Assay system using validated probeshuman MT1-MMP (no. 4331182) and eukaryotic 18Sendogenous control (no. 4319413E).Statistical analysisAll assays were repeated at least 3 times in 3 separateexperiments. For data involving continuous variable, themeans and standard errors of the mean were calculatedand where appropriate, analysis of variance was used toexamine differences between multiple groups.ResultsBSP induces non-specific quenching of proMMP-2Due to variations of BSP phosphorylation of serines andO- and N-linked glycosylation, recombinant or native ratBSP (purified from long bones of adult rats) were used inbinding studies to assess binding between proMMP-2 andBSP. Intrinsic tryptophan fluorescence measurementsdemonstrated that titration of proMMP-2 with BSPresulted in a proportional quenching of MMP tryptophanemission spectra (Fig. 1), suggestive of direct protein-pro-tein interactions. Since proMMP-2 contains 15 tryp-tophan residues, whereas BSP contains none, thequenching of the tryptophan fluorescence signal suggeststhat proMMP-2 undergoes significant conformationalchanges, exposing internal tryptophan residues to a morepolar environment in the presence of BSP with an appar-ent Kd of 0.27 ± 0.11 μM. However, control studies usingosteopontin and RNase A in the same system also yieldeda similar quenching of the proMMP-2 tryptophan emis-sion spectra as well as the derivation of similar Kd values.The human recombinant BSP that was used previously todetect binding between BSP and proMMP-2 [13] mayhave included modifications necessary for measurementof potential interactions. Accordingly, the effect of post-translational modifications on the proposed interactionbetween BSP and MMP-2 was investigated using humanrecombinant BSP obtained from N. Fedarko (Fig. 2). Theemission peak in intrinsic fluorescence was observed at~335 nm, which is in contrast to the previous study [13]that reported an emission peak at 360 nm and an interac-tion between proMMP-2 and BSP with a kd in thenanomolar range. When the MMP-binding site withinBSP was studied by intrinsic fluorescence using BSP pep-tides (Fig. 3), each BSP fragment showed quenching of theMMP-2 tryptophan fluorescence signal, similar to theemission spectra obtained using the full-length BSP mol-ecule and the control proteins suggesting non-specificPage 3 of 11(page number not for citation purposes)included no MMP-2 and no BSP. interactions.BMC Cancer 2009, 9:121 http://www.biomedcentral.com/1471-2407/9/121BSP does not modify proMMP-2 activityTo examine potential activation induced by the additionof BSP to proMMP-2, zymography was employed to esti-mate the amount of mature enzyme of smaller molecularweight (59 or 62 kDa). In concentrations where BSP is inexcess of proMMP-2, there was no evidence for significantremoval of the pro-domain (Fig. 4) although in positivecontrols, proMMP-2 that had been activated in concanav-alin A-treated cells showed lower molecular mass MMP-2(Fig. 4, lanes 9, 10), consistent with cleavage of the pro-peptide and enzyme activation. When zymography bandswere further assessed, each BSP-treated proMMP-2 sampleresulted in an identical migration pattern as that ofuntreated enzyme. This is consistent with previous find-ings indicating that BSP binding does not induce signifi-cant cleavage of the pro-peptide [13]. Therefore, BSP-treated proMMP-2 migrates as an intact molecule (Mr of~66 kDa) on zymograms since the pro-peptide remainsattached.The effect of BSP on proMMP-2 activity was examinedusing fluorescent labeled gelatin substrate. Treatment ofproMMP-2 with increasing concentrations of recom-binant BSP or fetal porcine BSP did not alter enzymaticactivity compared to latent enzyme alone (Fig. 5). Usingthe same substrate, the ability of OPN to activateproMMP-3 was assayed, but activity above control valueswas also not observed. Since BSP may interact withproMMP-2 so that the inhibitory pro-peptide is removedfrom the active site [13], hence exposing the active site, weconsidered that the presence of BSP would lead to signifi-cant cleavage (auto-activation) to the lower molecularweight, active MMP-2. However, we