Osteopontin (OPN), also known as bone sialoprotein I (BSP-1 or BNSP), early T-lymphocyte activation (ETA-1), secreted phosphoprotein 1 (SPP1), 2ar and Rickettsia resistance (Ric), is a human gene product, which is also conserved in other species. Osteopontin is a SIBLINGglycoprotein that was first identified in 1986 in osteoblasts.
The prefix of the word "osteo" indicates that the protein is expressed in bone, although it is also expressed in other tissues. The suffix "-pontin" is derived from "pons," the Latin word for bridge, and signifies osteopontin's role as a linking protein. Osteopontin is an extracellular structural protein and therefore an organic component of bone. Synonyms for this protein include sialoprotein I and 44K BPP (bone phosphoprotein).
OPN is a highly negatively charged, extracellular matrix protein that lacks an extensive secondary structure. It is composed of about 300 amino acids (297 in mouse; 314 in human) and is expressed as a 33-kDa nascent protein; there are also functionally important cleavage sites. OPN can go through posttranslational modifications which increase its apparent molecular weight to about 44 kDa. The OPN gene is composed of 7 exons, 6 of which contain coding sequence. The first two exons contain the 5' untranslated region (5' UTR). Exons 2, 3, 4, 5, 6, and 7 code for 17, 13, 27, 14, 108 and 134 amino acids, respectively. All intron-exon boundaries are of the phase 0 type, thus alternative exon splicing maintains the reading frame of the OPN gene.
Figure 1. Proteolytic cleavage sites for full length osteopontin (OPN-FL). Thrombin exposes the cleaved epitope SVVYGLR (OPN-R), and then CPB removes the c-terminal arginine from OPN-R. The cleaved epitope has a non-RGD domain, which binds to integrin receptors (α4β1, α9β1, and α9β4). Next to the cleaved epitope, there is a RGD domain which interacts with other integrin receptors (αvβ1,3,5, and α5β1).
Full length OPN (OPN-FL) can be modified by thrombin cleavage, which exposes a cryptic sequence, SVVYGLR on the cleaved form of the protein known as OPN-R (Fig. 1). This thrombin-cleaved OPN (OPN-R) exposes an epitope for integrin receptors of α4β1, α9β1, and α9β4. These integrin receptors are present on a number of immune cells such as mast cells, neutrophils, and T cells. It is also expressed by monocytes and macrophages. Upon binding these receptors, cells use several signal transduction pathways to elicit immune responses in these cells (See Section 3 for more detail). OPN-R can be further cleaved by Carboxypeptidase B (CPB) by removal of C-terminal arginine and become OPN-L (Fig. 2). The function of OPN-L is largely unknown.
It appears an intracellular variant of OPN (iOPN) is involved in a number of cellular processes including migration, fusion and motility. Intracellular OPN is generated using an alternative translation start site on the same mRNA species used to generate the extracellular isoform. This alternative translation start site is downstream of the N-terminal endoplasmic reticulum-targeting signal sequence, thus allowing cytoplasmic translation of OPN.
Various human cancers, including breast cancer, have been observed to express splice variants of OPN. The cancer specific splice variants are osteopontin-a, osteopontin-b and osteopontin-c. Exon 5 is lacking from osteopontin-b, whereas osteopontin-c lacks exon 4. Osteopontin-c has been suggested to facilitate the anchorage-independent phenotype of some human breast cancer cells due to its inability to associate with the extracellular matrix.
Osteopontin seen in a lung tissue sample from a patient with ideopathic pulmonary fibrosis.
Regulation of the osteopontin gene is incompletely understood. Different cell types may differ in their regulatory mechanisms of the OPN gene. OPN expression in bone predominantly occurs by osteoblasts and osteocyctes (bone-forming cells) as well as osteoclasts (bone-resorbing cells). Runx2 (aka Cbfa1) and osterix (Osx) transcription factors are required for the expression of OPN  Runx2 and Osx bind promoters of osteoblast-specific genes such as Col1α1, Bsp, and Opn and upregulate transcription.
Hypocalcemia and hypophosphatemia (instances that stimulate kidney proximal tubule cells to produce calcitriol (1α,25-dihydroxyvitamin D3)) lead to increases in OPN transcription, translation and secretion. This is due to the presence of a high-specificity vitamin D response element (VDRE) in the OPN gene promoter.
