Bone sialoprotein does not interact with pro-gelatinase A (MMP-2) or mediate MMP-2 activation
- Queena Hwang1,
- Sela Cheifetz†1,
- Christopher M Overall2,
- Christopher A McCulloch1Email author and
- Jaro Sodek†1
© Hwang et al; licensee BioMed Central Ltd. 2009
Received: 23 September 2008
Accepted: 22 April 2009
Published: 22 April 2009
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 protein bone sialoprotein (BSP) and MMP-2 leads to conformational change in MMP-2 that initiates the conversion of the pro-enzyme into a catalytically active form. This model is particularly relevant to cancer 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.
We critically assessed the relationship between BSP and proMMP-2 and its activation using 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 to test the model.
Studies with a fluorogenic substrate for MMP-2 showed no activation of proMMP-2 by BSP. Binding and pull-down assays demonstrated no interaction between MMP-2 and BSP. While BSP-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 a BSP substratum showed that the BSP-αvβ3 integrin interaction does not induce the expression of MT1-MMP.
These studies do not support a role for BSP in promoting metastasis through interactions with pro-MMP-2.
Bone sialoprotein (BSP) is a highly glycosylated and sulfated phosphoprotein that is expressed largely in mineralizing tissues  but is also associated with cancer metastasis. Elevated levels of BSP have been reported in tumors and serum from patients with breast, lung, prostate, or thyroid cancer . Expression of BSP in cancer has been associated with metastasis of tumor cells to bone  as well as hydroxyapatite crystal formation in tumor tissues and breast cancer cell lines .
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that cooperate to modulate homeostasis of the extracellular environment by regulating oncogenic signaling networks and degrading extracellular matrix components, thereby contributing to tumor cell progression [5–7]. MMP-2 (also known as gelatinase A) is made up of five structural domains including an inhibitory pro-domain [8–10]. Functional activity is regulated by enzymatic removal of the inhibitory pro-domain. A primary mechanism of proMMP-2 activation involves formation of a tri-molecular complex on cell surfaces involving tissue inhibitor of metalloproteinase-2 and membrane type 1-MMP (MT1-MMP) [11, 12]. An alternative mechanism for controlling MMP-2 activity has invoked apparent structural changes that arise from specific interactions between BSP and proMMP-2 [13, 14]. It was reported that upon binding to BSP the proteolytic activity of proMMP-2 increased significantly, but paradoxically without removal of the inhibitory pro-peptide . It was suggested that BSP-mediated conformational changes upon partnering with proMMP-2 may facilitate removal of the inhibitory pro-peptide by another protease, which is similar to the binding and activation of proMMP-2 by MT1-MMP. A 26 amino acid domain of BSP appears to be involved in the displacement of MMP-2's propeptide from the active site of MMP2, thereby enhancing protease activity .
Since BSP and MMP-2 are associated with tumor progression [2, 15–17, 7], the potential modulation of proMMP-2 activity by BSP is particularly relevant to tumor metastasis. We critically assessed potential interactions between BSP and proMMP-2 that mediate proMMP-2 activation.
Recombinant proMMP-2 was produced as described . Bacterial recombinant rat BSP, rat native BSP, and BSP fragments were produced by Harvey Goldberg (University of Western Ontario). The BSP fragments contained amino acids 1–100, 99–200, 200–301, 51–150, and 99–250) of BSP. Recombinant human BSP expressed in human bone marrow stromal cells was from N.S. Fedarko (Johns Hopkins). Porcine BSP, G2 BSP, human BSP, and pig OPN were purified from 0.5 M EDTA and 4 M guanidine-HCL (G2) extracts of bone tissues. OPN was purified from bovine milk.
Human breast cancer cell lines MDA-MB231, MCF7, T47D, and fibrosarcoma HT1080 cells were obtained from ATCC. Rat bone marrow stromal cells were from S. Pitaru (Tel Aviv, Israel). Human gingival fibroblasts were grown in primary culture and for production of activated MMP2, cells were treated with concanavalin A. Cells were maintained in α-minimum essential medium (MEM) containing 10% fetal bovine serum. T47D cells were maintained in a monolayer culture in RPM1 containing 1% Glutamax 1 and10% FBS.