found no increase inthe amount of pro-peptide-free MMP-2 by zymographyconfirming the fluorescent gelatin cleavage assays. Fur-ther, BSP did not mediate proMMP-2 catalytic activity asshown with the fluorescent substrates.Fluorescence emission spectrum of BSP-treated proMMP-2igure 1Fluorescence emission spectrum of BSP-treated proMMP-2. proMMP-2 (333 nM) was incubated with increasing con-centrations of native or recombinant BSP, OPN or RNase A (negative controls). Emission scans were obtained after each addi-tion of BSP (excitation wavelength of 295 nm). In all cases, titrations of proMMP-2 yielded proportional quenching of the proMMP-2 tryptophan emission spectra.Tryptophan fluorescence profileFigure 2Tryptophan fluorescence profile. proMMP-2 (333 nM) was incubated with nM amounts of native BSP. Emission scans were obtained after each addition of BSP (excitation Page 4 of 11(page number not for citation purposes)wavelength = 295 nm). Emission peak was at 335 nm.BMC Cancer 2009, 9:121 http://www.biomedcentral.com/1471-2407/9/121Analysis of bound and unbound MMP-2Despite the lack of significant pro-peptide cleavage whenproMMP-2 dose response curves to BSP were examined,we hypothesized that BSP-induced activation mightinvolve only a fraction of the total amount of enzyme. Weused ELISA plates to resolve BSP-bound and unboundfractions, which allows for higher resolution examina-tions of the BSP-proMMP-2 interactions. Previous find-ings have suggested a 1:1 stoichiometry of bindingbetween BSP and proMMP-2 and a Kd value of 2.9 ± 0.9nM [13]. Such a strong affinity should allow for detectionof the interaction. However, our data showed no bindingbetween the proMMP-2 and BSP as detected when theBSP-bound (extract) and unbound (supernatant) frac-tions were analyzed by zymography (Fig. 6).We considered that the lack of association betweenproMMP-2 and BSP could be a consequence of disruptionof a binding motif from fixing BSP to a hydrophobic sur-face. Accordingly, we assessed the ability of BSP to associ-ate with proMMP-2 in solution. Bone proteins werebiotinylated, incubated with proMMP-2 and isolatedusing streptavidin beads. When bead-bound entities wereassessed by zymography, each bead-purified bone proteinshowed no evidence of MMP-2 binding (data not shown).Further, MMP-2 was recovered entirely in the latent form(Mr of 66 kDa) in the supernatant. Alternatively, whenbiotinylated bone proteins were pre-bound to streptavi-din beads, followed by the addition of proMMP-2, similarresults were observed.Interactions between BSP peptides and proMMP-2Figure 3Interactions between BSP peptides and proMMP-2. proMMP-2 (333 nM) was incubated with nM amounts of BSP pep-tides. Emission scans were obtained after each addition of BSP (excitation wavelength = 295 nm). Emission spectra show that titration of proMMP-2 with each BSP peptide yielded proportional quenching of the proMMP-2 emission spectra.Zymography analysis of BSP-treated proMMP-2Figure 4Zymography analysis of BSP-treated proMMP-2. ProMMP-2 was incubated with (lanes 5–8) or without (lanes 1–4) increasing amounts of BSP for 4 hours at 21°C, and resolved by zymography. ConA cell-activated MMP-2 were used as stand-ards (lanes 9–10). Lane 1, 2 ng proMMP-2; lane 2, 0.5 ng proMMP-2; lane 3, 0.2 ng proMMP-2; lane 4, 0.05 ng proMMP-2; lane 5, 2 ng proMMP-2 + 2 ng BSP; lane 6, 0.5 ng proMMP-2 + 2 ng BSP; lane 7, 0.2 ng proMMP-2 + 2 ng BSP; lane 8, 0.05 ng 1        2       3        4        5        6     7       8        9      10       proMMP-2MMP-2Page 5 of 11(page number not for citation purposes)proMMP-2 + 2 ng BSP; lanes 9 and 10, 0.05 ng conA activated MMP-2.BMC Cancer 2009, 9:121 http://www.biomedcentral.com/1471-2407/9/121Analysis of potential adaptor moleculesBecause of the lack of any evidence of specific binding ofproMMP-2 to BSP, the need for potential adaptor mole-cules in this interaction was examined using solid phasebinding assay on ELISA plates followed by zymography.Serum-free conditioned medium collected from MDA-MB231, rat bone marrow cells, HT1080 or human gingi-val fibroblasts were used as a source of MMP-2 and addedto