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Dentineogenesis starts on odontoblasts, which synthesise and secrete non-collagenous proteins (NCPs) and collagen. When dentine is injured, dental pulp progenitors/mesenchymal stem cells (MSCs) can migrate to the injured area, differentiate into odontoblasts and facilitate formation of reactionary dentine. Dental pulp progenitor cell/MSC differentiation is controlled at given niches. Among dental NCPs, dentine sialophosphoprotein (DSPP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family, whose members share common biochemical characteristics such as an Arg-Gly-Asp (RGD) motif. DSPP expression is cell- and tissue-specific and highly seen in odontoblasts and dentine. DSPP mutations cause hereditary dentine diseases. DSPP is catalysed into dentine glycoprotein (DGP)/sialoprotein (DSP) and phosphoprotein (DPP) by proteolysis. DSP is further processed towards active molecules. DPP contains an RGD motif and abundant Ser-Asp/Asp-Ser repeat regions. DPP-RGD motif binds to integrin αVβ3 and activates intracellular signalling via mitogen-activated protein kinase (MAPK) and focal adhesion kinase (FAK)-ERK pathways. Unlike other SIBLING proteins, DPP lacks the RGD motif in some species. However, DPP Ser-Asp/Asp-Ser repeat regions bind to calcium-phosphate deposits and promote hydroxyapatite crystal growth and mineralisation via calmodulin-dependent protein kinase II (CaMKII) cascades. DSP lacks the RGD site but contains signal peptides. The tripeptides of the signal domains interact with cargo receptors within the endoplasmic reticulum that facilitate transport of DSPP from the endoplasmic reticulum to the extracellular matrix. Furthermore, the middle- and COOH-terminal regions of DSP bind to cellular membrane receptors, integrin β6 and occludin, inducing cell differentiation. The present review may shed light on DSPP roles during odontogenesis.

The development of biomaterials for the interface between tendon and bone is important for realizing functional tendon replacements. Toward the development of new materials for such applications, engineered recombinant spider silk proteins were modified with peptide tag sequences derived from noncollagenous proteins in bone, so-called SIBLING proteins, such as osteopontin and sialoprotein, which are known to interact with collagen and to initiate mineralization. Materials made of these spider silk-SIBLING hybrids were analyzed concerning mineralization and interaction with cells. They showed enhanced calcium phosphate formation upon incubation in mineralization agents. In gradient films, MC3T3-E1 mouse preosteoblasts adhered preferentially along the gradient toward the variant with a collagen binding motif.

Significance: Since protein disulfide isomerase (PDI) was first described in 1963, researchers have shown conclusively that PDI and sibling proteins are quintessential for thrombus formation. PDI, ...

Background Previously we found that a group of phosphorylated proteins (SIBLINGs) in bone binds with the Ti-device, and increases the early bone formation around the Ti-implants remarkably. From these results, we explained the biochemical mechanism of a strong bond between living bone and Ti, which was discovered by Branemark and colleagues. For the clinical application of our findings, we need a large amount of these proteins or their substitutes. Objective We aimed to create a new molecule that equips with essential functions of SIBLINGs, Ti-binding, and bone enhancement around the Ti implant. Methods We chemically phosphorylated chitin and obtained a soluble form of phosphorylated chitin (P-chitin). In this solution, we immersed the Ti-devices of web-form (TW) which we previously developed and obtained the P-chitin coated TWs. Then we tested the P-chitin coated TWs for their calcification ability in vitro, and bone enhancing ability in vivo, by implanting them into rat calvaria. We compared the P-chitin coated TW and the non-coated TW in regard to their calcification and bone enhancing abilities. Results Ti-devices coated with phosphorylated-chitin induced a ten times higher calcification in vitro at 20 days, and four times more elevated amount of bone formation in vivo at two weeks than the uncoated Ti-device. Conclusions Phosphorylated chitin could be a partial substitute of bone SIBLING proteins and are clinically applicable to accelerate bone formation around the Ti implants, thereby achieving the strong bond between living bone and Ti.

Eventhough it is well established that materials can promote stem cell differentiation, hard tissue formation is a templated process for which little is known regarding the in vitro process. We have found that surface curvature enables self-assembly of triple helical collagen fibrils into banded bundle structures from rat tail and human collagen secreted by dental pulp stem cells. Collagen fibrils were adsorbed at 4 °C on spun cast flat P4VP films and electrospun fibers. Protein adsorption was observed on both surfaces, but large banded bundles with a uniform spacing of approximately 55 nm were present only on the fiber surfaces. SEM/EDS mapping showed that dental pulp stem cells plated on the same surfaces biomineralized copiously only along the electrospun fibers. Raman spectroscopy indicated that despite the presence of adsorbed collagen on the flat surfaces, only the deposits present on the fibrous surface had a protein to hydroxyl apatite ratio similar to natural dentin from human teeth. RT-PCR indicated up regulation of collagen, osteocalcin and dental sialophosphate protein, confirming that odontogenic differentiation is promoted only on the fiber scaffolds. Taken together the results indicate that, in addition to surface chemistry, the supermolecular structure of ECM collagen, which is essential in directing DPSCs differentiation and templating biomineralization, can be modified by the underlying surface morphology. Statement of Significance The past decade has been focused efforts in the use of dental pulp stem cells (DPSC) for dental regeneration. Eventhough the factors required for DPSCs differentiation have been well studied, actual mineral deposition, positively identified as dentin, has not been achieved in vitro. Hard tissue is known to be a templated process in vivo where the mineral to protein ratio is tightly controlled via proteins which aid in collagen conformation and mineral sequestration. Here we show that one can mimic this process in vitro via the combination of materials selection and morphology. The material chemistry is shown to induce genetic upregulation the genes responsible for collagen and osteocalcin, while Raman spectroscopy confirms the translation and adsorption the proteins on the substrate. But, we show that the simple presence of collagen is not enough to template actual biomineral deposition similar to that found in vivo. Mineral deposition is a complicated process templated on collagen bundles and mediated by specific sibling proteins that determine the protein to mineral ratio. Here we show that surface curvature can reduce the barrier to collagen bundle formation, directing DPSC differentiation along odontogenic lineage, and subsequently templating actual dentin, comparable to that found in vivo in human teeth.

The two SIBLING (Small Integrin Binding Ligand N-linked Glycoproteins), bone sialoprotein (BSP) and osteopontin (OPN) are expressed in osteoblasts and osteoclasts. In mature BSP knockout (KO, -/-) mice, both bone formation and resorption as well as mineralization are impaired. OPN-/- mice display impaired resorption, and OPN is described as an inhibitor of mineralization. However, OPN is overexpressed in BSP-/- mice, complicating the understanding of their phenotype. We have generated and characterized mice with a double KO (DKO) of OPN and BSP, to try and unravel their respective contributions. Despite the absence of OPN, DKO bones are still hypomineralized. The SIBLING, matrix extracellular phosphoglycoprotein with ASARM motif (MEPE) is highly overexpressed in both BSP-/- and DKO and may impair mineralization through liberation of its ASARM (Acidic Serine-Aspartate Rich MEPE associated) peptides. DKO mice also display evidence of active formation of trabecular, secondary bone as well as primary bone in the marrow-ablation repair model. A higher number of osteoclasts form in DKO marrow cultures, with higher resorption activity, and DKO long bones display a localized and conspicuous cortical macroporosity. High bone formation and resorption parameters, and high cortical porosity in DKO mice suggest an active bone modeling/remodeling, in the absence of two key regulators of bone cell performance. This first double KO of SIBLING proteins thus results in a singular, non-trivial phenotype leading to reconsider the interpretation of each single KO, concerning in particular matrix mineralization and the regulation of bone cell activity.