Extracellular inorganic phosphate (ePi) has also been identified as a modulator of OPN expression.
Stimulation of OPN expression also occurs upon exposure of cells to pro-inflammatory cytokines, classical mediators of acute inflammation (e.g. tumour necrosis factor α [TNFα], infterleukin-1β [IL-1β]), angiotensin II, transforming growth factor β (TGFβ) and parathyroid hormone (PTH), although a detailed mechanistic understanding of these regulatory pathways are not yet known. Hyperglycemia and hypoxia are also known to increase OPN expression.
Role in biomineralization
OPN belongs to a family of secreted acidic proteins whose members have an abundance of negatively charged amino acids such as Asp and Glu. OPN also has a large number of consensus sequence sites for post-translational phosphorylation of Ser residues to form phosphoserine, providing additional negative charge. Contiguous stretches of high negative charge in OPN have been identified and named the polyAsp motif (poly-aspartic acid) and the ASARM motif (acidic serine- and asparate-rich motif), with the latter sequence having multiple phosphorylation sites. This overall negative charge of OPN, along with its specific acidic motifs and the fact that OPN is an intrinsically disordered protein allowing for open and flexible structures, permit OPN to bind strongly to calcium atoms available at crystal surfaces in various biominerals. Such binding of OPN to various types of calcium-based biominerals ‒ such as calcium-phosphate mineral in bones and teeth, calcium-carbonate mineral in inner ear otoconia and avian eggshells, and calcium-oxalate mineral in kidney stones – acts as a mineralization inhibitor to regulate crystal growth. OPN is a substrate protein for a number of enzymes whose actions may modulate the mineralization-inhibiting function of OPN, a prominent one being PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) which can extensively degrade OPN and whose inactivating gene mutations lead to altered processing of OPN and osteomalacia (soft hypomineralized bones) in X-linked hypophosphatemia (XLH). Along with its role in the regulation of normal mineralization within the extracellular matrices of bones and teeth, OPN is also upregulated at sites of pathologic, ectopic calcification – such as for example, in urolithiasis and vascular calcification ‒ presumably at least in part to inhibit debilitating mineralization in these soft tissues.
As discussed, OPN binds to several integrin receptors including α4β1, α9β1, and α9β4 expressed by leukocytes. These receptors have been well-established to function in cell adhesion, migration, and survival in these cells. Therefore, recent research efforts have focused on the role of OPN in mediating such responses.
Osteopontin (OPN) is expressed in a range of immune cells, including macrophages, neutrophils, dendritic cells, and T and B cells, with varying kinetics. OPN is reported to act as an immune modulator in a variety of manners. Firstly, it has chemotactic properties, which promote cell recruitment to inflammatory sites. It also functions as an adhesion protein, involved in cell attachment and wound healing. In addition, OPN mediates cell activation and cytokine production, as well as promoting cell survival by regulating apoptosis. The following examples are found.
OPN plays an important role in neutrophil recruitment in alcoholic liver disease. OPN is important for the migration of neutrophil in vitro. In addition, OPN recruits inflammatory cells to arthritis joints in the collagen-induced arthritis model of rheumatoid arthritis. A recent in vitro study in 2008 has found that OPN plays a role in mast cell migration. Here OPN knock-out mast cells were cultured and they observed a decreased level of chemotaxis in these cells compared to wildtype mast cells. OPN was also found to act as a macrophage chemotactic factor. In this study, researchers looked at the accumulation of macrophages in the brain of rhesus monkeys and found that OPN prevented macrophages from leaving the accumulation site, indicating an increased level of chemotaxis.
Activated T cells are promoted by IL-12 to differentiate towards the Th1 type, producing cytokines including IL-12 and IFNγ. OPN inhibits production of the Th2 cytokine IL-10, which leads to enhanced Th1 response. OPN influences cell-mediated immunity and has Th1 cytokine functions. It enhances B cell immunoglobulin production and proliferation. Recent studies in 2008 suggest that OPN also induces mast cell degranulation. The researchers here observed that IgE-mediated anaphylaxis was significantly reduced in OPN knock-out mice compared to wild type mice. The role of OPN in activation of macrophages has also been implicated in a cancer study, when researchers discovered that OPN-producing tumors were able to induce macrophage activation compared to OPN-deficient tumors.