BSP that was conjugated to an ELISA plate. Since thereported binding between BSP and proMMP-2 was ini-tially identified by a co-purification of proMMP-2 andrecombinant BSP expressed in bone marrow cells [13], wehypothesized that given the absence of a direct interactionthen complexes with other proteins might be required,similar to the TIMP-2 bridge between the physiologicalactivator MT1-MMP and MMP-2 [18]. Nonetheless,zymography analysis of BSP-bound (extract) andunbound (supernatant) fractions revealed that latent andactive MMP-2 secreted by bone marrow cells (Fig. 7), aswell as the other cell lines, were recovered entirely in theProMMP-2 activation is unaffected by cellular adhesion to BSPDespite the lack of direct or indirect interaction observedbetween BSP and proMMP-2, clustering of the α2β1integrins in cancer cells stimulated by fibrillar collagenhas been shown to promote tyrosine kinase-mediatedevents that result in expression of MT1-MMP andproMMP-2 activation [21]. To investigate the conse-quences of integrin αvβ3 clustering by BSP, the levels ofproMMP-2 activation in MDA-MB231, MCF7, and T47Dcells grown on BSP substrata were compared to that ofcells grown on plastic. There was a similar level ofproMMP-2 activation in cells after attachment to BSP incomparison to cells grown on plastic (Fig. 8). SinceproMMP-2 activation is directly associated with the levelof MT1-MMP activity, these results indicated that cellularbinding to BSP via integrin αvβ3 does not modify MT1-MMP activity on the cell surface. Previously we haveshown that proMMP-2 does not directly bind αvβ3[22].ProMMP-2 activity after incubation with BSPFigure 5ProMMP-2 activity after incubation with BSP. ProMMP-2 (1.4 or 2.8 nM) was incubated with recombinant BSP (rBSP) or native BSP (nBSP) (4.9 or 9.8 nM) and 12.5 μg/mL fluorescent substrate. Results are values calibrated with fluorescence from substrate + BSP controls. Fluorescence levels of other controls, including substrate only, substrate + proMMP-2 and substrate + APMA-activated enzyme, are also shown.25004500650085001050012500145000 50 100 150 200Time (min.)FluorescenceSubstrate onlySubstrate + proMMP-2Substrate + activated MMP-21.4 nM MMP-2 + 4.9 nM nBSP2.8 nM MMP-2 + 9.8 nM nBSP1.4 nM MMP-2 + 4.9 nM rBSP2.8 nM MMP-2 + 9.8 nM rBSPPage 6 of 11(page number not for citation purposes)supernatant, unbound fraction as observed for recom-binant proMMP-2.BMC Cancer 2009, 9:121 http://www.biomedcentral.com/1471-2407/9/121Cellular adhesion to BSP does not alter MT1-MMP transcript levelSince the activity of MT1-MMP is regulated at multiplesteps, differences in MT1-MMP expression may not bedetected by analysis of proMMP-2 activation. Accordingly,MT1-MMP mRNA levels were analyzed by real time RT-PCR to investigate quantitatively whether MT1-MMPmRNA levels are different between cancer cells grown ona BSP substratum and on poly-L-Lysine. Real-time PCRresults (Fig. 9) did not detect any significant changes inthe MT1-MMP transcript level by stimulation with BSP (p> 0.2), which was consistent with an unaltered level ofMT1-MMP activity as observed by zymography.DiscussionCellular invasion in metastasis is a coordinated event thatinvolves multiple metabolic processes and cellular com-ponents, including deployment and activation of celladhesion molecules and proteolytic enzymes. Frequently,multimers of proteases show increased catalytic efficiencyand in the plasma membrane, enabling focal proteolysisunder cellular control. MMPs have traditionally beenassociated with tumor cell invasion and metastasis, in par-ticular MMP-2 and its activator MT1-MMP. MMP-2 isuniquely activated on the cell surface by MT-MMPs in ahighly regulated process after complex formation of pro-and active MMP-2 with MT1-MMP and TIMP-2 [23,12].Extracellularly, clustering by heparin or ConA [24] andhave been reported that resulted in activation of proMMP-2 [13]. We assessed here the ability of various forms ofBSP to bind and activate proMMP-2. We investigated thepossibility that BSP activated proMMP-2 by analysis ofgelatinase activity using a fluorescent substrate, but theanalysis showed no activation