Background Dentin sialophosphoprotein (DSPP) belongs to the family of small integrin-binding ligand N -linked glycoproteins (SIBLINGs), which share common biochemical features such as an arginine-glycine-aspartic acid (RGD) integrin-binding site. However, amino acid sequence analyses suggest that DSPP has lost some common features, but acquired other unique features, such as repeat sequences of serine-serine-aspartic acid (SDrr) that are not observed in other SIBLINGs proteins. Highlight We review the biochemical features of DSPP using genetically modified mice and proteomic analyses. DSPP of some species lack the RGD sites unlike other SIBLING proteins such as dentin matrix protein-1 (DMP-1) and bone sialoprotein (BSP). We previously identified that mouse and human RGD domains in DSPP required the cleavage of an Ala-Ser peptide bond, next to the RGD domains, to become active. Other species such as bovine, sheep, and bears, possess a Thr-Ser bond next to the RGD domain, which is intrinsically unable to sequester the ability of the RGD domain. To predict the functional importance of certain proteins/domains based on evolutionary conservation rates, the RGD domain of DSPP did not appear to have pivotal roles compared to other SIBLINGs. However, upon investigating the peptide bond next to the RGD domains of DSPP in 37 species, we found most catarrhini, in which humans are classified, possess the Ala-Ser bond. Conclusion The functions of DSPP for integrin-mediated signaling possibly arize from the proteolytic cleavage of the peptide bonds close to the RGD domain and induce reactionary dentinogenesis in vivo .

Levels of serum phosphate are controlled by the peptide hormone FGF23, secreted from bone osteocytes. Elevated levels of circulating FGF23 are a key factor in several hypophosphatemic disorders and play a role in chronic kidney disease. Posttranslational processing of FGF23 includes multi-site O-glycosylation, which reduces intracellular cleavage by proprotein convertases. The FGF23 protein also contains four serine phosphorylation consensus sequences (S-X-D/E); in this work, we asked whether FGF23 is a substrate for secretory phosphorylation. Both HEK cells as well as IDG-SW3 cells, an osteocyte model, incorporated radiolabeled orthophosphate into intact FGF23, as well as into the 14-kDa carboxy-terminal-but not the 17-kDa N-terminal-fragment. Sequential serine-to-alanine site-directed mutagenesis of four kinase consensus sites showed that labeling occurred on three serines within the carboxy-terminal fragment, Ser180 (adjacent to the cleavage site), Ser207, and Ser212. Liquid chromatography-coupled mass spectroscopy indicated the presence of phosphate at Ser212 in recombinant R&D mouse FGF23(R179Q) , confirming labeling results. A phosphopeptide-specific antibody was raised against phospho-Ser212 and exhibited immunoreactivity in osteocytes present in mouse long bone, providing further evidence that FGF23 is naturally phosphorylated in bone. Bone SIBLING proteins are serine-phosphorylated by the ubiquitous Golgi secretory kinase FAM20C. Cotransfection of HEK and MC3T3 cells with FGF23 and active, but not inactive, FAM20C kinase increased the storage and release of FGF23 in radiolabeling experiments, indicating potential effects of phosphorylation on FGF23 stability. Collectively, these data point to an important role for phosphorylation of FGF23 in bone.

Bone tissue is comprised of collagen, non-collagenous proteins, and hydroxyapatite and the SIBLING (small integrin binding, N-linked glycoprotein) family of proteins is the primary group of non-collagenous proteins. By replicating the native interactions between collagen and the SIBLING family of proteins at the interface of an implant, it is believed that a bone scaffold will more easily integrate with the surrounding tissue. In this work bone sialoprotein (BSP), osteopontin (OPN), dentin sialoprotein (DSP), dentin phosphoprotein (DPP), C-terminal fragment of dentin matrix protein 1 (DMP1-C) and proteoglycan versions of DSP (DSP-PG) and DMP1 (DMP1-PG) were tested individually to determine their roles in collagen fibrillogenesis and the prevention of denaturation. It was shown that DSP and DPP slowed down fibrillogenesis, while other SIBLINGs had limited impact. In addition, the denaturation time was faster in the presence of DSP and OPN, indicating a negative impact. The role of calcium ions in these processes was also investigated. The presence of calcium ions sped up fibrillogenesis in all scenarios tested, but it had a negative impact by reducing the extent. Calcium also sped up the denaturation in most cases, with the exception of DMP1-C and DSP where the opposite was seen. Calcium had a similar effect on the proteoglycan variants in the fibrillogenesis process, but had no impact on the denaturation process in the presence of these two. It is believed that incorporating DMP1-C or DSP on the surface of a bone implant may improve the collagen interactions with the implant, thereby facilitating improved osteointegration.

The small integrin-binding ligand N-linked glycoprotein (SIBLING) family consists of osteopontin (OPN), bonesialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE). These proteins, initially identified in bone and teeth, share many structural characteristics. It is now well established that they are over expressed in many tumors and play a critical role at different steps of cancer development. In this review, we describe the roles of SIBLING proteins at different stages of cancer progression including cancer cell adhesion, proliferation, migration, invasion, metastasis and angiogenesis.

X-linked hypophosphatemia (XLH/HYP)—with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscesses—is caused by mutations in the zinc-metallopeptidase PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome). PHEX is highly expressed by mineralized tissue cells. Inactivating mutations in PHEX lead to distal renal effects (implying accumulation of a secreted, circulating phosphaturic factor) and accumulation in bone and teeth of mineralization-inhibiting, acidic serine- and aspartate-rich motif (ASARM)-containing peptides, which are proteolytically derived from the mineral-binding matrix proteins of the SIBLING family (small, integrin-binding ligand N-linked glycoproteins). Although the latter observation suggests a local, direct matrix effect for PHEX, its physiologically relevant substrate protein(s) have not been identified. Here, we investigated two SIBLING proteins containing the ASARM motif—osteopontin (OPN) and bone sialoprotein (BSP)—as potential substrates for PHEX. Using cleavage assays, gel electrophoresis, and mass spectrometry, we report that OPN is a full-length protein substrate for PHEX. Degradation of OPN was essentially complete, including hydrolysis of the ASARM motif, resulting in only very small residual fragments. Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts having no PHEX activity clearly showed accumulation of an ∼35 kDa OPN fragment that was not present in wild-type mouse bone. Immunohistochemistry and immunogold labeling (electron microscopy) for OPN in Hyp bone likewise showed an accumulation of OPN and/or its fragments compared with normal wild-type bone. Incubation of Hyp mouse bone extracts with PHEX resulted in the complete degradation of these fragments. In conclusion, these results identify full-length OPN and its fragments as novel, physiologically relevant substrates for PHEX, suggesting that accumulation of mineralization-inhibiting OPN fragments may contribute to the mineralization defect seen in the osteomalacic bone characteristic of XLH/HYP. © 2013 American Society for Bone and Mineral Research.