Cell Culture on BSP substratum
Cells were seeded on to 24-well plates coated with 30 nM rat recombinant or rat native BSP. For analysis of proMMP-2 activation, conditioned medium was collected after 24 hours in serum-free medium, concentrated, and analyzed for gelatinase activity by zymography. To analyze MT1-MMP mRNA, cells were seeded at 1.0 × 106 cells/mL on a non-tissue culture 96-well ELISA plates coated with rat native BSP (0.15 μM) or poly-L-Lysine (0.1%).
Tryptophan fluorescence binding assay
Since BSP contains no tryptophans, the binding of BSP to proMMP-2 was measured from the shift in tryptophan fluorescence of proMMP-2 (15 tryptophan residues) (excitation = 295 nm; emission = 300–400 nm) after addition of BSP, BSP peptides, or control proteins (from 17 nM-1165 nM) to proMMP-2 (333 nM). All spectra were corrected for buffer and dilution effects. Under these conditions, fluorescence observed was attributed exclusively to tryptophans from proMMP-2 as described previously . To estimate dissociation constants (Kd), a saturation curve for BSP-proMMP-2 complex formation was obtained. K d values were calculated using the Scatchard equation r/[free BSP] = n/K d - r/K d where n represents the number of binding sites and r = [bound BSP]/[total proMMP-2]. Each experiment was carried out in triplicate.
Analysis of proMMP-2 auto-activation
Gelatinase activities were determined by gelatin zymography . BSP-mediated proMMP-2 activation was monitored by incubating 0.2 ng/2 μL of BSP with proMMP-2 (0.05, 0.2, 0.5, or 2 ng) and adding collagenase assay buffer (total volume of 8 μL). For positive controls, activated MMP-2 was obtained from concanavalin A-treated fibroblast-conditioned medium. After 4 hours of incubation at 21°C, samples extracted in SDS-PAGE sample buffer (without DTT) were analyzed by gelatin zymography.
Gelatinase substrate assay
ProMMP-2 activity was measured with a highly quenched, fluorescein-labeled (DQ) gelatin substrate at 21°C. Upon proteolytic digestion, its fluorescence is revealaed and can be used to measure enzymatic activity. Each assay was conducted at 21°C in collagenase assay buffer. ProMMP-2 (1.4 nM or 2.8 nM) was added to 12.5 μg/mL substrate in the presence or absence of BSP (4.9 nM or 9.8 nM). Cleavage of the substrate was monitored using a micro-plate based multi-detection reader (485 nm excitation, 520 nm emission filters; FLUOstar OPTIMA, BMG Labtech, Offenburg, Germany). Changes in fluorescence intensity were monitored in relation to controls: substrate + BSP, substrate or proMMP-2 as negative controls, and substrate + proMMP-2 activated with aminophenylmercuric acetate (APMA) as a positive control.
For analysis of bound and unbound proMMP-2, 25 ng/50 μL of the pro-enzyme was added to ELISA plates coated with various concentrations of pBSPE (porcine BSP-extract), pBSPG2 (G2-extract), human bone proteins (hBP), pig bone OPN (OPN), and incubated for 1 hour at 21°C. Supernatants and bound proteins were analyzed by gelatin zymography. For analysis of potential adaptor molecules, conditioned medium from cells was added to ELISA plates coated with 40 nM rat recombinant BSP, BSA, or gelatin and incubated for 1 hour at 21°C. Supernatant and bound proteins extracted with sample buffer were analyzed for gelatinase activity by zymography. For analysis of potential adaptor molecules, 200 μL of conditioned medium collected from MDA-MB231, rat bone marrow stromal cells, HT1080 cells, and human gingival fibroblasts at 60 hours after seeding was added to a 96-well ELISA plated coated with 40 nM rat recombinant BSP, BSA or gelatin. Each mixture was incubated for 1 hour at 21°C. Supernatant and bound proteins extracted with sample buffer were analyzed for gelatinase activity using zymography.
Biotinylation of bone proteins
For biotinylation, 22 moles of biotin were used per mole of bone proteins. Correspondingly, appropriate amount s of biotin (1 mg of biotin dissolved in mL DMSO) were added to each protein preparation. The mixtures were stirred for 2 hr at 4°C. To remove free biotin, the mixtures were desalted on a 10 mL desalting column equilibrated in 50 mM ammonium bicarbonate buffer, pH 8.5. Biotinylation of the eluate fractions was assessed using dot blot analysis, where 2 μL of each fraction was taken and probed with streptavidin horseradish peroxidase. Finally, the highly biotinylated fractions were pooled, speed vacuumed and reconstituted in water.