of proMMP-2. Further,when OPN, another SIBLING protein, was assessed inproMMP-3 activation using the same substrate, no activa-tion could be detected. Therefore, BSP does not appear tobe involved in the activation of proMMP-2.After careful examination of the conditions used for acti-vation in the previous study [13], we noticed that despitea reported Kd value of 2.9 ± 0.9 nM, a 500-fold molarexcess of BSP was necessary to demonstrate proMMP acti-vation. We repeated these experiments using the humanBSP at this same ratio but again found no activation.Given the potential ability of BSP to promote displace-ment of pro-peptides from active sites of proMMP-2 [13],we considered that there may be auto-activation of thelatent enzyme in the presence of BSP. However, whenproMMP-2 was treated with BSP, the proMMP-2 migratedas an intact molecule on zymograms, indicating that BSPdoes not activate proMMP-2.Activation of MMP-2 requires unidentified protein-pro-tein interactions, one of which might involve BSP. Extra-cellularly, one of the known interactors is native type IProMMP-2 recovery in BSP-unbound sampleFigure 6ProMMP-2 recovery in BSP-unbound sample. ProMMP-2 (0.5 mM) was added to decreasing concentrations of indicated SIBLING proteins (40 mM, 20 mM, 10 mM, 5 mM, 2.5 mM, 1.2 mM, 0.6 mM, and 0 mM) coated on an ELISA plate, and incu-bated at 21°C for 4 hours. Samples of the unbound (Supernatant) and bound (Extract) proteins were extracted in SDS sample buffer, and analyzed by zymography. proMMP-2 was recovered completely in the latent form in the unbound (Supernatant) fractions.Page 7 of 11(page number not for citation purposes)claudin [25], increases MMP-2 activation. Recently, spe-cific interactions between BSP and latent forms of MMP-2collagen, which results in the lateral association of MT1-MMP to accelerate activation of progelatinase A [26,27].BMC Cancer 2009, 9:121 http://www.biomedcentral.com/1471-2407/9/121As a result of our inability to detect BSP-induced activa-tion of proMMP-2, we examined the interaction of thesetwo proteins using binding assays. Since previous findings[13] have suggested a 1:1 stoichiometric binding betweenBSP and proMMP-2 with a Kd value in the low nanomolarrange, such an affinity presumably allows detection of theinteraction using less sensitive assays such as affinityadsorption. However, we found no evidence of interac-tion between BSP and proMMP-2 using these assays.To address the possibility of cell-derived adaptor mole-cules required for the BSP-proMMP-2 interaction, BSP wasincubated with conditioned medium collected frombreast cancer cells, bone marrow cells or human gingivalnot bind to BSP. Notably, BSP is highly heterogeneous asa result of variations in the phosphorylation of serinesand O- and N-linked glycosylation [28]. Presumably, BSPexpressed by diverse cells types is modified differently,and variations in post-translational modifications maydetermine the activity of these proteins and the bindingand activation of proMMP-2. Accordingly we employedrecombinant BSP, BSP purified from bone, or recom-binant human BSP to assess binding to proMMP-2. As wewere unable to detect binding of any of the BSPs toproMMP-2, there is evidently a need to re-assess thepotential ability of BSP to bind to and activate proMMP-2in the context of cancer cell metastasis although we can-not rule out the possibility that much more highly glyco-MMP-2 from conditioned medium recovery in BSP-unbound fractionFigure 7MMP-2 from conditioned medium recovery in BSP-unbound fraction. Serum-free conditioned media collected from: 1) MDA-MB231, 2) rat bone marrow cells, 3) HT1080, and 4) human gingival fibroblasts were added to ELISA plates coated with indicated proteins (35 μM) and incubated at RT for one hour. Samples of bound (extract) and unbound (supernatant) pro-teins were extracted in SDS sample buffer and analyzed by zymography. Zymography shows that when MMP-2 is added to BSP-coated plates, both latent and active enzymes are recovered completely in supernatants. BSA and gelatin were used as negative and positive MMP-2-binding controls respectively.Page 8 of 11(page