There is substantial practical interest in the mechanism by which the carbonated apatite of bone mineral can be initiated specifically in a matrix. The current literature is replete with studies aimed at mimicking the properties of vertebrate bone, teeth, and other hard tissues by creating organic matrices that can be mineralized in vitro and either functionally substitute for bone on a permanent basis or serve as a temporary structure that can be replaced by normal remodeling processes. A key element in this is mineralization of an implant with the matrix and mineral arranged in the proper orientations and relationships. This review examines the pathway to crystallization from a supersaturated calcium phosphate solution in vitro, focusing on the basic mechanistic questions concerning mineral nucleation and growth. Since bone and dentin mineral forms within collagenous matrices, we consider how the in vitro crystallization mechanisms might or might not be applicable to understanding the in vivo processes of biomineralization in bone and dentin. We propose that the pathway to crystallization from the calcium phosphate-supersaturated tissue fluids involves the formation of a dense liquid phase of first-layer bound-water hydrated calcium and phosphate ions in which the crystallization is nucleated. SIBLING proteins and their in vitro analogs, such as polyaspartic acids, have similar dense liquid first-layer bound-water surfaces which interact with the dense liquid calcium phosphate nucleation clusters and modulate the rate of crystallization within the bone and dentin collagen fibril matrix.

The eggshell is an ancient innovation that helped the vertebrates' transition from the oceans and gain dominion over the land. Coincident with this conquest, several new eggshell and noncollagenous bone-matrix proteins (NCPs) emerged. The protein ovocleidin-116 is one of these proteins with an ancestry stretching back to the Triassic. Ovocleidin-116 is an avian homolog of Matrix Extracellular Phosphoglycoprotein (MEPE) and belongs to a group of proteins called Small Integrin-Binding Ligand Interacting Glycoproteins (SIBLINGs). The genes for these NCPs are all clustered on chromosome 5q in mice and chromosome 4q in humans. A unifying feature of the SIBLING proteins is an Acidic Serine Aspartate-Rich MEPE (ASARM)-associated motif. The ASARM motif and the released ASARM peptide play roles in mineralization, bone turnover, mechanotransduction, phosphate regulation and energy metabolism. ASARM peptides and motifs are physiological substrates for phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX), a Zn metalloendopeptidase. Defects in PHEX are responsible for X-linked hypophosphatemic rickets. PHEX interacts with another ASARM motif containing SIBLING protein, Dentin Matrix Protein-1 (DMP1). DMP1 mutations cause bone-renal defects that are identical with the defects caused by loss of PHEX function. This results in autosomal recessive hypophosphatemic rickets (ARHR). In both X-linked hypophosphatemic rickets and ARHR, increased fibroblast growth factor 23 (FGF23) expression occurs, and activating mutations in FGF23 cause autosomal dominant hypophosphatemic rickets (ADHR). ASARM peptide administration in vitro and in vivo also induces increased FGF23 expression. This review will discuss the evidence for a new integrative pathway involved in bone formation, bone-renal mineralization, renal phosphate homeostasis and energy metabolism in disease and health.

Increased acidic serine aspartate-rich MEPE-associated motif (ASARM) peptides cause mineralization defects in X-linked hypophosphatemic rickets mice (HYP) and “directly” inhibit renal phosphate uptake in vitro. However, ASARM peptides also bind to phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and are a physiological substrate for this bone-expressed, phosphate-regulating enzyme. We therefore tested the hypothesis that circulating ASARM peptides also “indirectly” contribute to a bone-renal PHEX-dependent hypophosphatemia in normal mice. Male mice ( n = 5; 12 wk) were fed for 8 wk with a normal phosphorus and vitamin D3diet (1% Pidiet) or a reduced phosphorus and vitamin D3diet (0.1% Pidiet). For the final 4 wk, transplantation of mini-osmotic pumps supplied a continuous infusion of either ASARM peptide (5 mg·day−1·kg−1) or vehicle. HYP, autosomal recessive hypophosphatemic rickets (ARHR), and normal mice (no pumps or ASARM infusion; 0.4% Pidiet) were used in a separate experiment designed to measure and compare circulating ASARM peptides in disease and health. ASARM treatment decreased serum phosphate concentration and renal phosphate cotransporter (NPT2A) mRNA with the 1% Pidiet. This was accompanied by a twofold increase in serum ASARM and 1,25-dihydroxy vitamin D3[1,25 (OH)2D3] levels without changes in parathyroid hormone. For both diets, ASARM-treated mice showed significant increases in serum fibroblast growth factor 23 (FGF23; +50%) and reduced serum osteocalcin (−30%) and osteopontin (−25%). Circulating ASARM peptides showed a significant inverse correlation with serum Piand a significant positive correlation with fractional excretion of phosphate. We conclude that constitutive overexpression of ASARM peptides plays a “component” PHEX-independent part in the HYP and ARHR hypophosphatemia. In contrast, with wild-type mice, ASARM peptides likely play a bone PHEX-dependent role in renal phosphate regulation and FGF23 expression. They may also coordinate FGF23 expression by competitively modulating PHEX/DMP1 interactions and thus bone-renal mineral regulation.

The protease that cleaves the most abundant non-collagenous protein of dentin matrix, dentin sialophosphoprotein (DSPP), into its two final dentin matrix products, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), has not been directly identified. In this study, full-length recombinant mouse DSPP was made for the first time in furin-deficient mammalian LoVo cells and used to test the ability of three different isoforms of one candidate protease, bone morphogenetic protein-1 (BMP1) to cleave DSPP at the appropriate site. Furthermore, two reported enhancers of BMP1/mTLD activity (procollagen C-endopeptidase enhancer-1, PCPE-1, and secreted frizzled-related protein-2, sFRP2) were tested for their abilities to modulate BMP1-mediated processing of both DSPP and another SIBLING family member with a similar cleavage motif, dentin matrix protein-1 (DMP1). Three splice variants of BMP1 (classic BMP1, the full-length mTolloid (mTLD), and the shorter isoform lacking the CUB3 domain, BMP1-5) were all shown to cleave the recombinant DSPP in vitro although mTLD was relatively inefficient at processing both DSPP and DMP1. Mutation of the MQGDD peptide motif to IEGDD completely eliminated the ability of all three recombinant isoforms to process full-length recombinant DSPP in vitro thereby verifying the single predicted cleavage site. Furthermore when human bone marrow stromal cells (which naturally express furin-activated BMP1) were transduced with the adenovirus-encoding either wild-type or mutant DSPP, they were observed to fully cleave wild-type DSPP but failed to process the mutant DSPP(MQDeltaIE) during biogenesis. All three BMP1 isoforms were shown to process type I procollagen as well as DSPP and DMP1 much more efficiently in low-salt buffer (< or = 50 mM NaCl) compared to commonly used normal saline buffers (150 mM NaCl). Neither PCPE-1 nor sFRP2 were able to enhance any of the three BMP1 isoforms in cleaving either DSPP or DMP1 under either low or normal saline conditions. Interestingly, we were unable to reproduce sFRP2's reported ability to enhance the processing of type I procollagen by BMP1/mTLD. In summary, three isoforms of BMP1 process both DSPP and DMP1 at the MQX/DDP motif, but the identity of a protein that can enhance the cleavage of the two SIBLING proteins remains elusive.