Solution phase binding assay
Biotinylated BSP was utilized to examine the potential interaction between BSP and proMMP-2 in solution and in these experiments 25 ng proMMP-2 was incubated with 5 μg biotinylated protein in 50 μL Tris-Tween (0.05%; pH ~7.6) for 1 hour at 21°C. To isolate BSP along with bound proteins, streptavidin beads were added, incubated for 30 minute at 21°C, centrifuged, and supernatants were collected. Beads were rinsed and supernatants and bead eluates were analyzed by gelatin zymography. Controls included no MMP-2 and no BSP.
RNA was extracted from cells using a Stratagene RNA miniprep kit. Total RNA (1 μg) was reverse transcribed and real-time PCR for MT1 was performed using the TaqMan® Gene Expression Assay system using validated probes human MT1-MMP (no. 4331182) and eukaryotic 18S endogenous control (no. 4319413E).
All assays were repeated at least 3 times in 3 separate experiments. For data involving continuous variable, the means and standard errors of the mean were calculated and where appropriate, analysis of variance was used to examine differences between multiple groups.
BSP induces non-specific quenching of proMMP-2
BSP does not modify proMMP-2 activity
Analysis of bound and unbound MMP-2
We considered that the lack of association between proMMP-2 and BSP could be a consequence of disruption of a binding motif from fixing BSP to a hydrophobic surface. Accordingly, we assessed the ability of BSP to associate with proMMP-2 in solution. Bone proteins were biotinylated, incubated with proMMP-2 and isolated using streptavidin beads. When bead-bound entities were assessed by zymography, each bead-purified bone protein showed no evidence of MMP-2 binding (data not shown). Further, MMP-2 was recovered entirely in the latent form (M r of 66 kDa) in the supernatant. Alternatively, when biotinylated bone proteins were pre-bound to streptavidin beads, followed by the addition of proMMP-2, similar results were observed.
Analysis of potential adaptor molecules
ProMMP-2 activation is unaffected by cellular adhesion to BSP
Cellular adhesion to BSP does not alter MT1-MMP transcript level
Cellular invasion in metastasis is a coordinated event that involves multiple metabolic processes and cellular components, including deployment and activation of cell adhesion molecules and proteolytic enzymes. Frequently, multimers of proteases show increased catalytic efficiency and in the plasma membrane, enabling focal proteolysis under cellular control. MMPs have traditionally been associated with tumor cell invasion and metastasis, in particular MMP-2 and its activator MT1-MMP. MMP-2 is uniquely activated on the cell surface by MT-MMPs in a highly 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  and claudin , increases MMP-2 activation. Recently, specific interactions between BSP and latent forms of MMP-2 have been reported that resulted in activation of proMMP-2 . We assessed here the ability of various forms of BSP to bind and activate proMMP-2. We investigated the possibility that BSP activated proMMP-2 by analysis of gelatinase activity using a fluorescent substrate, but the analysis showed no activation of proMMP-2. Further, when OPN, another SIBLING protein, was assessed in proMMP-3 activation using the same substrate, no activation could be detected. Therefore, BSP does not appear to be involved in the activation of proMMP-2.
After careful examination of the conditions used for activation in the previous study , we noticed that despite a reported Kd value of 2.9 ± 0.9 nM, a 500-fold molar excess of BSP was necessary to demonstrate proMMP activation. We repeated these experiments using the human BSP at this same ratio but again found no activation. Given the potential ability of BSP to promote displacement of pro-peptides from active sites of proMMP-2 , we considered that there may be auto-activation of the latent enzyme in the presence of BSP. However, when proMMP-2 was treated with BSP, the proMMP-2 migrated as an intact molecule on zymograms, indicating that BSP does not activate proMMP-2.
Activation of MMP-2 requires unidentified protein-protein interactions, one of which might involve BSP. Extracellularly, one of the known interactors is native type I collagen, which results in the lateral association of MT1-MMP to accelerate activation of progelatinase A [26, 27]. As a result of our inability to detect BSP-induced activation of proMMP-2, we examined the interaction of these two proteins using binding assays. Since previous findings  have suggested a 1:1 stoichiometric binding between BSP and proMMP-2 with a Kd value in the low nanomolar range, such an affinity presumably allows detection of the interaction using less sensitive assays such as affinity adsorption. However, we found no evidence of interaction between BSP and proMMP-2 using these assays.
