Panel A, staining of a human osteosarcoma with a PAR-1 antibody reveals strong staining in solid tumor areas with osteoblastic differentiation (open arrow), whereas single tumor cells are within a chondroblastic component (black arrow) (×200, lower panel). Panel B, negative control of case shown in A (×100) substituting IgG1 for the primary antibody (×200, lower panel). Panel C, trichrome stain to detect collagenous matrix of patient shown in A reveals blue staining of the chondroid matrix (red arrow) and red staining of collagenous matrix (yellow arrow). Tumor cells (brown dots with white halo) are scattered within both matrices (×200, lower panel). Panel D, summary of the immunohistochemical score from 25 primary osteosarcomas. 24 of 25 tissues from patients with osteosarcomas were PAR-1 positive. The number of patients in each score category is indicated below the score. Panel E, RT-PCR. Total RNA extracted either from snap frozen human osteosarcomas or U2-OS cells were reverse-transcribed, and the cDNA was subjected to PCR using primers for PAR-1.

U2-OS cells were stimulated with the indicated amounts of thrombin with or without the PI 3-kinase inhibitor Ly294002 and then subjected to zymography (A) or Western blotting (B) with a phosphospecific AKT (Ser-473) antibody. The membrane was reprobed with an antibody recognizing total AKT.

Panel A, FACS: the expression level of α- and β-integrins was measured in U2-OS cells with and without thrombin stimulation. Cells were detached, incubated with the indicated primary monoclonal antibodies, followed by a phosphatidylethanolamine-labeled secondary antibody, and then subjected to FACS analysis. Isotypic mouse IgG was used as a negative control. Panel B, Western blot analysis of β1-integrin in U2-OS cells after thrombin stimulation with or without the PI 3-kinase inhibitor Ly294002. The membrane was re-probed with an antibody detecting actin. Panel C, quantitative RT-PCR. Messenger RNA was extracted from U2-OS cells treated with thrombin or with thrombin and LY294002. Relative expression of β1-integrin normalized to GAPDH was measured by SYBR Green quantitative real time PCR. Panel D, Matrigel invasion assay. U2-OS cells (50,000/well) were plated on the top chamber with serum-free media in the presence of the indicated concentrations of blocking antibodies against β1- or αvβ5-integrin antibody or control IgG and allowed to invade through the Matrigel barrier for 24 h. Invading cells on the underside of the filter were enumerated using an inverted microscope after cells were removed from the top aspect of the filter. Experiments were performed in triplicates with a minimum of 10 grids (×40 magnification) per filter counted (* indicates p < 0.005).

Panel A, protein binding assay. The MMP-9 hemopexin domain was coated to ELISA plates and incubated with an α5β1-integrin-Fc fusion protein. Bound protein was detected via the Fc part of the fusion protein using horseradish peroxidase-conjugated protein A. The values for the protein detected on coated SA as negative control were subtracted from the corresponding MMP-9-bound signals. α5β1-integrin binds to the MMP-9 hemopexin domain in a dose-dependent manner. Panel B, immunoprecipitation, U2-OS cells were preincubated with or without a peptide spanning the LRSG sequence in the hemopexin domain of MMP-9, a scrambled control peptide, or a MMP-9 antibody raised against a sequence in the hemopexin domain adjacent to LRSG followed by stimulation with thrombin. Cell lysates (250 µg) were immunoprecipitated with a β1-integrin antibody and immunoblotted with a MMP-9 antibody raised against the hemopexin domain. Panel C, gelatinase assay. U2-OS cells were seeded in a 96-well plate, cultured in serum-free media, and stimulated with thrombin, or with thrombin and treated with the LRSG-peptide. uenched gelatin was added and fluorescence measured at the indicated times as described in the legend to Fig. 2E. Panels D–F, invasion assays. U2-OS cells were plated on a Matrigel-coated modified Boyden chamber and then incubated with or without thrombin, the LRSG, or a scrambled control peptide (D) or panel E using the antibody against the MMP-9 hemopexin domain. Panel F, TE-85 osteosarcoma cells were plated on a Matrigel-coated modified Boyden chamber and then incubated with or without 4 units/ml of thrombin. Invasion assays (D–F) were performed in triplicate with a minimum of 10 grids (40× magnification) per filter counted (n.s., not significant, * indicates p < 0.05, *** indicates p < 0.001). All experiments were repeated at least three times.