Oral Diseases (2010) 16, 453–464 Objectives: Human X-linked hypophosphatemia (XLH) and its murine homologue, Hyp are caused by inactivating mutations in PHEX gene. The protein encoded by PHEX gene is an endopeptidase whose physiological substrate(s) has not been identified. Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP), two members of the Small Integrin-Binding LIgand, N-linked Glycoprotein (SIBLING) family are proteolytically processed. It has been speculated that PHEX endopeptidase may be responsible for the proteolytic cleavage of DMP1 and DSPP. To test this hypothesis and to analyse the distribution of SIBLING proteins in the predentin/dentin complex and mandible of Hyp mice, we compared the expression of four SIBLING proteins, DMP1, DSPP, bone sialoprotein (BSP) and osteopontin (OPN) between Hyp and wild-type mice. Methods: These SIBLING proteins were analysed by protein chemistry and immunohistochemistry. Results: (1) Dentin matrix protein 1 and DSPP fragments are present in the extracts of Hyp predentin/dentin and bone; (2) the level of DMP1 proteoglycan form, BSP and OPN is elevated in the Hyp bone. Conclusions: The PHEX protein is not the enzyme responsible for the proteolytic processing of DMP1 and DSPP. The altered distribution of SIBLING proteins may be involved in the pathogenesis of bone and dentin defects in Hyp and XLH.

Dentin is a collagen-based mineralized tissue consisting of inorganic apatite crystallites embedded in an extracellular matrix. In addition to type I collagen, the dentin organic matrix contains several proteins and proteoglycans, collectively referred to as non-collagenous proteins, that play fundamental roles in actively promoting, controlling, and regulating fibrillogenesis, crystal growth, and mineralization during dentinogenesis. Similarly to collagen fibrils, non-collagenous proteins are synthesized and secreted by odontoblasts and some of them are detectable in both predentin and mineralized dentin while others are only within the dentin layer. Non-collagenous components include proteoglycans (such as decorin, biglycan, fibromodulin, lumican, osteoadherin, and versican) and several glycoproteins such as osteocalcin, osteonectin, and the SIBLING proteins (i.e. osteopontin, bone sialoprotein, dentin matrix protein-1, dentin sialophosphoprotein, and matrix extracellular phosphoglycoprotein). The first part of this review offers an overview of the nature of the above-mentioned non-collagenous proteins of the dentin organic matrix and their postulated functions, since a better understanding of the biological dynamics of the extracellular macromolecules is fundamental to clarifying the properties and function of the human mature sound dentin.

Bone Sialoprotein (BSP) is a member of the "Small Integrin-Binding Ligand N-linked Glycoproteins" (SIBLING) extracellular matrix protein family of mineralized tissues. BSP has been less studied than other SIBLING proteins such as Osteopontin (OPN), which is coexpressed with it in several skeletal cell types. Here we review the contribution of genetically engineered mice (BSP gene knockout and overexpression) to the understanding of the role of BSP in the bone organ. The studies made so far highlight the role of BSP in skeletal mineralization, as well as its importance for proper osteoblast and osteoclast differentiation and activity, most prominently in primary/repair bone. The absence of BSP also affects the local environment of the bone tissue, in particular hematopoiesis and vascularization. Interestingly, lack of BSP induces an overexpression of OPN, and the cognate protein could be responsible for some aspects of the BSP gene knockout skeletal phenotype, while replacing BSP for some of its functions. Such interplay between the partly overlapping functions of SIBLING proteins, as well as the network of cross-regulations in which they are involved should now be the focus of further work.

© 2016 International Society of Matrix Biology. Tendons/ligaments insert into bone via a transitional structure, the enthesis, which is susceptible to injury and difficult to repair. Fibrocartilaginous entheses contain fibrocartilage in their transitional zone, part of which is mineralized. Mineral-associated proteins within this zone have not been adequately characterized. Members of the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family are acidic phosphoproteins expressed in mineralized tissues. Here we show that two SIBLING proteins, bone sialoprotein (BSP) and osteopontin (OPN), are present in the mouse enthesis. Histological analyses indicate that the calcified zone of the quadriceps tendon enthesis is longer in Bsp-/- mice, however no difference is apparent in the supraspinatus tendon enthesis. In an analysis of mineral content within the calcified zone, micro-CT and Raman spectroscopy reveal that the mineral content in the calcified fibrocartilage of the quadriceps tendon enthesis are similar between wild type and Bsp-/- mice. Mechanical testing of the patellar tendon shows that while the tendons fail under similar loads, the Bsp-/- patellar tendon is 7.5% larger in cross sectional area than wild type tendons, resulting in a 16.5% reduction in failure stress. However, Picrosirius Red staining shows no difference in collagen organization. Data collected here indicate that BSP is present in the calcified fibrocartilage of murine entheses and suggest that BSP plays a regulatory role in this structure, influencing the growth of the calcified fibrocartilage in addition to the weakening of the tendon mechanical properties. Based on the phenotype of the Bsp-/- mouse enthesis, and the known in vitro functional properties of the protein, BSP may be a useful therapeutic molecule in the reattachment of tendons and ligaments to bone.

International audience; Bone sialoprotein (BSP) belongs to the "small integrin-binding ligand N-linked glycoprotein" (SIBLING) family, whose members interact with bone cells and bone mineral. BSP is strongly expressed in bone and we previously showed that BSP knockout (BSP-/-) mice have a higher bone mass than wild type (BSP+/+) littermates, with lower bone remodelling. Because baseline bone formation activity is constitutively lower in BSP-/-mice, we studied the impact of the absence of BSP on in vitro osteogenesis in mouse calvaria cell (MCC) cultures. MCC BSP-/-cultures exhibit fewer fibroblast (CFU-F), preosteoblast (CFU-ALP) and osteoblast colonies (bone nodules) than wild type, indicative of a lower number of osteopro-genitors. No mineralized colonies were observed in BSP-/-cultures, along with little/no expression of either osteogenic markers or SIBLING proteins MEPE or DMP1. Osteopontin (OPN) is the only SIBLING expressed in standard density BSP-/-culture, at higher levels than in wild type in early culture times. At higher plating density, the effects of the absence of BSP were partly rescued, with resumed expression of osteoblast markers and cognate SIBLING proteins, and mineralization of the mutant cultures. OPN expression and amount are further increased in high density BSP-/-cultures, while PHEX and CatB expression are differentiatlly regulated in a manner that may favor mineralization. Altogether, we found that BSP regulates mouse calvaria osteoblast cell clonogenicity, differentiation and activity in vitro in a cell density dependent manner, consistent with the effective skeletogenesis but the low levels of bone formation observed in vivo. The BSP knockout bone microenviron-ment may alter the proliferation/cell fate of early osteoprogenitors.

