DAVID T. DENHARDT |
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A personal overview of osteopontin:
OPN is an O-glycosylated phosphoprotein that is synthesized in a variety
of tissues and cells and secreted into body fluids. It was identified
both as a major non-collagenous bone matrix protein and as a cytokine
(Eta-1) produced by activated T cells and transformed cell lines. Receptors
for OPN include certain integrins and CD44 variants. These receptors
mediate cell adhesion, migration and survival in many cell types. OPN?s
potential to interact with ubiquitously expressed multiple cell surface
receptors makes it an active player in many physiological and pathological
processes including wound healing, bone turnover, tumorigenesis, inflammation,
ischemia, stress, and immune responses.
In the immune system, OPN is expressed by many different cell types, including macrophages, neutrophils, dendritic cells, NK cells, and T and B lymphocytes; it is up-regulated in response to injury and inflammation in every organ examined; for example, cardiac tissue, kidney, lung, bone, brain, the gastrointestinal tract, joints, liver, adipose tissue and most tumors. OPN has been identified as a biomarker for various types of cancers and inflammatory diseases. Excessive or dysregulated OPN expression has been linked to the pathogenesis of both autoimmune disorders such as multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, atherosclerosis and other inflammatory diseases including cardiovascular disease, chronic obstructive pulmonary disease, inflammatory bowel disease, liver disease and asthma. Although initially regarded as an RGD-containing adhesive bone matrix protein because of its presence in the extracellular matrix of mineralized tissues, it is now established as a soluble cytokine/hormone capable of stimulating signal transduction pathways in many different cell types. Structure of OPN: OPN is a highly negatively charged protein that lacks extensive secondary structure. It is encoded by a single gene in a cluster of ?SIBLING? family proteins (Small Integrin Binding Ligand N-linked Glycoprotein, though not all are ?N-linked?) located on chromosome 4 in humans. Its promoter is responsive to a number of different transcription factors. Full-length OPN is composed of some 300 amino acids (297 in mouse; 314 in human); there are also functionally important cleavage products and occasional splice variants. Although expressed as a ~33-kDa nascent protein, extensive posttranslational modifications increase its apparent MW to about 44 kDa; in SDS-PAGE gels it migrates in the range of 50-75 kDa depending on conditions. Both highly conserved sequence motifs and post-translational modifications contribute to different functional activities of OPN. Integrin Receptors: Located near the center of the OPN protein is an arginine-glycine-aspartate (RGD) domain, a motif common to many extracellular matrix proteins and known to engage many integrins. OPN also contains an aspartate-rich, mineral-binding region, two heparin-binding sites, a thrombin cleavage site and somewhere near the C-terminus a region that binds specific CD44 variants. Via the RGD sequence, OPN interacts with the avb1, avb3, avb5, avb6, and a5b1 integrins. A cryptic integrin binding site (SVVYGLR in human, SLAYGLR in mouse) is exposed after thrombin cleavage between the L and R residues; OPN is important in the regulation of cell adhesion, spreading and migration by promoting the adherence of cells expressing a4 and a9 integrins (a9b1, a4b7), which are preferentially expressed by leukocytes. Both intact OPN and the N-terminal fragment of OPN promote leukocyte adhesion to a4b1; interestingly, there are two different binding sites for a4b1 present in a 38-amino acid domain within the N-terminal thrombin fragment. The interaction of OPN with the a8b1 integrin is necessary for normal kidney morphogenesis. CD44: OPN interacts with CD44 v6- and v7-containing isoforms, stimulating (in a human tumor cell) transcription of the CD44 gene and enhancing the abundance of CD44s, v6 and v9 at the cell surface. The interaction appears to be RGD-independent and to require the presence of ß1 integrins. The ligation of CD44 variant isoforms by OPN, likely involving a site in the C-terminal region of OPN, mediates chemotaxis and adhesion of fibroblasts, T cells and bone marrow cells. CD44 engagement with OPN down-regulates IL-10 expression in peritoneal macrophages. OPN promotes proliferation and survival of IL-3-dependent bone marrow cells; anti-CD44 antibody attenuates these actions. A monoclonal antibody recognizing the C-terminal region of OPN blocks the ability of cells to attach to OPN, suggesting that attachment of OPN to CD44 modulates the cells? ability to bind to the RGD binding motif via the RGD sequence. Post-translational modifications (PTM): PTMs of OPN influence OPN function. The OPN protein is highly modified, including ser/thr phosphorylation, O-linked glycosylation, tyrosine sulfation and sialylation. Many sites of PTMs are conserved across species; however the degree of modification of the protein varies depending on the source tissue and cell type or differentiation stage. For example, both bovine and human milk OPN have a large number of phosphorylated serine residues (28 and 32 respectively, mostly in motifs implicating casein kinase 2). These are located in clusters that are distant from the RGDSVVY and glycosylated regions. In contrast, rat bone OPN (at least as isolated) contains only 10-11 phosphorylated residues. Phosphorylation of OPN appears necessary for various physiological functions, including migration of cancer cells, adhesion and bone resorption by osteoclasts, inhibition of smooth muscle cell calcification and regulation of mineralization. The phosphorylation of OPN is usually heterogeneous, and it is not known whether certain specific sites are critical for a given function. Intracellular OPN: An intracellular form
