Matrix metalloproteinases (MMPs) degrade the extracellular matrix (ECM) and play critical roles in tissue repair, tumor invasion, and metastasis. MMPs are regulated by different cytokines, ECM proteins, and other factors. However, the molecular mechanisms by which osteopontin (OPN), an ECM protein, regulates ECM invasion and tumor growth and modulates MMP activation in B16F10 cells are not well defined. We have purified OPN from human milk and shown that OPN induces pro-MMP-2 production and activation in these cells. Moreover, our data revealed that OPN-induced membrane type 1 (MT1) MMP expression correlates with translocation of p65 (nuclear factor-B (NF-B)) into the nucleus. However, when the super-repressor form of IB␣ (inhibitor of NF-B) was transfected into cells followed by treatment with OPN, no induction of MT1-MMP expression was observed, indicating that OPN activates pro-MMP-2 via an NF-B-mediated pathway. OPN also enhanced cell migration and ECM invasion by interacting with ␣ v  3 integrin, but these effects were reduced drastically when the MMP-2-specific antisense S-oligonucleotide was used to suppress MMP-2 expression. Interestingly, when the OPN-treated cells were injected into nude mice, the mice developed larger tumors, and the MMP-2 levels in the tumors were significantly higher than in controls. The proliferation data indicate that OPN increases the growth rate in these cells. Both tumor size and MMP-2 expression were reduced dramatically when anti-MMP-2 antibody or antisense S-oligonucleotide-transfected cells were injected into the nude mice. To our knowledge, this is the first report that MMP-2 plays a direct role in OPN-induced cell migration, invasion, and tumor growth and that demonstrates that OPN-stimulated MMP-2 activation occurs through NF-B-mediated induction of MT1-MMP.Cell migration and extracellular matrix invasion are some of the major steps in embryonic development (1, 2) and wound healing and cancer cell metastasis (3, 4). However, the exact molecular mechanisms that regulate these processes are not well understood. In the past, several investigators have shown that matrix metalloproteinases (MMPs) 1 and the tissue inhibitor of matrix metalloproteinase (TIMP) play a major role in the regulation of cancer cell migration, extracellular matrix (ECM) invasion, and metastasis by degrading the ECM proteins (3-5). Current investigations have focused on the understanding of molecular mechanism(s) by which osteopontin (OPN), an ECM protein, regulates MMP expression both in vitro and in vivo and controls invasiveness and tumor growth in B16F10 cells.OPN is a noncollagenous, sialic acid-rich, and glycosylated phosphoprotein (6, 7). It has an N-terminal signal sequence, a highly acidic region consisting of nine consecutive aspartic acid residues, and a GRGDS cell adhesion sequence predicted to be flanked by the -sheet structure (8). This protein has a functional thrombin cleavage site and is a substrate for tissue transglutaminase (7). OPN binds with type I collagen (9), fibronectin (...