Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly Osteopontin (OPN)1 is an arginine-glycine-aspartic acid (RGD)-containing glycoprotein that interacts with integrins and CD44 as major receptors. OPN has been shown to be multifunctional, with activities in cell migration, cell survival, inhibition of calcification, regulation of immune cell function, and control of tumor cell phenotype (1-4). Targeting of the gene encoding OPN, spp1, has revealed that while OPN is not necessary for normal embryonic development, fertility, and health under pathogen-free conditions (5, 6), loss of the protein has significant consequences in several models of injury/disease as diverse as renal injury, viral and bacterial infection, bone remodeling, and tumor growth (7-12). The fact that no other proteins seem to share a redundant activity with OPN under these conditions suggests that OPN has a unique functional role during tissue injury and stress. Interestingly, several members of the matrix metalloproteinase (MMP) family are also induced during injury/disease processes in patterns overlapping that of OPN (13). In particular, we have found that during squamous cell carcinoma progression, OPN and MMP-3 expression correspond both in a temporal and cell-specific fashion (9, 14). We have also identified overlapping expression patterns of OPN and MMP-3 in the stroma during skin incisional wound healing (5) and OPN and MMP-7 during involution of the postpartum uterus (15).OPN is known to be a substrate for proteolytic cleavage by the proteases thrombin (16, 17) and enterokinase (18). Thrombin cleavage of OPN (Arg 168 -Ser 169 in humans, Arg 153 -Ser 154 in rats) is of interest, since hydrolysis of this peptide bond reveals a binding site for the integrins ␣ 9 ␤ 1 (19) and ␣ 4 ␤ 1 (20), SV-VYGLR, not present in the full-length molecule (16). In addition, functional properties of thrombin-cleaved OPN differ from the intact protein (21-24), demonstrating that proteolytic cleavage is one mechanism of regulating the bioactivity of OPN.In the present study, we have identified and characterized novel cleavage sites in OPN for two members of the MMP family, MMP-3 and MMP-7. Furthermore, we show that lower molecular weight forms of OPN corresponding to predicted MMP cleavage fragments are present in cell lines in vitro and in tissues in vivo. Biological assays demonstrate that the MMPcleaved OPN has increased activity in promoting both cell adhesion and migration compared with full-length OPN. In addition, using inhibitory reagents, we have determined that the same receptors that interact with OPN also mediate interaction of MMP-c...

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