Panel A, zymography: U2-OS osteosarcoma cells were stimulated with thrombin or TRAP peptide at the indicated concentrations. HT-1080 cells were used as a positive control. Panel B, TE85 osteosarcoma cells were stimulated with 2 units/ml of thrombin. For A and B conditioned media normalized for differences in cell number was subjected to gelatin zymography using a 10% SDS-PAGE containing 0.1% gelatin. Panel C, U2-OS cells were stimulated with thrombin or thrombin and the PI 3-kinase inhibitor LY294002, cells were extracted using RIPA buffer and subjected to Western blotting using aMMP-9 antibody. Panel D, the membrane was reprobed with an antibody against MT1-MMP and actin. Panel E, gelatinase assay: cell-associated gelatinolytic activity was measured in U2-OS cells using quenched fluorescent-labeled gelatin. Cells were stimulated with thrombin and where indicated human recombinant TIMP-1 protein (25µM) or the MMP inhibitor GM 6001 (50µM) added. Fluorescence was measured (excitation = 488 nm, emission = 528 nm) with a fluorescence spectrophotometer. Panel F, quantitative real time RT-PCR: total RNA was extracted from U2-OS cells and the relative expression of MMP-9 normalized to GAPDH was measured using TaqMan quantitative real time RT-PCR. Panel G, transfection. U2-OS were transiently transfected with the MMP-9–670 promoter chloramphenicol acetyltransferase construct (MMP-9) followed by stimulation with thrombin. Co-transfection with an expression plasmid for H-ras was in cluded a sa positive control. Cells were lysed 24h after stimulation. Chloramphenicol acetyltransferase activity was measured by incubating cell lysates with 4µm [¹⁴C]chloramphenicol and 1 mg/ml acetyl coenzyme A. The mixture was separated by extraction with ethyl acetate and acetylated products separated on thin layer chromatography plates using chloroform/methanol as the mobile phase. Reactions were visualized by autoradiography. Panel H, staining of a human osteosarcoma, fibroblastic type with a MMP-9 antibody. Pleomorphic spindle cells, organized in sheets reveal strong staining (×200 and 400).

Panel A, invasion assay: U2-OS cells were plated in a modified Boyden chamber coated with Matrigel and incubated for 24 h. Cells were stimulated with thrombin followed by treatment with the PI 3-kinase inhibitor LY294002 or the MMP inhibitor GM 6001 or in panel B with either a blocking antibody against MMP-9 (#6-6B, 2µg/ml) or with a blocking antibody against urokinase (uPA) (394, 10 µg/ml). Experiments were performed in triplicate with a minimum of 10 grids (×40 magnification) per filter counted (*** indicates p < 0.001). Panel C, the inhibitory effect of the anti-MMP-9 antibody on recombinant human MMP-9 (squares) and MMP-2 (triangles) was tested using quenched gelatin as described in the legend to Fig. 2E.

Panel A, FACS. U2-OS cells were stimulated with 2 units/ml of thrombin and MMP-9 expression on the cell surface measured. Panel B, immunoprecipitation. U2-OS cells were stimulated with thrombin alone or with thrombin and the PI 3-kinase inhibitor LY294002. Cells were lysed and 250µg of protein subjected to immunoprecipitation (IP) with an antibody against MMP-9 or β1-integrin, respectively. The proteins were separated from the agarose, resolved on a SDS-PAGE gel, and transferred to a nitrocellulose membrane, which was immunoblotted (IB) with an antibody against β1-integrin or MMP-9. As a control, lysates were immunoprecipitated and blotted with antibodies against MMP-9 and β1-integrin. Lower panel, control immunoprecipitation with anMMP-9isotype-specific IgG. Panel C, confocal laser scanning immunofluorescence for MMP-9 and β1-integrin. U2-OS cells were plated in 8-well culture chambers precoated with collagen I and stimulated with thrombin, fixed, and triple staining performed with MMP-9 antibody (green), β1-integrin antibody (red), and 4′,6-diamidino-2-phenylindole (blue) to visualize the nuclei. The yellow signal indicates co-localization of MMP-9 and β1-integrin. The images are representative of the most prevalent cells (bar, 20µm).

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