Fig 2. Known immunologic functions of OPN. OPN binds to several integrin receptors including α4β1, α9β1, and α9β4 expressed by leukocytes and are known to induce cell adhesion, migration, and survival in immune cells including neutrophils, macrophages, T cells, mast cells, and osteoclasts.
OPN is an important anti-apoptotic factor in many circumstances. OPN blocks the activation-induced cell death of macrophages and T cells as well as fibroblasts and endothelial cells exposed to harmful stimuli. OPN prevents non-programmed cell death in inflammatory colitis.
Potential clinical application
The fact that OPN interacts with multiple cell surface receptors which are ubiquitously expressed makes it an active player in many physiological and pathological processes including wound healing, bone turnover, tumorigenesis, inflammation, ischemia and immune responses1. Therefore, manipulation of plasma (or local) OPN levels may be useful in the treatment of autoimmune diseases, cancer metastasis, bone (and tooth) mineralization diseases, osteoporosis and some forms of stress.
Role in autoimmune diseases
OPN has been implicated in pathogenesis of rheumatoid arthritis. For instance, researchers found that OPN-R, the thrombin-cleaved form of OPN, was elevated in the rheumatoid arthritis joint. However, the role of OPN in rheumatoid arthritis is still unclear. One group found that OPN knock-out mice were protected against arthritis. while others were not able to reproduce this observation. OPN has been found to play a role in other autoimmune diseases including autoimmune hepatitis, allergic airway disease, and multiple sclerosis.
Research has implicated osteopontin in excessive scar-forming and a gel has been developed to inhibit its effect.
Role in allergy and asthma
Osteopontin has recently been associated with allergic inflammation and asthma. Using a murine model of allergic inflammation, it was demonstrated that OPN-s, the secreted form of OPN, exerts opposing effects on mouse Th2 effector responses and subsequent allergic airway disease: pro-inflammatory at primary systemic sensitization, and anti-inflammatory during secondary pulmonary antigenic challenge, mainly through the regulation of different dendritic cell subsets. OPN deficiency was also reported to protect against remodeling and bronchial hyperresponsiveness (BHR), again using a chronic allergen-challenge model of airway remodeling. Furthermore, it was recently demonstrated that OPN expression is upregulated in human asthma, is associated with remodeling changes and its subepithelial expression correlates to disease severity. OPN has also been reported to be increased in the sputum supernatant of smoking asthmatics, as well as the BALF and bronchial tissue of smoking controls and asthmatics.
Role in muscle disease and injury
Evidence is accumulating that suggests that osteopontin plays a number of roles in diseases of skeletal muscle, such as Duchenne muscular dystrophy. Osteopontin has been described as a component of the inflammatory environment of dystrophic and injured muscles, and has also been shown to increase scarring of diaphragm muscles of aged dystrophic mice. A recent study has identified osteopontin as a determinant of disease severity in patients with Duchenne muscular dystrophy. This study found that a mutation in the osteopontin gene promoter, known to cause low levels of osteopontin expression, is associated with a decrease in age to loss of ambulation and muscle strength in patients with Duchenne muscular dystrophy.
^ abCrosby, A.H., Edwards, S.J., Murray, J.C., Dixon, M.J. (May 1995). "Genomic organization of the human osteopontin gene: exclusion of the locus from a causative role in the pathogenesis of dentinogenesis imperfecta type II". Genomics27 (1): 155–160. doi:10.1006/geno.1995.1018. PMID7665163.
^ abBanerjee A, Apte UM, Smith R, Ramaiah SK (March 2006). "Higher neutrophil infiltration mediated by osteopontin is a likely contributing factor to the increased susceptibility of females to alcoholic liver disease". J. Pathol.208 (4): 473–85. doi:10.1002/path.1917. PMID16440289.
^Suzuki K, Zhu B, Rittling SR, Denhardt DT, Goldberg HA, McCulloch CA, Sodek J (August 2002). "Colocalization of intracellular osteopontin with CD44 is associated with migration, cell fusion, and resorption in osteoclasts". J Bone Miner Res17 (1): 1486–1497. doi:10.1359/jbmr.2002.17.8.1486. PMID12162503.