number not for citation purposes)fibroblasts. As observed for recombinant proMMP-2,latent and active MMP-2 secreted by cancer cells also didsylated BSP than the bovine BSP we used here couldconceivably mediate an interaction with proMMP2.BMC Cancer 2009, 9:121 http://www.biomedcentral.com/1471-2407/9/121MMP-2 binds to the surface of cancer cells via thefibronectin type II module repeats of the enzyme [29,30].Despite the lack of an interaction between BSP andproMMP-2, it is possible that an interaction between BSPand the αvβ3 integrin itself may trigger downstream sign-aling events that affect the expression, processing, andactivity of MMP-2. Thus, the requirement of an activeRGD sequence in BSP-mediated cancer cell invasion sug-gests that BSP binding to the αvβ3 integrin may promoteclustering of integrin molecules, which could activatedownstream signaling events. Notably, ECM proteins canpromote raft formation and type I collagen activatesMMP-2 through β1-integrins, which increases MT1-MMPlevels [21], and by direct binding of pericellular nativetype I collagen with the MT1-MMP hemopexin domain[26]. MT1-MMP enhances focal proteolysis [31] andexperimental metastasis [32], is associated with MMP-2activation in lung carcinoma [33] and invasive humanbreast cancer cell lines [34,35], and is over-expressed inhigh-grade gliomas, fibrosarcomas [36] and in carcino-mas of the lung, stomach, head and neck [37]. However,in our studies there was no evidence of integrin-mediatedenhancement in the level of MT1-MMP transcript level,nor in MT1-MMP activity. Evidently, a more completeunderstanding of integrin-mediated signaling events willbe important for defining the significance of BSP bindingto the αvβ3 integrin in vivo.ConclusionCollectively, using the methods reported here, our studiesdo not support a role for BSP in promoting pro-MMP-2activation.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsQYJH conducted the experiments and the analyses andwrote the first drafts of the manuscript. SC designed theRT-PCR experiments and probes. CMO designed theEffect of cell attachment to BSP on MT1-MMP-mediated activation of proMMP-2Figure 8Effect of cell attachment to BSP on MT1-MMP-mediated activation of proMMP-2. Serum-free conditioned medium was collected from the indicated breast cancer cell lines seeded on BSP (30 μM) coated on ELISA plates, concentrated, and analyzed on zymograms. There were no significant differences in the level of proMMP-2 activation between cells grown on recombinant (r)BSP or native (n)BSP compared to cells grown on plastic.Plastic    rBSP   nBSP   Plastic   rBSP    nBSP    Plastic   rBSP    nBSPproMMP-2MMP-2MDA-MB-231                            MCF7                             T47DMT1-MMP transcript levels after BSP stimulationFigure 9MT1-MMP transcript levels after BSP stimulation. MDA, MCF7, T47D, or HT1080 cells were seeded on native BSP (blue bars) or poly-L-Lysine (grey bars) coated on an ELISA plate. Total RNA was reverse transcribed and sub-jected to qPCR analysis using specific primers for MT1-MMP. Results were normalized as fold increase over cells seeded on poly-L-Lysine and expressed as mean ± SEM (n = 3). From the comparison no significant differences (p > 0.2) in the MT1-MMP transcript level were observed between cells 012Relative expression(fold over control)MDA               MCF7                T47D              HT1080Page 9 of 11(page number not for citation purposes)proMMP2 activation experiments and contributed to thepenultimate draft manuscript. CAM drafted the manu-grown on BSP and cells grown on poly-L-Lysine.BMC Cancer 2009, 9:121 http://www.biomedcentral.com/1471-2407/9/121script and wrote the final draft. JS designed the experi-ments and helped to write the initial drafts.AcknowledgementsThe research was supported by CIHR Operating, Group and Research Resource grants to SC, CMO, CAM and JS. We thank W. Houry (Univer-sity of Toronto) with technical assistance and use of equipment for spec-troscopy analyses. This research was completed prior to the death of Dr. J. Sodek in August, 2007 and of Dr. S. Cheifetz in May, 2008.References1. Oldberg A, Franzen A, Heinegard D: The primary structure of acell-binding bone sialoprotein.  