Transglutaminase 2 (TG2), a protein-crosslinking enzyme, participates in extracellular matrix maturation and cell adhesion in cartilage and bone. We hypothesized that TG2 has similar roles in teeth. A TG activity assay and immunoblotting of rat tooth extracts showed TG activity and the presence of high-molecular-weight forms of the SIBLING (Small Integrin-Binding LIgand N-linked Glycoprotein) proteins: dentin matrix protein 1 (DMP1), dentin phosphoprotein (DPP), and bone sialoprotein (BSP). DMP1 and BSP, each containing both glutamine and lysine residues critical for crosslink formation, readily formed polymers in vitro when incubated with TG2. The ability of glutamine-lacking DPP to form polymers in vitro and in vivo demonstrates that it could act as a lysine donor for crosslinking, potentially having protein crosslinking partner(s) in teeth. Consistent with a role in cell adhesion, the TG2 isoform was co-localized by immunohistochemistry with its substrates at cell-matrix adhesion sites, including along odontoblast tubules (DMP1 and DPP), in the pericellular matrix of cementocytes (DMP1), and in predentin (BSP).

Several proteins associated with mineralised tissue (teeth and bone) or involved in calcium phosphate stabilisation in the body fluids, milk and saliva have been mapped to the q arm of human chromosome 4. These include the dentine/bone proteins dentine sialophosphoprotein (DSPP), dentine matrix protein 1 (DMP1), bone sialoprotein (BSP), matrix extracellular phosphoglycoprotein, osteopontin (OPN), enamelin, ameloblastin, milk caseins, salivary statherin, and proline-rich proteins. The proposed function of those that are multiphosphorylated is: (i) the stabilisation of calcium phosphate in solution (e.g. casein, statherin) preventing spontaneous precipitation and seeded-crystal growth or (ii) promoting biomineralisation (e.g. the phosphophoryn domain of DSPP), where the protein described as a template macromolecule, is proposed to act as a nucleator/promoter of crystal growth. The genes of these proteins have been subjected to conserved chromosomal synteny during mammalian evolution. The multiphosphorylated proteins statherin, caseins, phosphophoryn, BSP and OPN have been characterised as intrinsically disordered. The codon usage patterns for the amino acid serine reveal a bias for AGC and AGT codons within the human genes dspp, dmp1 and bsp, mouse dspp and dmp1 but not significantly for statherin or caseins. This pattern was also observed in the gene encoding hen phosvitin that also contains stretches of multiphosphorylated serines and in the dmp1 gene sequences of mammalian, reptilian and avian classes. In conclusion, these intrinsically disordered multiphosphorylated proteins are the translation products of genes displaying examples of codon usage bias, internal repeats and conserved chromosomal synteny within the mammalian class.

Matrix proteins play important roles in tissue morphogenesis. We have studied the expression of genes encoding the related SIBLING glycoproteins osteopontin (OPN), bone sialoprotein (BSP), and dentin matrix protein (DMP) during the development of male and female gonads during mouse embryogenesis. Opn mRNA was expressed specifically by Sertoli cells of the developing testis cords, in the mesonephric tubules of both sexes, and, transiently, in the Müllerian ducts of both sexes, as determined by whole-mount and section in situ hybridization. OPN protein was detected in the cytoplasm of Sertoli cells and luminal cells of the mesonephric tubules, with small amounts associated with the plasma membrane of germ cells. We found no defects in developing testes of Opn-/- mice using a range of cell type-specific markers, suggesting that other SIBLING proteins may function in testis development. Dmp and Bsp mRNA was also expressed in the developing testis cords, supporting the view that all three SIBLING proteins may contribute to testis differentiation.

The extracellular matrix (ECM) of bone and dentin contains several non-collagenous proteins. One category of non-collagenous protein is termed the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family, that includes osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE). These polyanionic SIBLING proteins are believed to play key biological roles in the mineralization of bone and dentin. Although the specific mechanisms involved in controlling bone and dentin formation are still unknown, it is clear that some functions of the SIBLING family members are dependent on the nature and extent of post-translational modifications (PTMs), such as phosphorylation, glycosylation, and proteolytic processing, since these PTMs would have significant effects on their structure. OPN and BSP are present in the ECM of bone and dentin as full-length forms, whereas amino acid sequencing indicates that DMP1 and DSPP exist as proteolytically processed fragments that result from scission of X-Asp bonds. We hypothesized that the processing of DMP1 and DSPP is catalyzed by the PHEX enzyme, since this protein, an endopeptidase that is predominantly expressed in bone and tooth, has a strong preference for cleavage at the NH2-terminus of aspartyl residue. We envision that the proteolytic processing of DMP1 and DSPP may be an activation process that plays a significant, crucial role in osteogenesis and dentinogenesis, and that a failure in this processing would cause defective mineralization in bone and dentin, as observed in X-linked hypophosphatemic rickets.

Full-length cDNA coding for dentin matrix protein 1 (DMP1) has been cloned and sequenced, but the corresponding complete protein has not been isolated. In searching for naturally occurring DMP1, we recently discovered that the extracellular matrix of bone contains fragments originating from DMP1. Shortened forms of DMP1, termed 37K and 57K fragments, were treated with alkaline phosphatase and then digested with trypsin. The resultant peptides were purified by a two-dimensional method: size exclusion followed by reversed-phase high performance liquid chromatography. Purified peptides were sequenced by Edman degradation and mass spectrometry, and the sequences compared with the DMP1 sequence predicted from cDNA. Extensive sequencing of tryptic peptides revealed that the 37K fragments originated from the NH2-terminal region, and the 57K fragments were from the COOH-terminal part of DMP1. Phosphate analysis indicated that the 37K fragments contained 12 phosphates, and the 57K fragments had 41. From 37K fragments, two peptides lacked a COOH-terminal lysine or arginine; instead they ended at Phe173 and Ser180 and were thus COOH termini of 37K fragments. Two peptides were from the NH2 termini of 57K fragments, starting at Asp218 and Asp222. These findings indicated that DMP1 is proteolytically cleaved at four bonds, Phe173–Asp174, Ser180–Asp181, Ser217–Asp218, and Gln221–Asp222, forming eight fragments. The uniformity of cleavages at the NH2-terminal peptide bonds of aspartyl residues suggests that a single proteinase is involved. Based on its reported specificity, we hypothesize that these scissions are catalyzed by PHEX protein. We envision that the proteolytic processing of DMP1 plays a crucial role during osteogenesis and dentinogenesis.

Bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), enamelin (ENAM), matrix extracellular phosphoglycoprotein (MEPE), and osteopontin (OPN) are glycophosphoproteins expressed in bones and/or teeth. Direct comparison of their amino acid sequences do not suggest that they belong to a single genetic family, but a detailed analysis of their chromosomal location and gene structure does. Analysis of human brain mRNA by RT-PCR has led to the discovery of two additional exons thereby making it more convincing that MEPE is a member of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family. We propose that the members of this SIBLING family are extended, flexible proteins in solution that can facilitate the formation of a number of different complexes. For example, OPN can bridge complement Factor H to either an RGD-dependent integrin or to CD44 forming a membrane-bound complex that actively suppresses the alternate complement pathway. Two possible mechanisms for inhibiting the lytic pathway of alternate complement are presented.