To address the possibility of cell-derived adaptor molecules required for the BSP-proMMP-2 interaction, BSP was incubated with conditioned medium collected from breast cancer cells, bone marrow cells or human gingival fibroblasts. As observed for recombinant proMMP-2, latent and active MMP-2 secreted by cancer cells also did not bind to BSP. Notably, BSP is highly heterogeneous as a result of variations in the phosphorylation of serines and O- and N-linked glycosylation . Presumably, BSP expressed by diverse cells types is modified differently, and variations in post-translational modifications may determine the activity of these proteins and the binding and activation of proMMP-2. Accordingly we employed recombinant BSP, BSP purified from bone, or recombinant human BSP to assess binding to proMMP-2. As we were unable to detect binding of any of the BSPs to proMMP-2, there is evidently a need to re-assess the potential ability of BSP to bind to and activate proMMP-2 in the context of cancer cell metastasis although we cannot rule out the possibility that much more highly glycosylated BSP than the bovine BSP we used here could conceivably mediate an interaction with proMMP2.
MMP-2 binds to the surface of cancer cells via the fibronectin type II module repeats of the enzyme [29, 30]. Despite the lack of an interaction between BSP and proMMP-2, it is possible that an interaction between BSP and the αvβ3 integrin itself may trigger downstream signaling events that affect the expression, processing, and activity of MMP-2. Thus, the requirement of an active RGD sequence in BSP-mediated cancer cell invasion suggests that BSP binding to the αvβ3 integrin may promote clustering of integrin molecules, which could activate downstream signaling events. Notably, ECM proteins can promote raft formation and type I collagen activates MMP-2 through β1-integrins, which increases MT1-MMP levels , and by direct binding of pericellular native type I collagen with the MT1-MMP hemopexin domain . MT1-MMP enhances focal proteolysis  and experimental metastasis , is associated with MMP-2 activation in lung carcinoma  and invasive human breast cancer cell lines [34, 35], and is over-expressed in high-grade gliomas, fibrosarcomas  and in carcinomas of the lung, stomach, head and neck . However, in our studies there was no evidence of integrin-mediated enhancement in the level of MT1-MMP transcript level, nor in MT1-MMP activity. Evidently, a more complete understanding of integrin-mediated signaling events will be important for defining the significance of BSP binding to the αvβ3 integrin in vivo.
Collectively, using the methods reported here, our studies do not support a role for BSP in promoting pro-MMP-2 activation.
The research was supported by CIHR Operating, Group and Research Resource grants to SC, CMO, CAM and JS. We thank W. Houry (University of Toronto) with technical assistance and use of equipment for spectroscopy analyses. This research was completed prior to the death of Dr. J. Sodek in August, 2007 and of Dr. S. Cheifetz in May, 2008.
- Oldberg A, Franzen A, Heinegard D: The primary structure of a cell-binding bone sialoprotein. J Biol Chem. 1988, 263 (36): 19430-19432.PubMedGoogle Scholar
- Bellahcene A, Merville MP, Castronovo V: Expression of bone sialoprotein, a bone matrix protein, in human breast cancer. Cancer Res. 1994, 54 (11): 2823-2826.PubMedGoogle Scholar
- Bellahcene A, Kroll M, Liebens F, Castronovo V: Bone sialoprotein expression in primary human breast cancer is associated with bone metastases development. J Bone Miner Res. 1996, 11 (5): 665-670.View ArticlePubMedGoogle Scholar
- Bellahcene A, Menard S, Bufalino R, Moreau L, Castronovo V: Expression of bone sialoprotein in primary human breast cancer is associated with poor survival. Int J Cancer. 1996, 69 (4): 350-353.View ArticlePubMedGoogle Scholar