^Zhu B, Suzuki K, Goldberg HA, Rittling SR, Denhardt DT, McCulloch CA, Sodek J (January 2004). "Osteopontin modulates CD44-dependent chemotaxis of peritoneal macrophages through G-protein-coupled receptors: evidence of a role for an intracellular form of osteopontin". Journal of Cellular Physiology198 (1): 155–167. doi:10.1002/jcp.10394. PMID14584055.
^Junaid A, Moon MC, Harding GE, Zahradka P (February 2007). "Osteopontin localizes to the nucleus of 293 cells and associates with polo-like kinase-1". Am J Physiol Cell Physiol292 (1): 919–926. doi:10.1152/ajpcell.00477.2006. PMID17005603.
^ abcHe B, Mirza M, Weber GF (April 2006). "An osteopontin splice variant induces anchorage independence in human breast cancer cells". Oncogene25 (1): 2192–2202. doi:10.1038/sj.onc.1209248. PMID16288209.
^Mirza M, Shaughnessy E, Hurley JK, Vanpatten KA, Pestano GA, He B, Weber GF (February 2008). "Osteopontin-c is a selective marker of breast cancer". Int J Cancer122 (1): 889–897. doi:10.1002/ijc.23204. PMID17960616.
^ abUaesoontrachoon K, Yoo HJ, Tudor E, Pike RN, Mackie EJ, Pagel CN (April 2008). "Osteopontin and skeletal muscle myoblasts: Association with muscle regeneration and regulation of myoblast function in vitro". Int. J. Biochem. Cell Biol.40 (10): 2303–14. doi:10.1016/j.biocel.2008.03.020. PMID18490187.
^Merry, K., Dodds, R., Littlewood, A., Gowen, M. (April 1993). "Expression of Osteopontin mRNA by osteoclasts and osteoblasts in modelling adult human bone". J Cell Sci104 (4): 1013–1020. PMID8314886.
^Nakashima, K., Zhou, X., Kunkel, G., Zhang, Z., Deng, J.M., Behringer, R.R., de Crombrugghe, B. (January 2002). "The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation". Cell108 (1): 17–29. doi:10.1016/S0092-8674(01)00622-5. PMID11792318.
^Hullinger TG, Pan Q, Viswanathan HL, Somerman MJ (January 2001). "TGFbeta and BMP-2 activation of the OPN promoter: roles of smad- and hox-binding elements". Exp Cell Res262 (1): 69–74. doi:10.1006/excr.2000.5074. PMID11120606.
^Sodhi CP, Phadke SA, Batlle D, Sahai A (April 2001). "Hypoxia and high glucose cause exaggerated mesangial cell growth and collagen synthesis: role of osteopontin". Am J Physiol Renal Physiol280 (1): 667–674. PMID11249858.
^Fisher LW, Fedarko NS (2003). "Six genes expressed in bones and teeth encode the current members of the SIBLING family of proteins". Connect. Tissue Res. 44 Suppl 1: 33–40. PMID12952171.
^Addison WN, Nakano Y, Loisel T, Crine P, McKee MD (October 2008). "MEPE-ASARM peptides control extracellular matrix mineralization by binding to hydroxyapatite: an inhibition regulated by PHEX cleavage of ASARM". J. Bone Miner. Res.23 (10): 1638–49. doi:10.1359/jbmr.080601. PMID18597632.
^ abAddison WN, Masica DL, Gray JJ, McKee MD (April 2010). "Phosphorylation-dependent inhibition of mineralization by osteopontin ASARM peptides is regulated by PHEX cleavage". J. Bone Miner. Res.25 (4): 695–705. doi:10.1359/jbmr.090832. PMID19775205.
^Kurzbach D, Platzer G, Schwarz TC, Henen MA, Konrat R, Hinderberger D (July 2013). "Cooperative Unfolding of Compact Conformations of the Intrinsically Disordered Protein Osteopontin". Biochemistry52 (31): 5167–75. doi:10.1021/bi400502c. PMID23848319.
