Next we examined MMP-3, a member of the matrix metalloproteinase family that has the ability degrade many components of the extracellular matrix, such as collagen III-V, and IX-XI, as well as laminins, elastins, fibronectin, vitronectins and proteoglycans . Mouse epithelial mammary cells cultured with MMP-3 had decreased expression of cytokeratin markers and increased expression of vimentin, a clear sign of the epithelial-to-mesenchymal transition (EMT), in which epithelial cells morph into a mesenchymal-type cell to eliminate their connection to the basement membrane and initiate migration towards subsequent intravasation into blood vessels . MMP-3 can also activate other MMPs, and high levels of MMP-3 correlate with poor prognosis in breast cancer patients . MDA cells were treated with cmvIL-10 and then total MMP-3 levels were measured by ELISA. We were unable to detect any MMP-3 in cell supernatants, but cell-associated MMP-3 was detected by analysis of cell lysates. Relatively low levels of MMP-3 were produced by untreated MDA cells, but this amount increased significantly after 48 h of treatment with cmvIL-10 (Fig. 4a). Since MMPs are generally secreted as inactive pro-enzymes that require cleavage to become activated, we further examined MMP-3 by western blotting and zymography. Consistent with the ELISA results, an increase in total MMP-3 protein was observed over time with exposure to cmvIL-10 (Fig. 4b). The amount of active MMP-3 enzyme was also increased by cmvIL-10 treatment, as evidence by increased digestion of casein in the zymogen gel. Taken together, these results indicate that cmvIL-10 promotes increased expression and activation of MMP-3 by breast cancer cells, which is likely to contribute to increased degradation of the ECM and greater risk of metastasis.Fig. 4MMP-3 expression and activity are increased by cmvIL-10. a MDA-MB-231 cells were cultured in the presence or absence of 10 ng/ml cmvIL-10 for the indicated times and then cell lysates were analyzed by ELISA. Error bars SEM, *p < 0.05. b MDA cells cultured with 100 ng/ml cmvIL-10 for the indicated times were harvested and lysates examined by western blotting with anti-MMP3 or anti-MAPK as a protein loading control. Lysates from cells receiving the same treatment were analyzed under non-reducing conditions on a 4–16% Zymogram gel (lower panel). Arrow indicates active MMP-3. These results are representative of three independent experiments