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Journal of Dental Research, Vol. 81, No. 12,
831-35 (2002)
DOI: 10.1177/154405910208101207
MMP-9 Activation by Tumor Trypsin-2 Enhances in vivo Invasion of Human Tongue Carcinoma Cells
P. Nyberg1,
M. Moilanen1,
A. Paju2,
A. Sarin1,
U.-H. Stenman2,
T. Sorsa3 and
T. Salo1,*
1 Departments of Diagnostics and Oral Medicine, Institute of Dentistry, University of Oulu, PO Box 5281, FIN-90014, Oulu, Finland;
2 Department of Clinical Chemistry, Helsinki University Central Hospital, FIN-00029, Helsinki, Finland; and
3 Biomedicum and Oral Pathology Unit, Institute of Dentistry, Laboratory Diagnostics, Helsinki University Central Hospital, University of Helsinki, FIN-00014, Helsinki, Finland;
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Figure 1. TAT-2 protein production in human oral mucosal keratinocytic cell lines increased after TAT-2 gene transfection. The amount of secreted TAT-2 protein in HSC-3, SCC-25, and IHGK clones after transfection of a control vector or TAT-2 vector was measured from serum-free conditioned cell culture media (48 hrs) of 3 independent clones per cell line (two measurements for each clone) by immunofluorometric assay specific for TAT-2 (Itkonen et al., 1990). The data represent mean + SD.
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Figure 2. Activation of proMMP-9 increased after TAT-2 transfection of HSC-3 cells. (A) The activity of MMP-9 was measured by gelatin zymography of serum-free conditioned culture media of HSC-3 + TAT-2 (two clones with the highest increase in TAT-2 production) and control HSC-3 cells. The cell culture media treated with 50 ng/mL enterokinase was collected after 48 hrs of incubation. The upper 92-kDa gelatinolytic band represents the latent form of MMP-9 (proMMP-9), the middle 77-kDa band the active form (aMMP-9), and the lowest band the 72-kDa latent form of MMP-2 (proMMP-2). The proportion of active MMP-9 was significantly increased in TAT-2-transfected cells (lanes 5, 6) compared with control HSC-3 cells (lanes 1, 2). The addition of TATI (10 µg/mL) abolished the increased activation of MMP-9 (lanes 3, 4). (B) The ratio of active MMP-9 (aMMP-9) to latent MMP-9 (proMMP-9) in TAT-2-transfected HCS-3 cell media, in the control HSC-3 cell media and in TATI-treated TAT-2-transfected cell media. Gelatinolytic bands (n > 4) from zymography were quantitated with ScionImage software. Statistical analysis was performed by Scheffé's test, **p < 0.01. (C) ECL-Western immunoblot using polyclonal anti-MMP-9 antibody (Kjeldsen et al., 1993): conditioned cell culture media from control HSC-3 cells (lane 1), control cells treated with TATI (lane 2), TAT-2-transfected cells (lane 3), TAT-2-transfected cells with TATI (lane 4), control HSC-3 cells with enterokinase (lane 5), control cells treated with TATI and enterokinase (lane 6), TAT-2-transfected cells with enterokinase (lane 7), TAT-2-transfected cells with TATI and enterokinase (lane 8). (D) ECL-Western immunoblot using a specific antibody (Duncan et al., 1998) that recognizes only the activated form of MMP-9. The amount of MMP-9 in serum-free conditioned cell culture media increased after transfection of TAT-2 (lanes 2, 3) to HSC-3 cells (lane 1). The 77-kDa immunoreactive bands represent the active form of MMP-9. The sizes of molecular-weight standards (kDa) are shown in the left in A, C, and D.
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Figure 3. Intravasation of TAT-2-transfected HCS-3 cells was significantly more efficient than control HSC-3 cells in the CAM model. (A) 2 x 106 HCS-3 control cells or TAT-2-transfected clones (with the highest increase in TAT-2 production as measured previously with immunofluorometric assay according to Itkonen et al. [1990]) were inoculated in quadruplicate on upper CAMs with or without enterokinase. Fifty hrs after inoculation, the human DNA content from control and TAT-2-transfected cells in the lower CAM was determined by radioactive Alu-PCR. The resulting 220-bp PCR bands were quantitated by ScionImage. The band intensity and thus the number of intravasated cells changed from almost no intravasation in untreated HSC-3 control cells (lane 1), to a mild increase in HSC-3 control cells inoculated with enterokinase (lane 2), to a moderate increase in TAT-2-transfected HSC-3 cells without addition of enterokinase (lane 3), to a very significant increase in TAT-2-transfected HSC-3 cells in the presence of enterokinase (lane 4).
(B) To examine whether TAT-2 was really the reason for the increased intravasation, we inoculated 600 ng of TATI onto the CAMs along with the TAT-2-transfected HSC-3 cells (lane 3), together with enterokinase (lane 4). The intravasation efficiency was compared with the TAT-2-transfected HSC-3 cells with (lane 2) or without enterokinase (lane 1).
(C) Quantitated CAM assay results. Alu-PCR bands from four experiments were scanned, and the results were expressed as the percentage of change in band intensity compared with untreated HSC-3 control band intensities, mean + SD. The statistical differences among all groups were evaluated with Scheffé's test: *p < 0.05, **p < 0.01, and ***p < 0.001.
(D) TAT-2 overproduction only slightly increased the intravasation efficiency of SCC-25 cells (lane 3) compared with control SCC-25 cells (lane 1) and enterokinase-treated control cells (lane 2). Enterokinase intensified the effect of TAT-2 (lane 4).
(E) Pre-malignant IHGK cells did not intravasate (lane 1), and enterokinase (lane 2), TAT-2 (lane 3), or the combination of them (lane 4) caused only a very slight increase.
(F) The effect of MMP-9 inhibition by a specific inhibitor, CTT-peptide (Koivunen et al., 1999), on intravasation efficiency was determined with CAM assay: HSC-3 control cells (lane 1), HSC-3 cells with 2 µg/CAM CTT-peptide (lanes 2, 3), 20 µg/CAM CTT-peptide (lanes 4, 5), and 100 µg/CAM CTT-peptide (lanes 6, 7).
(G) The effect of negative control peptide on intravasation: HSC-3 control cells (lane 1), HSC-3 cells with 2 µg/CAM control peptide (lanes 2, 3), 20 µg/CAM control peptide (lanes 4, 5), and 100 µg/CAM control peptide (lanes 6, 7).
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