- Egeblad M, Werb Z: New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer. 2002, 2 (3): 161-174.View ArticlePubMedGoogle Scholar
- Sternlicht MD, Werb Z: How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol. 2001, 17: 463-516.View ArticlePubMedPubMed CentralGoogle Scholar
- Overall CM, Kleifeld O: Towards third generation matrix metalloproteinase inhibitors for cancer therapy. Br J Cancer. 2006, 94 (7): 941-946.View ArticlePubMedPubMed CentralGoogle Scholar
- Murphy G, Knauper V: Relating matrix metalloproteinase structure to function: why the "hemopexin" domain?. Matrix Biol. 1997, 15 (8–9): 511-518.View ArticlePubMedGoogle Scholar
- Nagase H, Woessner JF: Matrix metalloproteinases. J Biol Chem. 1999, 274 (31): 21491-21494.View ArticlePubMedGoogle Scholar
- Overall CM: Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains, modules, and exosites. Mol Biotechnol. 2002, 22 (1): 51-86.View ArticlePubMedGoogle Scholar
- Seiki M: Membrane-type matrix metalloproteinases. APMIS. 1999, 107 (1): 137-143.View ArticlePubMedGoogle Scholar
- Overall CM, King AE, Sam DK, Ong AD, Lau TT, Wallon UM, DeClerck YA, Atherstone J: Identification of the tissue inhibitor of metalloproteinases-2 (TIMP-2) binding site on the hemopexin 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 III and IV. J Biol Chem. 1999, 274 (7): 4421-4429.View ArticlePubMedGoogle Scholar
- Fedarko NS, Jain A, Karadag A, Fisher LW: Three small integrin binding ligand N-linked glycoproteins (SIBLINGs) bind and activate specific matrix metalloproteinases. FASEB J. 2004, 18 (6): 734-736.PubMedGoogle Scholar
- Jain A, Karadag A, Fisher LW, Fedarko NS: Structural requirements for bone sialoprotein binding and modulation of matrix metalloproteinase-2. Biochemistry. 2008, 47 (38): 10162-10170.View ArticlePubMedPubMed CentralGoogle Scholar
- Polette M, Nawrocki B, Gilles C, Sato H, Seiki M, Tournier JM, Birembaut P: MT-MMP expression and localisation in human lung and breast cancers. Virchows Arch. 1996, 428 (1): 29-35.View ArticlePubMedGoogle Scholar
- Ishigaki S, Toi M, Ueno T, Matsumoto H, Muta M, Koike M, Seiki M: Significance of membrane type 1 matrix metalloproteinase expression in breast cancer. Jpn J Cancer Res. 1999, 90 (5): 516-522.View ArticlePubMedGoogle Scholar
- Kanayama H, Yokota K, Kurokawa Y, Murakami Y, Nishitani M, Kagawa S: Prognostic values of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 expression in bladder cancer. Cancer. 1998, 82 (7): 1359-1366.View ArticlePubMedGoogle Scholar
- Morrison CJ, Butler GS, Bigg HF, Roberts CR, Soloway PD, Overall CM: Cellular activation of MMP-2 (gelatinase A) by MT2-MMP occurs via a TIMP-2-independent pathway. J Biol Chem. 2001, 276 (50): 47402-47410.View ArticlePubMedGoogle Scholar
- Mainardi CL, Hasty KA, Hibbs MS: Antibody to rabbit macrophage type V collagenase/gelatinase and its use to further characterize the enzyme. Coll Relat Res. 1984, 4 (3): 209-217.View ArticlePubMedGoogle Scholar
- Overall CM, Sodek J, McCulloch CA, Birek P: Evidence for polymorphonuclear leukocyte collagenase and 92-kilodalton gelatinase in gingival crevicular fluid. Infect Immun. 1991, 59 (12): 4687-4692.PubMedPubMed CentralGoogle Scholar
- Ellerbroek SM, Wu YI, Overall CM, Stack MS: Functional interplay between type I collagen and cell surface matrix metalloproteinase activity. J Biol Chem. 2001, 276 (27): 24833-24842.View ArticlePubMedGoogle Scholar
- Nisato RE, Hosseini G, Sirrenberg C, Butler GS, Crabbe T, Docherty AJ, Wiesner M, Murphy G, Overall CM, Goodman SL, et al: Dissecting the role of matrix metalloproteinases (MMP) and integrin alpha(v)beta3 in angiogenesis in vitro: absence of hemopexin C domain bioactivity, but membrane-Type 1-MMP and alpha(v)beta3 are critical. Cancer Res. 2005, 65 (20): 9377-9387.View ArticlePubMedGoogle Scholar
- Strongin AY, Marmer BL, Grant GA, Goldberg GI: Plasma membrane-dependent activation of the 72-kDa type IV collagenase is prevented by complex formation with TIMP-2. J Biol Chem. 1993, 268 (19): 14033-14039.PubMedGoogle Scholar
