Остеопонтин: структура, гены и функции в организме человека.

Mammalian protein found in Homo sapiens

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 protein that in humans is encoded by the SPP1 gene (secreted phosphoprotein 1). The murine ortholog is Spp1. Osteopontin is a SIBLING (glycoprotein) that was first identified in 1986 in osteoblasts. The prefix 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. The gene has 7 exons, spans 5 kilobases in length and in humans it is located on the long arm of chromosome 4 region 22 (4q1322.1). The protein is composed of ~300 amino acids residues and has ~30 carbohydrate residues attached, including 10 sialic acid residues, which are attached to the protein during post translational modification in the Golgi apparatus. The protein is rich in acidic residues: 30 36% are either aspartic or glutamic acid.

OPN is a highly negatively charged, heavily phosphorylated extracellular matrix protein that lacks an extensive secondary structure as an intrinsically disordered protein. The OPN gene is composed of 7 exons, 6 of which containing 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.]]

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. OPN R can be further cleaved by Carboxypeptidase B (CPB) by removal of C terminal arginine and become OPN L. 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. preosteoblasts, osteoblasts, osteocytes, odontoblasts, some bone marrow cells, hypertrophic chondrocytes, dendritic cells, macrophages, smooth muscle, skeletal muscle myoblasts, endothelial cells, and extraosseous (non bone) cells in the inner ear, brain, kidney, deciduum, and placenta. Synthesis of osteopontin is stimulated by calcitriol (1,25 dihydroxy vitamin D3).

Regulation of the osteopontin gene expression 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. Osteopontin (OPN) expression is modulated by Schistosoma mansoni egg antigen. Schistosoma mansoni egg antigens directly stimulate the expression of the profibrogenic molecule osteopontin (OPN), and systemic OPN levels strongly correlate with disease severity, suggesting its use as a potential morbidity biomarker. Investigation into the impact of Praziquantel use on systemic OPN levels and on liver collagen deposition in chronic murine schistosomiasis revealed that Praziquantel treatment significantly reduced systemic OPN levels and liver collagen deposition, indicating that OPN could be a reliable tool for monitoring PZQ efficacy and fibrosis regression. 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.

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.

OPN belongs to a family of secreted acidic proteins (SIBLINGs, Small Integrin Binding LIgand N Glycosylated 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 aspartate 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 by stabilizing transient mineral precursor phases and by binding directly to crystal surfaces, all of which regulate crystal growth. OPN is a substrate protein for a number of enzymes whose actions may modulate the mineralization inhibiting function of OPN. PHEX (phosphate regulating endopeptidase homolog X linked) is one such enzyme, which extensively degrades OPN, and whose inactivating gene mutations (in X linked hypophosphatemia, XLH) lead to altered processing of OPN such that inhibitory OPN cannot be degraded and accumulates in the bone (and tooth) extracellular matrix, contributing locally to the osteomalacia (soft hypomineralized bones, and odontomalacia soft teeth) characteristic of XLH. OPN is also upregulated at sites of pathologic, ectopic calcification – such as for example, in urolithiasis Specifically, OPN anchors osteoclasts to the surface of bones where it is immobilized by its mineral binding properties allowing subsequent usage of its RGD motif for osteoclast integrin binding for cell attachment and migration. The organic part of bone is about 20% of the dry weight, and counts in, other than osteopontin, collagen type I, osteocalcin, osteonectin, and alkaline phosphatase. Collagen type I counts for 90% of the protein mass. The inorganic part of bone is the mineral hydroxyapatite, Ca10(PO4)6(OH)2. Loss of bone may lead to osteoporosis, as the bone is depleted for calcium if this is not supplied in the diet. OPN serves to initiate the process by which osteoclasts develop their ruffled borders to begin bone resorption. OPN contains and RGD integrin binding motif

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.

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 rhesus monkey, OPN prevents macrophages from leaving the accumulation site in brains, indicating an increased level of chemotaxis.

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. Osteopontin (OPN) is expressed in a range of immune cells, including macrophages, neutrophils, dendritic cells, microglia 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. 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.'''

Osteopontin has recently been associated with allergic inflammation and asthma. Expression of Opn is significantly increased in lung epithelial and subepithelial cells of asthmatic patients in comparison to healthy subjects. Opn expression is also upregulated in lungs of mice with allergic airway inflammation. 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.