J Biol Chem 1988,263(36):19430-19432.2. Bellahcene A, Merville MP, Castronovo V: Expression of bone sia-loprotein, a bone matrix protein, in human breast cancer.Cancer Res 1994, 54(11):2823-2826.3. Bellahcene A, Kroll M, Liebens F, Castronovo V: Bone sialoproteinexpression in primary human breast cancer is associatedwith bone metastases development.  J Bone Miner Res 1996,11(5):665-670.4. Bellahcene A, Menard S, Bufalino R, Moreau L, Castronovo V:Expression of bone sialoprotein in primary human breastcancer is associated with poor survival.  Int J Cancer 1996,69(4):350-353.5. Egeblad M, Werb Z: New functions for the matrix metallopro-teinases in cancer progression.  Nat Rev Cancer 2002,2(3):161-174.6. Sternlicht MD, Werb Z: How matrix metalloproteinases regu-late cell behavior.  Annu Rev Cell Dev Biol 2001, 17:463-516.7. Overall CM, Kleifeld O: Towards third generation matrix met-alloproteinase inhibitors for cancer therapy.  Br J Cancer 2006,94(7):941-946.8. Murphy G, Knauper V: Relating matrix metalloproteinasestructure to function: why the "hemopexin" domain?  MatrixBiol 1997, 15(8–9):511-518.9. Nagase H, Woessner JF Jr: Matrix metalloproteinases.  J Biol Chem1999, 274(31):21491-21494.10. Overall CM: Molecular determinants of metalloproteinasesubstrate specificity: matrix metalloproteinase substratebinding domains, modules, and exosites.  Mol Biotechnol 2002,22(1):51-86.11. Seiki M: Membrane-type matrix metalloproteinases.  APMIS1999, 107(1):137-143.12. Overall CM, King AE, Sam DK, Ong AD, Lau TT, Wallon UM,DeClerck YA, Atherstone J: Identification of the tissue inhibitorof metalloproteinases-2 (TIMP-2) binding site on the hemo-pexin carboxyl domain of human gelatinase A by site-directed mutagenesis. The hierarchical role in binding TIMP-2 of the unique cationic clusters of hemopexin modules IIIand IV.  J Biol Chem 1999, 274(7):4421-4429.13. Fedarko NS, Jain A, Karadag A, Fisher LW: Three small integrinbinding ligand N-linked glycoproteins (SIBLINGs) bind andactivate specific matrix metalloproteinases.  FASEB J 2004,18(6):734-736.14. Jain A, Karadag A, Fisher LW, Fedarko NS: Structural require-ments for bone sialoprotein binding and modulation ofmatrix metalloproteinase-2.  Biochemistry 2008,47(38):10162-10170.15. Polette M, Nawrocki B, Gilles C, Sato H, Seiki M, Tournier JM, Birem-baut P: MT-MMP expression and localisation in human lungand breast cancers.  Virchows Arch 1996, 428(1):29-35.16. Ishigaki S, Toi M, Ueno T, Matsumoto H, Muta M, Koike M, Seiki M:Significance of membrane type 1 matrix metalloproteinaseexpression in breast cancer.  Jpn J Cancer Res 1999,90(5):516-522.17. Kanayama H, Yokota K, Kurokawa Y, Murakami Y, Nishitani M,Kagawa S: Prognostic values of matrix metalloproteinase-2and tissue inhibitor of metalloproteinase-2 expression inbladder cancer.  Cancer 1998, 82(7):1359-1366.MMP occurs via a TIMP-2-independent pathway.  J Biol Chem2001, 276(50):47402-47410.19. Mainardi CL, Hasty KA, Hibbs MS: Antibody to rabbit macro-phage type V collagenase/gelatinase and its use to furthercharacterize the enzyme.  Coll Relat Res 1984, 4(3):209-217.20. Overall CM, Sodek J, McCulloch CA, Birek P: Evidence for poly-morphonuclear leukocyte collagenase and 92-kilodaltongelatinase in gingival crevicular fluid.  Infect Immun 1991,59(12):4687-4692.21. Ellerbroek SM, Wu YI, Overall CM, Stack MS: Functional interplaybetween type I collagen and cell surface matrix metallopro-teinase activity.  J Biol Chem 2001, 276(27):24833-24842.22. Nisato RE, Hosseini G, Sirrenberg C, Butler GS, Crabbe T, DochertyAJ, Wiesner M, Murphy G, Overall CM, Goodman SL, et al.: Dissect-ing the role of matrix metalloproteinases (MMP) andintegrin alpha(v)beta3 in angiogenesis in vitro: absence ofhemopexin C domain bioactivity, but membrane-Type 1-MMP and alpha(v)beta3 are critical.  Cancer Res 2005,65(20):9377-9387.23. Strongin AY, Marmer BL, Grant GA, Goldberg GI: Plasma mem-brane-dependent activation of the 72-kDa type IV colla-genase is prevented by complex formation with TIMP-2.  