Osteopontin

Osteopontin, also known as bone /sialoprotein I, early T-lymphocyte activation, secreted phosphoprotein 1, 2ar and Rickettsia resistance, is a protein that in humans is encoded by the SPP1 gene. The murine ortholog is Spp1. Osteopontin is a SIBLING 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. 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.

Structure

OPN is a highly negatively charged, heavily phosphorylated extracellular matrix protein that lacks an extensive secondary structure as an intrinsically disordered protein. It is composed of about 300 amino acids 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 containing coding sequence. The first two exons contain the 5' untranslated region. 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.
Image:OPN2.jpg|thumb|250px|right|Figure 1. Proteolytic cleavage sites for full length osteopontin. Thrombin exposes the cleaved epitope SVVYGLR, and then CPB removes the c-terminal arginine from OPN-R. The cleaved epitope has a non-RGD domain, which binds to integrin receptors. Next to the cleaved epitope, there is a RGD domain that interacts with other integrin receptors. Not shown here are the extensive number of cleavage sites along the full length of the protein as degraded by the enzyme PHEX expressed by mineralized tissue cells.
SPP1 structure corresponds to an osteopontin antibody

Isoforms

Full-length OPN can be modified by thrombin cleavage, which exposes a cryptic sequence, SVVYGLR on the cleaved form of the protein known as OPN-R. This thrombin-cleaved OPN 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 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 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.

Tissue distribution

Osteopontin is expressed in a variety of tissue types including cardiac fibroblasts, preosteoblasts, osteoblasts, osteocytes, odontoblasts, some bone marrow cells, hypertrophic chondrocytes, dendritic cells, macrophages, smooth muscle, skeletal muscle myoblasts, endothelial cells, and extraosseous cells in the inner ear, brain, kidney, deciduum, and placenta. Synthesis of osteopontin is stimulated by calcitriol.

Regulation

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 as well as osteoclasts. Runx2 and osterix 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 lead to increases in OPN transcription, translation and secretion. This is due to the presence of a high-specificity vitamin D response element in the OPN gene promoter.
Osteopontin expression is modulated by Schistosoma mansoni egg antigen.
Schistosoma mansoni egg antigens directly stimulate the expression of the profibrogenic molecule osteopontin, 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.
Extracellular inorganic phosphate 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, angiotensin II, transforming growth factor β and parathyroid hormone, although a detailed mechanistic understanding of these regulatory pathways are not yet known. Hyperglycemia and hypoxia are also known to increase OPN expression.

Function

Apoptosis

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.

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 and the ASARM 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 is one such enzyme, which extensively degrades OPN, and whose inactivating gene mutations lead to altered processing of OPN such that inhibitory OPN cannot be degraded and accumulates in the bone extracellular matrix, contributing locally to the osteomalacia characteristic of XLH. A relationship describing local, physiologic double-negative regulation of mineralization involving OPN has been termed the Stenciling Principle of mineralization, whereby enzyme-substrate pairs imprint mineralization patterns into the extracellular matrix by degrading mineralization inhibitors. In relation to mineralization diseases, the Stenciling Principle is particularly relevant to the osteomalacia and odontomalacia observed in hypophosphatasia and X-linked hypophosphatemia.
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.

Bone remodeling

Osteopontin has been implicated as an important factor in bone remodeling. 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. OPN at bone surfaces is located in a thin organic layer, the so-called lamina limitans. 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, Ca₁₀₆₂. 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