Fourier transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) were used to characterize the mineral in bones of two different lines of Opn-deficient (Opn-/-) mice and their background-matched wild-type controls (Opn+/+). Sections of tibia and femur from 12-week-old and 16-week-old mice were evaluated with a spatial resolution between 10 microm (FTIRM) and 7 microm (FTIRI). FTIRI was used to examine 400 microm x 400 microm areas in cortical bone and trabecular bone and FTIRM examined selected 20 microm x 20 microm areas at sites within these anatomically defined areas. Despite the absence of an obvious phenotype in Opn-deficient mice, being undetectable by radiographic and histological methods, FTIRM analyses revealed that the relative amount of mineral in the more mature areas of the bone (central cortical bone) of Opn-knockout mice was significantly increased. Moreover, mineral maturity (mineral crystal size and perfection) throughout all anatomic regions of the Opn-deficient bone was significantly increased. The 2-dimensional, color-coded data (images) produced by FTIRI showed similar increases in mineral maturity in the Opn-/- bone, however, the crystallinity parameters were less sensitive, and significance was not achieved in all areas analyzed. Nonetheless, the findings of increased mineral content and increased crystal size/perfection in both lines of Opn-deficient mice at both ages are consistent with in vitro data indicating that Opn is a potent inhibitor of mineral formation and mineral crystal growth and proliferation, and also support a role for Opn in osteoclast recruitment and function.

Four sialic acid-rich (SA-rich) proteins found in bone and dentin, osteopontin (OPN), bone sialoprotein (BSP), bone acidic glycoprotein-75 (BAG-75), and dentin matrix protein 1 (DMP1), share some common features. We used SDS-PAGE and Western immunoblots to analyze and compare SA-rich proteins in bone and dentin extracts from rats with a single chromatographic procedure. OPN was detected in dentin extracts, with a relative level less than one-seventieth of that in bone. Both bone and dentin BSP demonstrated an extremely broad distribution pattern, probably due to a high degree of heterogeneity in post-translational modifications. BAG-75 in both bone and dentin was detected as an 83 kDa band, dramatically distinct from that of DMP1. Using a polyclonal antibody raised against a purified bone 57 kDa protein (a portion of DMP1), we detected 150 kDa protein bands in bone fraction; the same bands were recognized by anti-recombinant rat DMP1 antibody. Bands from dentin migrating at about 150 kDa in earlier fractions and progressing to 200 kDa in later fractions showed a clear immunoreactivity to the anti-57 kDa antibody. We conclude that the majority of DMP1 in rat bone is processed into fragments, whereas that in dentin remains intact.

Dentin is the major mineralized extracellular matrix of the tooth. The organic components of dentin consist of type I collagen (90%) with 10% noncollagenous proteins, which are also components of bone. Two dentin proteins, dentin sialoprotein and dentin phosphoprotein, have been shown to be tooth-specific being expressed mostly by odontoblast cells. In this study, we screened a mouse molar tooth library for dentin sialoprotein and dentin phosphoprotein cDNA clones. Analysis of the clones resulted in characterization of a 4420-nucleotide cDNA that contained a 940-amino acid open reading frame. The signal peptide and NH2-terminal sequence was 75% homologous to the cDNA sequence of rat dentin sialoprotein. The continued open reading frame, however, contained a RGD sequence followed by a region of repeated aspartic acid and serine residues. This portion of the protein codes for amino acid sequence consistent with that of dentin phosphoprotein. The noncoding region contains three potential polyadenylation signals, two of which were shown to be utilized. Northern blot analysis indicated the presence of two major transcripts of 4.4 and 2.2 kilobases in odontoblasts. Chromosomal mapping localized the gene to human chromosome 4. These data suggest that the previously identified dentin extracellular matrix proteins, dentin sialoprotein and dentin phosphoprotein, are expressed as a single cDNA transcript coding for a protein that is specifically cleaved into two smaller polypeptides with unique physical-chemical characteristics. Therefore, we propose that the gene be named dentin sialophosphoprotein. The location of the human dentin sialophosphoprotein gene on chromosome 4 suggests that this gene may be a strong candidate gene for the genetic disease dentinogenesis imperfecta type II.

It has been widely shown that proteoglycans (PG) and their glycosaminoglycan (GAG) side-chains form supramolecular aggregates that interconnect the collagenous network in connective tissues and play a significant role in regulating the mechanical behavior of the extracellular matrix, particularly in soft tissues. However, collective evidence of the mechanical participation of PGs and GAGs in mineralized tissues remains poorly explored in the literature. Here, we address this knowledge gap and discuss the participation of PGs on the biomechanics of mineralized tissues including dentine, cementum and bone. We review evidence suggesting that, on a microscale, PGs regulate the hydrostatic and osmotic pressure, as well as the poroelastic behavior of dentine and bone. On the nanoscale, we review the so-called sliding filament theory and intramolecular stretching of GAGs. We also discuss recent interpretations whereby folding and unfolding of the PG protein core, potentially in association with SIBLING proteins, may be a contributing factor to the mechanical behavior of mineralized tissues. Finally, we review in vitro and in vivo studies of mineralized tissues with targeted disruption or digestion of specific PG family members, which provide further insights into their relevance to the mechanical properties of load bearing hard tissues. In summary, this review brings forth collective evidence suggesting that PGs and GAGs, although less than 5% of the tissue matrix, may play a role in the mechanical behavior and durability of mineralized tissues.

Human bone sialoprotein (BSP) is an essential component of the extracellular matrix of bone. It is thought to be the primary nucleator of hydroxyapatite crystallization, and is known to bind to hydroxyapatite, collagen, and cells. Mature BSP shows extensive post-translational modifications, including attachment of glycans, sulfation, and phosphorylation, and is highly flexible with no specific 2D or 3D structure in solution or the solid state. These features have severely limited the experimental characterization of the structure of this protein. We have therefore developed a 3D structural model for BSP, based on the available literature data, using molecular modelling techniques. The complete model consists of 301 amino acids, including six phosphorylated serines and two sulfated tyrosines, plus 92 N- and O-linked glycan residues. A notable feature of the model is a large acidic patch that provides a surface for binding Ca2+ ions. Density functional theory quantum calculations with an implicit solvent model indicate that Ca2+ ions are bound most strongly by the phosphorylated serines within BSP, along with reasonably strong binding to Asp and Glu, but weak binding to His and sulfated tyrosine. The process of early hydroxyapatite nucleation has been studied by molecular dynamics on an acidic surface loop of the protein; the results suggest that the cationic nature of the loop promotes nucleation by attracting Ca2+ ions, while its flexibility allows for their rapid self-assembly with PO43− ions, rather than providing a regular template for crystallization. The binding of a hydroxyapatite crystal at the protein's acidic patch has also been modelled. The relationships between hydroxyapatite, collagen and BSP are discussed.

As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth - where calcium phosphate crystals are deposited and grow within an extracellular matrix - is essential for dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition in which teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibers (Sharpey's fibers) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth-suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin-binding ligand N-linked glycoproteins and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here, with clinical examples given, namely tissue-nonspecific alkaline phosphatase and phosphate-regulating gene with homologies to endopeptidases on the X chromosome. Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia and X-linked hypophosphatemia, respectively, where the levels of local and systemic circulating mineralization determinants are perturbed. In X-linked hypophosphatemia, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization-regulating small integrin-binding ligand N-linked glycoproteins, such as matrix extracellular phosphoglycoprotein and osteopontin, and the phosphorylated peptides proteolytically released from them, such as the acidic serine- and aspartate-rich-motif peptide, may accumulate locally to impair mineralization in this disease.