- Overall CM, Sodek J: Concanavalin A produces a matrix-degradative phenotype in human fibroblasts. Induction and endogenous activation of collagenase, 72-kDa gelatinase, and Pump-1 is accompanied by the suppression of the tissue inhibitor of matrix metalloproteinases. J Biol Chem. 1990, 265 (34): 21141-21151.PubMedGoogle Scholar
- Miyamori H, Takino T, Kobayashi Y, Tokai H, Itoh Y, Seiki M, Sato H: Claudin promotes activation of pro-matrix metalloproteinase-2 mediated by membrane-type matrix metalloproteinases. J Biol Chem. 2001, 276 (30): 28204-28211.View ArticlePubMedGoogle Scholar
- Tam EM, Wu YI, Butler GS, Stack MS, Overall CM: Collagen binding properties of the membrane type-1 matrix metalloproteinase (MT1-MMP) hemopexin C domain. The ectodomain of the 44-kDa autocatalytic product of MT1-MMP inhibits cell invasion by disrupting native type I collagen cleavage. J Biol Chem. 2002, 277 (41): 39005-39014.View ArticlePubMedGoogle Scholar
- Tam EM, Moore TR, Butler GS, Overall CM: Characterization of the distinct collagen binding, helicase and cleavage mechanisms of matrix metalloproteinase 2 and 14 (gelatinase A and MT1-MMP): the differential roles of the MMP hemopexin c domains and the MMP-2 fibronectin type II modules in collagen triple helicase activities. J Biol Chem. 2004, 279 (41): 43336-43344.View ArticlePubMedGoogle Scholar
- Ganss B, Kim RH, Sodek J: Bone sialoprotein. Crit Rev Oral Biol Med. 1999, 10 (1): 79-98.View ArticlePubMedGoogle Scholar
- Steffensen B, Wallon UM, Overall CM: Extracellular matrix binding properties of recombinant fibronectin type II-like modules of human 72-kDa gelatinase/type IV collagenase. High affinity binding to native type I collagen but not native type IV collagen. J Biol Chem. 1995, 270 (19): 11555-11566.View ArticlePubMedGoogle Scholar
- Saad S, Gottlieb DJ, Bradstock KF, Overall CM, Bendall LJ: Cancer cell-associated fibronectin induces release of matrix metalloproteinase-2 from normal fibroblasts. Cancer Res. 2002, 62 (1): 283-289.PubMedGoogle Scholar
- Seiki M, Yana I: Roles of pericellular proteolysis by membrane type-1 matrix metalloproteinase in cancer invasion and angiogenesis. Cancer Sci. 2003, 94 (7): 569-574.View ArticlePubMedGoogle Scholar
- Tsunezuka Y, Kinoh H, Takino T, Watanabe Y, Okada Y, Shinagawa A, Sato H, Seiki M: Expression of membrane-type matrix metalloproteinase 1 (MT1-MMP) in tumor cells enhances pulmonary metastasis in an experimental metastasis assay. Cancer Res. 1996, 56 (24): 5678-5683.PubMedGoogle Scholar
- Tokuraku M, Sato H, Murakami S, Okada Y, Watanabe Y, Seiki M: Activation of the precursor of gelatinase A/72 kDa type IV collagenase/MMP-2 in lung carcinomas correlates with the expression of membrane-type matrix metalloproteinase (MT-MMP) and with lymph node metastasis. Int J Cancer. 1995, 64 (5): 355-359.View ArticlePubMedGoogle Scholar
- Gilles C, Polette M, Seiki M, Birembaut P, Thompson EW: Implication of collagen type I-induced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma. Lab Invest. 1997, 76 (5): 651-660.PubMedGoogle Scholar
- Pulyaeva H, Bueno J, Polette M, Birembaut P, Sato H, Seiki M, Thompson EW: MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells. Clin Exp Metastasis. 1997, 15 (2): 111-120.View ArticlePubMedGoogle Scholar
- Yamamoto M, Mohanam S, Sawaya R, Fuller GN, Seiki M, Sato H, Gokaslan ZL, Liotta LA, Nicolson GL, Rao JS: Differential expression of membrane-type matrix metalloproteinase and its correlation with gelatinase A activation in human malignant brain tumors in vivo and in vitro. Cancer Res. 1996, 56 (2): 384-392.PubMedGoogle Scholar
- Sato H, Seiki M: Membrane-type matrix metalloproteinases (MT-MMPs) in tumor metastasis. Journal of biochemistry. 1996, 119 (2): 209-215.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/121/prepub
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