Cell Type-specific Post-translational Modifications of Mouse Osteopontin Are Associated with Different Adhesive Properties

Abstract

Osteopontin (OPN) is a highly modified integrin-binding protein found in all body fluids. Expression of OPN is strongly correlated with poor prognosis in many different human cancers, suggesting an important but poorly understood role for this protein in tumorigenesis and metastasis. The protein exists in a number of different isoforms differing in the degree of post-translational modifications that are likely to exhibit different functional properties. This study examines for the first time the post-translational modifications of OPN from transformed cells and the effects of these modifications on cell biology. We have characterized the complete phosphorylation and glycosylation patterns of OPN expressed by murine ras-transformed fibroblasts (FbOPN) and differentiating osteoblasts (ObOPN) by a combination of mass spectrometric analyses and Edman degradation. Mass spectrometric analysis showed masses of 34.9 and 35.9 kDa for FbOPN and ObOPN, respectively. Enzymatic dephosphorylation, sequence, and mass analyses demonstrated that FbOPN contains approximately four phosphate groups distributed over 16 potential phosphorylation sites, whereas ObOPN contains ϳ21 phosphate groups distributed over 27 sites. Five residues are O-glycosylated in both isoforms. These residues are fully modified in FbOPN, whereas one site is partially glycosylated in ObOPN. Although both forms of OPN mediated robust integrin-mediated adhesion of mouse ras-transformed fibroblasts, the less phosphorylated FbOPN mediated binding of MDA-MD-435 human tumor cells almost 6-fold more than the heavy phosphorylated ObOPN. These results strongly support the hypothesis that the degree of phosphorylation of OPN produced by different cell types can regulate its function. Osteopontin (OPN)3 is a highly phosphorylated glycoprotein comprising ϳ300 amino acid residues. The protein was first purified from the mineralized matrix of bovine bone (1). However, the presence of OPN is not limited to mineralized tissues but extends to a variety of tissues, cell types, and physiological fluids, including blood, urine, and milk (2). The amino acid sequence of OPN is rich in acidic amino acids and contains an integrin-binding Arg-Gly-Asp (RGD) sequence. OPN is a pleiotropic protein involved in a variety of cellular processes such as migration, adhesion, and signaling (2, 3). OPN is a key molecule in bone remodeling and functions as an inhibitor of ectopic calcification by inhibiting the formation of hydroxylapatite and calcium oxalate (4 -6). Furthermore, OPN is implicated in diverse biological processes, including tumorigenesis, metastasis, cytokine production, wound healing, autoimmune disease, and stroke (3, 7-10). OPN has recently been demonstrated to be required for mucosal protection in acute inflammatory colitis (11).OPN-integrin interaction controls many aspects of cell behavior, including cell attachment, migration, chemotaxis, and immune modulation in various cell types (2, 12). The ␣ v ␤ 6 , ␣ 5 ␤ 1 , ␣ 8 ␤ 1 , ␣ v ␤ 1 , ␣ v ␤ 5 , and ␣ v ␤ 3 integrins recog...

Tumor-derived osteopontin isoforms cooperate with TRP53 and CCL2 to promote lung metastasis

“…As previously reported on the distinct adhesive properties of murine osteopontin that result from cell specific post-translational modifications, 38 we show that iSPP1 and sSPP1 are differentially expressed by tumor cells, with the former preventing TRP53 stabilization and apoptosis, and the latter signaling to host CCR2 receptors via CCL2 to promote lung homing. Our results corroborate previous work on the inhibition of apoptosis 39 and the involvement of TRP53 in iSPP1-dependent anti-apoptotic signaling 40 and show for the first time that the pro-survival effects of osteopontin in cancer cells are conditional on wild-type TRP53.…”

Osteopontin: Role in immune regulation and stress responses

“…In studies where I supplied OPN -/-mice with exogenous OPN, mice were injected with OPN from several sources: specifically preparations of OPN from RAStransformed mouse fibroblast cultures purified by Chris Kazanecki (Kazanecki, 2007) and a commercial mouse recombinant OPN expressed in a mouse myeloma cell line NS0 (R&D Systems, 441-OP). OPN was detectable in the plasma of all samples from these experiments and the amount of OPN correlated to the number of injections each animal received.…”

Pre- and post-translational regulation of osteopontin in cancer

“…Variation exists in the type of glycan structures which may consist of different combinations of N-acetylhexosamine, hexose, and sialic acid residues (Christensen et al 2007;Christensen et al 2008;Keykhosravani et al 2005). Corresponding regions in OPN from other sources likewise contain O-glycosylated threonines and serines.…”

“…These modifications can be cell type-specific (Christensen et al 2007), may depend on other physiological and pathological factors and may impact both OPN structure and function (Anborgh et al 2009;Zhang et al 2007). This review will focus on OPN's role in cancer and how splice variants and posttranslational modifications may influence its effects in cancer cells.…”