The small integrin-binding ligand N-linked glycoprotein (SIBLING) family consists of osteopontin, bone sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein and matrix extracellular phosphoglycoprotein. These proteins share many structural characteristics and are primarily located in bone and dentin. Accumulating evidence has implicated the SIBLING proteins in matrix mineralisation. Therefore, in this review, we discuss the individual role that each of the SIBLING proteins has in this highly orchestrated process. In particular, we emphasise how the nature and extent of their proteolytic processing and post-translational modification affect their functional role. Finally, we describe the likely roles of the SIBLING proteins in clinical disorders of hypophosphataemia and their potential therapeutic use. Journal of Endocrinology (2012) 214, 241-255

Native bone tissue is composed of a matrix of collagen, non-collagenous proteins, and calcium phosphate minerals, which are primarily hydroxyapatite (HA). The SIBLING (small integrin-binding ligand, N-linked glycoprotein) family of proteins is the primary non-collagenous protein group found in mineralized tissues. In this work, the mineralization induction capabilities of three of the SIBLING members, bone sialoprotein (BSP), osteopontin (OPN), and the calcium binding subdomain of dentin sialophosphoprotein, dentin phosphoprotein (DPP), are directly compared on a biomimetic collagen substrate. A self-assembled, loosely aligned collagen fibril substrate was prepared and then 125I radiolabeled adsorption isotherms were developed for BSP, OPN, and DPP. The results showed that BSP exhibited the highest binding capacity for collagen at lower concentrations, followed by DPP and OPN. However, at the highest concentrations all three proteins had similar adsorption levels. The adsorption isotherms were then used to identify conditions that resulted in identical amounts of adsorbed protein. These substrates were prepared and placed in simulated body fluid for 5 hours, 10 hours, and 24 hours at 37°C. The resulting mineral morphology was assessed by atomic force microscopy and the composition was determined using photochemical assays. Mineralization was seen in the presence of all of the proteins. However, DPP was seen to be the only protein that formed individual mineral nodules similar to those seen in developing bone. This suggests that DPP plays a significant role in the biomineralization process and that the incorporation of DPP into tissue engineering constructs may facilitate the induction of biomimetic mineral formation.

The manuscript tests the hypothesis that posttranslational modification of the SIBLING family of proteins in general and osteopontin in particular modify the abilities of these proteins to regulate in vitro hydroxyapatite (HA) formation. Osteopontin has diverse effects on hydroxyapatite (HA) mineral crystallite formation and growth depending on the extent of phosphorylation. We hypothesized that different regions of full-length OPN would also have distinct effects on the mineralization process. Thrombin fragmentation of milk OPN (mOPN) was used to test this hypothesis. Three fragments were tested in a de novo HA formation assay; an N-terminal fragment (aa 1-147), a central fragment (aa 148-204) denoted SKK-fragment and a C-terminal fragment (aa 205-262). Compared to intact mOPN the C- and N-terminal fragments behaved comparably, promoting HA formation and growth, but the central SKK-fragment acted as a mineralization inhibitor. In a seeded growth experiment all fragments inhibited mineral proliferation, but the SKK-fragment was the most effective inhibitor. These effects, seen in HA-formation and seeded growth assays in a gelatin gel system and in a pH-stat experiment were lost when the protein or fragments were dephosphorylated. Effects of the fully phosphorylated protein and fragments were also altered in the presence of fibrillar collagen. The diverse effects can be explained in terms of the intrinsically disordered nature of OPN and its fragments which enable them to interact with their multiple partners.

More than 300 million years ago, vertebrates emerged from the vast oceans to conquer gravity and the dry land. With this transition, new adaptations occurred that included ingenious changes in reproduction, waste secretion, and bone physiology. One new innovation, the egg shell, contained an ancestral protein (ovocleidin-116) that likely first appeared with the dinosaurs and was preserved through the theropod lineage in modern birds and reptiles. Ovocleidin-116 is an avian homolog of matrix extracellular phosphoglycoprotein (MEPE) and belongs to a group of proteins called short integrin-binding ligand-interacting glycoproteins (SIBLINGs). These proteins are all localized to a defined region on chromosome 5q in mice and chromosome 4q in humans. A unifying feature of SIBLING proteins is an acidic serine aspartate-rich MEPE-associated motif (ASARM). Recent research has shown that the ASARM motif and the released ASARM peptide have regulatory roles in mineralization (bone and teeth), phosphate regulation, vascularization, soft-tissue calcification, osteoclastogenesis, mechanotransduction, and fat energy metabolism. The MEPE ASARM motif and peptide are physiological substrates for PHEX, a zinc metalloendopeptidase. Defects in PHEX are responsible for X-linked hypophosphatemic rickets (HYP). There is evidence that PHEX interacts with another ASARM motif containing the SIBLING protein, dentin matrix protein-1 (DMP1). DMP1 mutations cause bone and renal defects that are identical with the defects caused by a loss of PHEX function. This results in autosomal recessive hypophosphatemic rickets (ARHR). In both HYP and ARHR, increased FGF23 expression plays a major role in the disease and in autosomal dominant hypophosphatemic rickets (ADHR), FGF23 half-life is increased by activating mutations. ASARM peptide administration in vitro and in vivo also induces increased FGF23 expression. FGF23 is a member of the fibroblast growth factor (FGF) family of cytokines, which surfaced 500 million years ago with the boney fish (i.e., teleosts) that do not contain SIBLING proteins. In terrestrial vertebrates, FGF23, like SIBLING proteins, is expressed in the osteocyte. The boney fish, however, are an-osteocytic, so a physiological bone-renal link with FGF23 and the SIBLINGs was cemented when life ventured from the oceans to the land during the Triassic period, approximately 300 million years ago. This link has been revealed by recent research that indicates a competitive displacement of a PHEX–DMP1 interaction by an ASARM peptide that leads to increased FGF23 expression. This review discusses the new discoveries that reveal a novel PHEX, DMP1, MEPE, ASARM peptide, and FGF23 bone-renal pathway. This pathway impacts not only bone formation, bone-renal mineralization, and renal phosphate homeostasis but also energy metabolism. The study of this new pathway is relevant for developing therapies for several diseases: bone–teeth mineral loss disorders, renal osteodystrophy, chronic kidney disease and bone mineralization disorders (CKD-MBD), end-stage renal diseases, ectopic arterial-calcification, cardiovascular disease renal calcification, diabetes, and obesity.

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) are expressed as a single mRNA transcript coding for a large precursor protein termed dentin sialophosphoprotein (DSPP). DSP, DPP, and DSPP have been considered to be tooth-specific. To test for the expression of the dspp gene in bone, we performed Western immunoblots and reverse-transcription polymerase chain-reaction (RT-PCR). With Western immunoblots, we detected DSP in the Gdm/EDTA extracts of rat long bone, at a level of about 1/400 of that in dentin. Using RT-PCR, we detected DSPP mRNA in mouse calvaria. Similar to Western immunoblots, the results of RT-PCR indicated that the dspp gene is expressed at a lower level in bone than in dentin and odontoblasts. Analysis of the data shows that DSPP is not a tooth-specific protein, and that dramatically different regulatory mechanisms governing DSPP expression are involved in the bone and dentin.