of OPN (iOPN, possibly the consequence of translation initiation downstream
of the usual start site) exists as an integral component of a CD44-ezrin/radixin/moesin
attachment complex on the inner surface of the plasma membrane; it is
involved in the migration of embryonic fibroblasts, activated macrophages,
and metastatic cells. In association with CD44 inside migratory cells,
iOPN modulates cytoskeletal-related functions including cell motility,
cell fusion and survival. The interaction of iOPN with CD44 presumably
involves a constant intracellular domain of CD44 in contrast with the
interaction of sOPN (secreted OPN) with extracellular variant domains
of CD44. Recent findings have revealed that iOPN also mediates IFN-a
expression in plasmacytoid dendritic cells by selectively coupling TLR9
signaling to expression of IFN-a. This indicates that OPN and apoptosis: Apoptosis plays an important role in many physiological/pathological processes because of its ability to inhibit the apoptotic response. OPN has long been regarded as a survival factor, in part by inhibiting apoptosis induced by a physical-chemical insult, growth factor deprivation for example. It has been demonstrated that OPN is an important anti-apoptotic factor in many circumstances; for example, OPN promotes cancer cell metastasis due to its anti-apoptotic properties that prevent programmed cell death and allows uncontrolled proliferation of tumor cells. OPN has also been found to block the activation-induced cell death of macrophages and T lymphocytes as well as fibroblasts and endothelial cells exposed to harmful stimuli. Interestingly, OPN appears to increase the survival of cardiac fibroblasts exposed to H2O2 and undergoing a caspase-3-independent form of necrosis. OPN prevention of non-programmed cell death was also observed in inflammatory colitis. OPN is a ligand for CD44 variants. Evidently, OPN may foster cell survival in a variety of contexts; for example, cancer metastasis, immune system stress, ischemia-reperfusion, and in some brain pathologies. In a sense it behaves as a soluble extracellular matrix molecule, engaging the same receptors as components of the extracellular matrix. Evidence suggests that OPN is a component of the hematopoietic stem cell niche, acting as a negative regulator of hematopoietic stem cell proliferation. OPN exhibits a highly restricted pattern of expression at the endosteal surface and contributes to HSC trans-marrow migration toward the endosteal region after transplantation. OPN-/- mice exhibit enhanced cell cycling and exogenous OPN suppresses the proliferation and differentiation of primitive stem cells in vitro indicating OPN?s negative role in HSC proliferation. Signaling pathways: OPN signaling through integrins can modulate (via activation of Ras and Src) the phosphorylation of kinases (NIK, IKKb) involved in NF?B activation; this results in the degradation of IB, an inhibitor of NF?B. NF?B regulates expression of many inflammatory cytokines. Several reports have concluded that OPN expression is increased by PI3K /Akt signaling. OPN signaling through CD44 engagement promotes cell survival by activating the PI3K/Akt pathway. A genetic profiling study documented that OPN is a downstream effector of the PI3K/Akt pathway, which is antagonized by PTEN; melanoma lines defective in PTEN expression exhibited increased OPN expression. Intracellular OPN is found to be localized together with the MyD88 and TLR9 complex near the inner cytoplasmic membrane; it activates nuclear translocation of transcription factor IRF7 to induce robust IFN-? production. OPN expression is responsive to many transcription factors. The OPN promoter can be activated by TGF-ß through Smad signaling pathways. An activator protein-1 (AP-1) consensus site in the OPN promoter has been identified that supports OPN transcription in macrophages. Liver X receptor agonists inhibit cytokine-induced OPN expression in macrophages through interference with AP-1 signaling pathways. AP-1 regulation is further demonstrated in that OPN transcription is suppressed by PPAR-a agonists through repression of AP-1-dependent transactivation of the OPN promoter.
Wang KX, Denhardt DT (2008) Osteopontin: Role in immune regulation and stress responses. Cytokine Growth Factor Reviews (in press) Bellahc?ne A, Castronovo V, Ogbureke KU, Fisher LW, Fedarko NS. (2008) Small integrin-binding ligand N-linked glycoproteins (SIBLINGs): multifunctional proteins in cancer. Nat Rev Cancer. 8:212-26. Bulfone-Paus S, Paus R. (2008) Osteopontin as a new player
in mast cell biology. Eur J Immunol. 38:338-41. |