Opn is up regulated in inflammatory bowel disease (IBD). Opn expression is highly up regulated in intestinal immune and non immune cells and in the plasma of patients with Crohn's disease (CD) and ulcerative colitis (UC), as well as in the colon and plasma of mice with experimental colitis. Increased plasma Opn levels are related to the severity of CD inflammation, and certain Opn gene (Spp1) haplotypes are modifiers of CD susceptibility. Opn has also a proinflammatory role in TNBS and dextran sulfate sodium (DSS) induced colitis, which are mouse models for IBD. Opn was found highly expressed by a specific dendritic cell (DC) subset derived from murine mesenteric lymph nodes (MLNs) and is highly proinflammatory for colitis. Dendritic cells are important for the development of intestinal inflammation in humans with IBD and in mice with experimental colitis. Opn expression by this inflammatory MLN DC subset is crucial for their pathogenic action during colitis. OPN is overexpressed in a variety of cancers, including lung cancer, breast cancer, colorectal cancer, stomach cancer, ovarian cancer, papillary thyroid carcinoma, melanoma and pleural mesothelioma; OPN contributes both glomerulonephritis and tubulointerstitial nephritis; and OPN is found in atheromatous plaques within arteries. Thus, manipulation of plasma OPN levels may be useful in the treatment of autoimmune diseases, cancer metastasis, osteoporosis and some forms of stress. It is expressed as one of three splice variants in PDAC, with osteopontin a expressed in nearly all PDAC, osteopontin b expression correlating with survival, and osteopontin c correlating with metastatic disease Because PDAC secretes alternatively spliced forms of osteopontin, it shows potential for tumor and disease stage specific targeting. Although the exact mechanisms of osteopontin signaling in PDAC are unknown, it binds to CD44 and integrins to trigger processes such as tumor progression and complement inhibition. Osteopontin also drives metastasis by triggering the release of vascular endothelial growth factor (VEGF) and matrix metalloprotease (MMP), which is inhibited by knocking down osteopontin. This process is also stimulated by nicotine, which is the proposed mechanism by which smokers experience elevated PC risk. Osteopontin is being explored as a marker for PC. It was found to perform better than CA19.9 in discerning IPMN [80] and resectable PDAC from pancreatitis Antiosteopontin antibodies are being developed, including hu1A12, which inhibited metastasis in an in vivo study and also when hybridized with the anti VEGF antibody bevacizumab At least one clinical trial is exploring the use of osteopontin as a marker of intratumoral hypoxia. However, this marker remains relatively unexplored. Osteopontin is also implicated in excessive scar formation and a gel has been developed to inhibit its effect. AOM1, an anti osteopontin monoclonal antibody drug developed by Pfizer, Inc. to inhibit osteopontin, showed promise at preventing progression of large metastatic tumors in mouse models of NSCLC. Even though Opn promotes metastasis and can be used as a cancer biomarker, latest studies described novel protecting functions of the molecule on innate cell populations during tumor development. Particularly, maintenance of a pool of natural killer (NK) cells with optimal immune function is crucial for host defense against cancerous tumor formation. A study in PNAS describes iOpn is an essential molecular component responsible for maintenance of functional NK cell expansion. Absence of iOPN results in failure to maintain normal NK cellularity and increased cell death following stimulation by cytokine IL 15. OPN deficient NK cells fail to successfully navigate the contraction phase of the immune response, resulting in impaired expansion of long lived NK cells and defective responses to tumor cells. In addition, plasmacytoid dendritic cells (pDCs) protect from melanoma, and this effect is mediated by type I IFNs. A study in JCB showed that a specific fragment (SLAYGLR) of the Opn protein can render pDCs more “fit” to protect from melanoma development. This was achieved by activation of a novel α4 integrin/IFN β axis which is MyD88 independent and operates via a PI3K/mTOR/IRF3 pathway.

Osteopontin is minimally expressed under normal conditions, but accumulates quickly as heart function declines. Specifically, it plays a central role in the remodeling response to myocardial infarction, and is dramatically upregulated in hypertrophic (HCM) and dilated cardiomyopathy (DCM).

OPN plays a role in oxidative and nitrosative stress, apoptosis, mitochondrial dysfunction, and excitotoxicity, which are also involved in the pathogenesis of Parkinson's disease. PD patients serum and cerebrospinal fluid (CSF) concentrations of OPN were studied, it shows that OPN levels in the body fluid is elevated in PD patients.

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.

An increase in Plasma OPN levels has been observed in patients with idiopathic hip OA. Furthermore, a correlation between OPN plasma levels and the severity of the disease has been noted.

OPN is expressed in endometrial cells during implantation. Due to the production of progesterone by the ovaries, OPN is up regulated immensely to aid in this process. The endometrium must undergo decidualization, the process in which the endometrium undergoes changes to prepare for implantation, which will lead to the attachment of the embryo. The endometrium houses stromal cells that will differentiate to produce an optimal environment for the embryo to attach (decidualization). OPN is a vital protein for stromal cell proliferation and differentiation as well as it binds to the receptor αvβ3 to assist with adhesion. OPN along with decidualization ultimately encourages the successful implantation of the early embryo. An OPN gene knock out results in attachment instability at the maternal fetal interface.