JBiol Chem 1993, 268(19):14033-14039.24. Overall CM, Sodek J: Concanavalin A produces a matrix-degra-dative phenotype in human fibroblasts. Induction and endog-enous activation of collagenase, 72-kDa gelatinase, andPump-1 is accompanied by the suppression of the tissueinhibitor of matrix metalloproteinases.  J Biol Chem 1990,265(34):21141-21151.25. Miyamori H, Takino T, Kobayashi Y, Tokai H, Itoh Y, Seiki M, Sato H:Claudin promotes activation of pro-matrix metalloprotein-ase-2 mediated by membrane-type matrix metalloprotein-ases.  J Biol Chem 2001, 276(30):28204-28211.26. Tam EM, Wu YI, Butler GS, Stack MS, Overall CM: Collagen bind-ing properties of the membrane type-1 matrix metallopro-teinase (MT1-MMP) hemopexin C domain. The ectodomainof the 44-kDa autocatalytic product of MT1-MMP inhibitscell invasion by disrupting native type I collagen cleavage.  JBiol Chem 2002, 277(41):39005-39014.27. Tam EM, Moore TR, Butler GS, Overall CM: Characterization ofthe distinct collagen binding, helicase and cleavage mecha-nisms of matrix metalloproteinase 2 and 14 (gelatinase Aand MT1-MMP): the differential roles of the MMP hemo-pexin c domains and the MMP-2 fibronectin type II modulesin collagen triple helicase activities.  J Biol Chem 2004,279(41):43336-43344.28. Ganss B, Kim RH, Sodek J: Bone sialoprotein.  Crit Rev Oral Biol Med1999, 10(1):79-98.29. Steffensen B, Wallon UM, Overall CM: Extracellular matrix bind-ing properties of recombinant fibronectin type II-like mod-ules of human 72-kDa gelatinase/type IV collagenase. Highaffinity binding to native type I collagen but not native typeIV collagen.  J Biol Chem 1995, 270(19):11555-11566.30. Saad S, Gottlieb DJ, Bradstock KF, Overall CM, Bendall LJ: Cancercell-associated fibronectin induces release of matrix metal-loproteinase-2 from normal fibroblasts.  Cancer Res 2002,62(1):283-289.31. Seiki M, Yana I: Roles of pericellular proteolysis by membranetype-1 matrix metalloproteinase in cancer invasion and ang-iogenesis.  Cancer Sci 2003, 94(7):569-574.32. Tsunezuka Y, Kinoh H, Takino T, Watanabe Y, Okada Y, ShinagawaA, Sato H, Seiki M: Expression of membrane-type matrix met-alloproteinase 1 (MT1-MMP) in tumor cells enhances pulmo-nary metastasis in an experimental metastasis assay.  CancerRes 1996, 56(24):5678-5683.33. Tokuraku M, Sato H, Murakami S, Okada Y, Watanabe Y, Seiki M:Activation of the precursor of gelatinase A/72 kDa type IVcollagenase/MMP-2 in lung carcinomas correlates with theexpression of membrane-type matrix metalloproteinase(MT-MMP) and with lymph node metastasis.  Int J Cancer 1995,64(5):355-359.34. Gilles C, Polette M, Seiki M, Birembaut P, Thompson EW: Implica-tion of collagen type I-induced membrane-type 1-matrixmetalloproteinase expression and matrix metalloprotein-Page 10 of 11(page number not for citation purposes)18. Morrison CJ, Butler GS, Bigg HF, Roberts CR, Soloway PD, OverallCM: Cellular activation of MMP-2 (gelatinase A) by MT2- ase-2 activation in the metastatic progression of breast car-cinoma.  Lab Invest 1997, 76(5):651-660.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central BMC Cancer 2009, 9:121 http://www.biomedcentral.com/1471-2407/9/12135. Pulyaeva H, Bueno J, Polette M, Birembaut P, Sato H, Seiki M, Thomp-son EW: MT1-MMP correlates with MMP-2 activation poten-tial seen after epithelial to mesenchymal transition in humanbreast carcinoma cells.  Clin Exp Metastasis 1997, 15(2):111-120.36. Yamamoto M, Mohanam S, Sawaya R, Fuller GN, Seiki M, Sato H,Gokaslan ZL, Liotta LA, Nicolson GL, Rao JS: Differential expres-sion of membrane-type matrix metalloproteinase and itscorrelation with gelatinase A activation in human malignantbrain tumors in vivo and in vitro.  Cancer Res 1996,56(2):384-392.37. Sato H, Seiki M: Membrane-type matrix metalloproteinases(MT-MMPs) in tumor metastasis.  Journal of biochemistry 1996,119(2):209-215.Pre-publication historyThe pre-publication history for this paper can be accessedhere:http://www.biomedcentral.com/1471-2407/9/121/prepubyours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 11 of 11(page number not for citation purposes)

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