312 (SppI) and early T-lymphocyte activation 1 (Eta-1) (Senger et al. 1989; Patarca et al. 1989). OPN is a member of the SIBLING (Small Integrin-Binding Ligand, N-linked Gly- coprotein) family of proteins, which map to human chromosome 4 (Fisher et al. 2001). OPN is synthesized as an approximately 32 kDa protein, but due to extensive post- translational modifications its apparent molecular mass ranges from 45 to 75 kDa (Kazanecki et al. 2007). OPN possesses a negative charge due to a preponderance of acidic amino acids and serine phosphorylation. OPN also contains calcium binding sites and two putative heparin binding domains (Kon et al. 2008). OPN can interact directly with extracellular matrix proteins including fibro- nectin (Mukherjee et al. 1995) and collagen type I (Chen et al. 1992; Martin et al. 2004). Adhesion motifs OPN has multiple functional adhesive motifs, which allows interactions with many cell types including smooth muscle cells, endothelial cells, and inflammatory cells, thus mediating a broad range of biological functions. The OPN protein is poorly conserved among species (63% human to mouse, 30% human to chicken) however its functional domains are conserved. The highly conserved motifs include: abundance of acidic residues, the RGD integrin binding domain, similar phosphorylation and glycosylation motifs, and at least one site of controlled proteolysis (Bellahcene et al. 2008). The adhesive RGD domain of OPN mediates interactions via αvβ1, αvβ3, αvβ5, αvβ6, α8β1, and α5β1 integrins (Liaw et al. 1995; Yokosaki et al. 2005; Denda et al. 1998; Hu et al. 1995). Immediately C- terminal to the RDG motif is a cryptic SVVYGLR (SLAYGLR in mice) sequence that becomes exposed upon cleavage with thrombin and mediates interactions with α9β1, α4β1, and α4β7 integrins (Yokosaki et al. 1999; Ito et al. 2009; Green et al. 2001). Finally, the ELVTDFTDLPAT (human OPN) domain of OPN has also been described to bind to α4β1 (Bayless and Davis 2001). In addition to interacting with integrins, OPN has also been reported to interact with CD44, the hyaluronic acid receptor (Weber et al. 1996). A number of different CD44 isoforms exist due to alternative splicing of the 10 variant exons and while OPN can bind some CD44 splice variants, notably v6 and v7, OPN does not bind to the standard isoform, CD44H (Smith et al. 1999; Katagiri et al. 1999). Further, interactions between OPN and CD44 appears to be mediated via β1 integrins in an RGD independent manner (Katagiri et al. 1999). Other studies indicate interactions between the C-terminal fragment of thrombin cleaved OPN and a CD44 variant (Weber et al. 2002). However, the precise domain of OPN that interacts with CD44 has not been identified. In addition, growing evidence suggests that S.A. Lund et al. OPN is a major regulator of CD44 surface expression, especially in osteoclasts (Chellaiah et al. 2003; Marroquin et al. 2004). Post-translational modifications OPN is subject to extensive post-translational modifica- tions including serine and threonine phosphorylation. Phosphorylation is cell specific with phosphorylation levels varying depending on the tissue type. OPN in milk is highly phosphorylated with human milk OPN containing 36 phosphate sites (Christensen et al. 2005). Phosphorylation tends to occur in clusters separated by stretches of unmodified residues. Normal rat kidney cells can secrete both phosphorylated and non-phosphorylated forms, indicating regulated control of phosphorylation (Singh et al. 1990). In some cases, OPN function is tightly controlled by phosphorylation state. Calcification of smooth muscle cells in vitro is inhibited by native OPN, but desphosphorylated or recombinant bacterially pro- duced OPN has no effect on calcification (Jono et al. 2000). Similarly, in vivo in OPN-null mice, phosphorylat- ed OPN, but not non-phosphorylated, prevents ectopic calcification in a subcutaneous model of bioprosthetic valve mineralization (Ohri et al. 2005). In contrast, the adhesive activity of OPN is not dependent on post- translational modifications, since bacterially derived re- combinant OPN has been shown to support cell adhesion in a wide variety of cell types (Gao et al. 2004; Xuan et al. 1994). OPN is also subject to sulfation (Nagata et al. 1989), glycosylation (Sorensen et al. 1995) and trans- glutamination (Beninati et al. 1994). Interestingly, poly- merization of OPN by transglutaminase 2 has been reported to increase the adhesive activity of OPN via the α9β1 integrin, independent of the SVVYGLR adhesion domain (Nishimichi et al. 2009). Precise regulation of OPN post-translational modifications may represent a mechanism to control OPN function. Proteolytic processing The bioactivity of OPN can be further regulated by proteolytic processing. OPN is a substrate for thrombin and the matrix metalloproteinases, MMP-3 (stromelysin-1), MMP-7 (matrilysin), MMP-2 and MMP-9 (Agnihotri et al. 2001; Dean and Overall 2007; Takafuji et al. 2007). Human OPN contains three cleavage sites for MMPs; Gly166- Leu167, Ala201-Tyr202, and Asp210-Leu211. The thrombin cleavage site generating a RGD and SVVYGLR (SLAYGLR in mice) is conserved in human and mice. Thrombin cleavage of OPN, Arg168-Ser169 in humans and Arg153-Ser154 in mice, reveals a cryptic SVVYGLR binding domain capable of fostering interactions with α9β1 (Smith