^Hunter GK, O'Young J, Grohe B, Karttunen M, Goldberg HA (December 2010). "The flexible polyelectrolyte hypothesis of protein-biomineral interaction". Langmuir26 (24): 18639–46. doi:10.1021/la100401r. PMID20527831.
^McKee MD, Nanci A (May 1995). "Postembedding colloidal-gold immunocytochemistry of noncollagenous extracellular matrix proteins in mineralized tissues". Microsc. Res. Tech.31 (1): 44–62. doi:10.1002/jemt.1070310105. PMID7626799.
^Takemura T, Sakagami M, Nakase T, Kubo T, Kitamura Y, Nomura S (September 1994). "Localization of osteopontin in the otoconial organs of adult rats". Hear. Res.79 (1-2): 99–104. PMID7806488.
^Hincke MT, Nys Y, Gautron J, Mann K, Rodriguez-Navarro AB, McKee MD (2012). "The eggshell: structure, composition and mineralization". Front. Biosci.17: 1266–80. PMID22201802.
^McKee MD, Nanci A, Khan SR (December 1995). "Ultrastructural immunodetection of osteopontin and osteocalcin as major matrix components of renal calculi". J. Bone Miner. Res.10 (12): 1913–29. doi:10.1002/jbmr.5650101211. PMID8619372.
^O'Young J, Chirico S, Al Tarhuni N, Grohe B, Karttunen M, Goldberg HA, Hunter GK (2009). "Phosphorylation of osteopontin peptides mediates adsorption to and incorporation into calcium oxalate crystals". Cells Tissues Organs (Print)189 (1-4): 51–5. doi:10.1159/000151724. PMID18728346.
^Barros NM, Hoac B, Neves RL, Addison WN, Assis DM, Murshed M, Carmona AK, McKee MD (March 2013). "Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine model of X-linked hypophosphatemia". J. Bone Miner. Res.28 (3): 688–99. doi:10.1002/jbmr.1766. PMID22991293.
^McKee MD, Addison WN, Kaartinen MT (2005). "Hierarchies of extracellular matrix and mineral organization in bone of the craniofacial complex and skeleton". Cells Tissues Organs (Print)181 (3-4): 176–88. doi:10.1159/000091379. PMID16612083.
^Choi ST, Kim JH, Kang EJ, Lee SW, Park MC, Park YB, Lee SK (December 2008). "Osteopontin might be involved in bone remodelling rather than in inflammation in ankylosing spondylitis". Rheumatology (Oxford)47 (12): 1775–9. doi:10.1093/rheumatology/ken385. PMID18854347.
^Apte UM, Banerjee A, McRee R, Wellberg E, Ramaiah SK (August 2005). "Role of osteopontin in hepatic neutrophil infiltration during alcoholic steatohepatitis". Toxicol. Appl. Pharmacol.207 (1): 25–38. doi:10.1016/j.taap.2004.12.018. PMID15885730.
^Ohshima S, Kobayashi H, Yamaguchi N, Nishioka K, Umeshita-Sasai M, Mima T, Nomura S, Kon S, Inobe M, Uede T, Saeki Y (April 2002). "Expression of osteopontin at sites of bone erosion in a murine experimental arthritis model of collagen-induced arthritis: possible involvement of osteopontin in bone destruction in arthritis". Arthritis Rheum.46 (4): 1094–101. doi:10.1002/art.10143. PMID11953989.
^Sakata M, Tsuruha JI, Masuko-Hongo K, Nakamura H, Matsui T, Sudo A, Nishioka K, Kato T (July 2001). "Autoantibodies to osteopontin in patients with osteoarthritis and rheumatoid arthritis". J. Rheumatol.28 (7): 1492–5. PMID11469452.
^Crawford HC, Matrisian LM, Liaw L (November 1998). "Distinct roles of osteopontin in host defense activity and tumor survival during squamous cell carcinoma progression in vivo". Cancer Res.58 (22): 5206–15. PMID9823334.
^Standal T, Borset M, Sundan A (September 2004). "Role of osteopontin in adhesion, migration, cell survival and bone remodeling". Exp. Oncol.26 (3): 179–84. PMID15494684.
