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Fig. 3. In fibroblasts, MMP-9 induction by tumor cells depends on the malignancy of the tumor cell line and on the integrity of TGF-ß-, EGFR- and MAPK-signaling. (A) MMP-9 was induced in cocultures of DFs and 10A, At.1k, CA1h or CA1a cells as early as at day 1. The level of its activity depends on the cell-type and also on the age of the culture. (B) Compared with S3WT and CA1a cells cultured together, the expression of MMP-9 is reduced in cocultures of S3KO and CA1a cells. Similar results were obtained for mammary fibroblasts (mS3KO, mS3WT). Notice, MMP-9-forms of a lower molecular weight, suggesting an activation by proteolytic processing in the cocultures. (C) DFs grown to 80% confluence in 24-well plates with DMEM compl. stimulated with CA1a cells (10,000/well) for 24 hours. The culture-supernatant was analyzed by gelatin zymography. Expression of MMP-9 was induced by CA1a cells within 12 hours. (D) Thirty minutes before addition of CA1a cells, DFs were treated with actinomycin D (10 ng/ml) or cycloheximide (10 ng/ml). Both substances blocked the induction of MMP-9 expression, indicating that it depends on an intact RNA- and protein synthesis. Experimental design as in C. (E) Induction of MMP-9 in serum-starved (0% serum) DFs by CA1A cells or CA1a lysate is modulated by several small-molecule inhibitors. DFs were incubated with inhibitors (LY294002, 20 µM; SB431542, 5 µM; manumycin, 5 µM; AG1478, 10 µM; or SB203580, 10 µM) or DMSO (negative control) for 30 minutes, and stimulated for 24 hours with CA1A cells (10,000/well) or lysates of the same number of cells. The culture-supernatant was analyzed by gelatin zymography. Zymograms shown here were optimized to show the effect of inhibitors on MMP-9 induction and show representative results of five to ten experiments. Notice, whereas MMP-9 levels are influenced by stimulation with inhibitors, MMP-2 levels remain constant. This indicates equal growth of fibroblasts, which was confirmed by fibroblast-monolayer staining as described in Materials and Methods. (F) SB431542 inhibits gelatinolysis in cocultures as shown by in-situ zymography. Cultures were treated with SB431542 (5 µM) or SB203580 (10 µM) for a 24-hour period and then again 1 hour before in-situ zymography. Media used for in-situ zymography were supplemented with SB431542 (5 µm) or SB203580 (10 µM). Bar, 80 µm.