^Da Silva AP, Pollett A, Rittling SR, Denhardt DT, Sodek J, Zohar R (September 2006). "Exacerbated tissue destruction in DSS-induced acute colitis of OPN-null mice is associated with downregulation of TNF-alpha expression and non-programmed cell death". J. Cell. Physiol.208 (3): 629–39. doi:10.1002/jcp.20701. PMID16741956.
^Jacobs JP, Pettit AR, Shinohara ML, et al. (August 2004). "Lack of requirement of osteopontin for inflammation, bone erosion, and cartilage damage in the K/BxN model of autoantibody-mediated arthritis". Arthritis Rheum.50 (8): 2685–94. doi:10.1002/art.20381. PMID15334485.
^Chabas D, Baranzini SE, Mitchell D, et al. (November 2001). "The influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease". Science294 (5547): 1731–5. doi:10.1126/science.1062960. PMID11721059.
^Steinman L (February 2007). "A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage". Nat. Med.13 (2): 139–45. doi:10.1038/nm1551. PMID17290272.
^Xanthou G, Alissafi T, Semitekolou M, Simoes DC, Economidou E, Gaga M, Lambrecht BN, Lloyd CM, Panoutsakopoulou V (May 2007). "Osteopontin has a crucial role in allergic airway disease through regulation of dendritic cell subsets". Nat. Med.13 (5): 570–9. doi:10.1038/nm1580. PMID17435770.
^Simoes DC, Xanthou G, Petrochilou K, Panoutsakopoulou V, Roussos C, Gratziou C (May 2009). "Osteopontin deficiency protects against airway remodeling and hyperresponsiveness in chronic asthma". Am J Respir Crit Care Med.179 (10): 894–902. doi:10.1164/rccm.200807-1081OC. PMID19234104.
^Samitas K, Zervas E, Vittorakis S, Semitekolou M, Alissafi T, Bossios A, Gogos H, Economidou E, Lötvall J, Xanthou G, Panoutsakopoulou V, Gaga M (2010). "Osteopontin expression and relation to disease severity in human asthma.". Eur. Respir. J.37 (2): 331–41. doi:10.1183/09031936.00017810. PMID20562127.
^Hillas G, Loukides S, Kostikas K, Simoes D, Petta V, Konstantellou E, Emmanouil P, Papiris S, Koulouris N, Bakakos P (Jan 2013). "Increased levels of osteopontin in sputum supernatant of smoking asthmatics.". Cytokine.61 (1): 251–5. doi:10.1016/j.cyto.2012.10.002. PMID23098767.
^Samitas K, Zervas E, Xanthou G, Panoutsakopoulou V, Gaga M (Feb 2013). "Osteopontin is increased in the bronchoalveolar lavage fluid and bronchial tissue of smoking asthmatics.". Cytokine61 (3): 713–5. doi:10.1016/j.cyto.2012.12.028. PMID23384656.
Fujisawa R (2002). "[Recent advances in research on bone matrix proteins]". Nippon Rinsho. 60. Suppl 3: 72–8. PMID11979972.
Denhardt DT, Mistretta D, Chambers AF, et al. (2003). "Transcriptional regulation of osteopontin and the metastatic phenotype: evidence for a Ras-activated enhancer in the human OPN promoter". Clin. Exp. Metastasis20 (1): 77–84. doi:10.1023/A:1022550721404. PMID12650610.
Konno S, Hizawa N, Nishimura M, Huang SK (2007). "Osteopontin: a potential biomarker for successful bee venom immunotherapy and a potential molecule for inhibiting IgE-mediated allergic responses". Allergology international : official journal of the Japanese Society of Allergology55 (4): 355–9. doi:10.2332/allergolint.55.355. PMID17130676.
Rodrigues LR, Teixeira JA, Schmitt FL, et al. (2007). "The role of osteopontin in tumor progression and metastasis in breast cancer". Cancer Epidemiol. Biomarkers Prev.16 (6): 1087–97. doi:10.1158/1055-9965.EPI-06-1008. PMID17548669.
Ramaiah SK, Rittling S (2007). "Role of osteopontin in regulating hepatic inflammatory responses and toxic liver injury". Expert opinion on drug metabolism & toxicology3 (4): 519–26. doi:10.1517/17425126.96.36.1999. PMID17696803.