Dentin sialophosphoprotein (DSPP) is a member of the SIBLING (small integrin binding N-linked glycoprotein) family of proteins commonly found in mineralized tissues. Dentin phosphoprotein (DPP) is a naturally occurring subdomain of DSPP that contains the cell binding RGD sequence. Previously, the orientation and conformation of other SIBLING family members specifically bound to collagen I have been investigated with respect to their cell adhesion properties. In this study, the orientation of DPP under similar circumstances is examined, and the results are discussed relative to the previous investigations. Radiolabeled adsorption isotherms were developed for DPP adsorbing to both tissue culture polystyrene (TCPS) and collagen coated TCPS. Then, a MC3T3-E1 cell adhesion assay was performed on TCPS and collagen coated TCPS in the presence of identical amounts of adsorbed DPP. It was discovered that there was a significant difference in the number of bound cells on the TCPS and collagen coated TCPS, with a preference for TCPS. Furthermore, a cell inhibition assay was conducted to confirm that the cell binding that occurred was due to specific integrin interactions with the RGD sequence of DPP. These results suggest that the orientation of DPP, rather than its conformation, dictates the accessibility of the cell binding RGD domains of DPP and that the RGD sequence in DPP is less accessible when DPP is specifically bound to collagen. The results obtained in this study are in stark contrast to previous studies with related SIBLING proteins, and suggest that DPP does not play a key role in cell binding to the collagen matrix of developing bone.

Osteopontin is a phosphorylated bone matrix sialoprotein, postulated to play a regulatory role in biomineralization. The effects of a crude preparation of rat bone osteopontin and a more highly purified bovine bone osteopontin were evaluated using a gel diffusion system to measure effects of 0.1-100 micrograms/ml of this matrix protein on hydroxyapatite formation and crystal proliferation. Bovine osteopontin at concentrations greater than 25 micrograms/ml inhibited both hydroxyapatite formation and growth in a dose-dependent manner. Osteopontin at concentrations lower than 25 micrograms/ml had no detectable effect on the amount of mineral accumulated in experiments with and without pre-formed hydroxyapatite seed crystals either when initial mineral deposition was assessed at 3.5 days, or when mineral formation and growth were assessed at 5 days. There was a statistically significant dose-dependent decrease in crystal length at all concentrations tested. The rat osteopontin preparation had similar inhibitory abilities. Partial dephosphorylation of bovine osteopontin with alkaline phosphatase removed its inhibitory ability, and reduced its ability to bind calcium. The affinity of bovine osteopontin for hydroxyapatite was determined based on a Langmuir adsorption isotherm, with values of K (binding affinity) and N (number of binding sites) being 0.026 ml/microgram and 1084 micrograms/m2, respectively. The data suggest that, in this system, osteopontin is an effective inhibitor of hydroxyapatite formation and growth due to its affinity for the hydroxyapatite crystals. In this system, osteopontin, distinct from other phosphoproteins which both promote and inhibit hydroxyapatite deposition, did not enhance mineral formation at any concentration tested.

Dentin Matrix Protein 1 (DMP1), the essential noncollagenous proteins in dentin and bone, is believed to play an important role in the mineralization of these tissues, although the mechanisms of its action are not fully understood. To gain insight into DMP1 functions in dentin mineralization we have performed immunomapping of DMP1 in fully mineralized rat incisors and in vitro calcium phosphate mineralization experiments in the presence of DMP1. DMP1 immunofluorescene was localized in peritubular dentin (PTD) and along the dentin-enamel boundary. In vitro phosphorylated DMP1 induced the formation of parallel arrays of crystallites with their c-axes co-aligned. Such crystalline arrangement is a hallmark of mineralized collagen fibrils of bone and dentin. Interestingly, in DMP1-rich PTD, which lacks collagen fibrils, the crystals are organized in a similar manner. Based on our findings we hypothesize, that in vivo DMP1 controls the mineral organization outside of the collagen fibrils and plays a major role in the mineralization of PTD.

Objective To determine the presence of small integrin-binding ligand N-linked glycoprotein (SIBLING) and bone components in juvenile dermatomyositis (DM) pathologic calcifications. Methods Calcifications were removed from 4 girls with juvenile DM symptoms for mean ± SD 36.9 ± 48.3 months and were stained for SIBLING proteins: full-length osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin phosphoprotein (DPP), and matrix extracellular phosphoglycoprotein (MEPE); bone markers: osteocalcin (OC), core-binding factor α 1 (CBFα1), and alkaline phosphatase (AP) for osteoblasts; tartrate-resistant acid phosphatase (TRAP) for osteoclasts; and the mineral regulators osteonectin (ON) and matrix Gla protein (MGP). The deposit center, periphery, adjacent connective tissue, and vascular endothelial cells were examined. Results Alizarin red stained calcified deposits that did not localize with collagen, like bone, under polarized light. Hematoxylin and eosin stain revealed a paucity of connective tissue and absence of bone-like structures. The deposits, connective tissue, and vascular endothelial cells were positive for BSP, DPP, DMP1, and AP; MEPE was not detected. OC, ON, and MGP were present in the deposits and vascular endothelial cells; OPN and CBFα1 were present in deposits and connective tissue. TRAP-positive osteoclasts were localized to the calcification periphery. Conclusion The disorganized juvenile DM calcifications differ in structure, composition, and protein content from bone, suggesting that they may not form through an osteogenic pathway. Osteoclasts at the deposit surface represent an attempt to initiate its resolution.

All animal embryos begin development by modifying the egg extracellular matrix. This protein-rich matrix protects against polyspermy, microbes and mechanical stress via enzyme-dependent transformations that alter the organization of its constituents. Using the sea urchin fertilization envelope,a well-defined extracellular structure formed within minutes of fertilization,we examine the mechanisms whereby limited permeability is established within this matrix. We find that the fertilization envelope acquires a barrier filtration of 40,000 daltons within minutes of insemination via a peroxidase-dependent mechanism, with dynamics that parallel requisite production of hydrogen peroxide by the zygote. To identify the molecular targets of this free-radical modification, we developed an in vivo technique to label and isolate the modified matrix components for mass spectrometry. This method revealed that four of the six major extracellular matrix components are selectively crosslinked, discriminating even sibling proteins from the same gene. Thus, specific free-radical chemistry is essential for establishing the embryonic microenvironment of early development.

Integrin-binding sialoprotein (IBSP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family; and the whole SIBLING family is further included in a larger secretory calcium-binding phosphoprotein (SCPP) family. SIBLING proteins are known to construct a part of the non-collagenous extracellular matrices of calcified tissues, and considered to have arisen by duplication and subsequent divergent evolution of a single ancient gene. To understand the alterations of SIBLING molecules associated with the evolution of calcified tissues in vertebrates, we initiated a search for lower vertebrate orthologs of SIBLING genes. In the present study, an IBSP ortholog from a reptile (caiman) and two distinct orthologs from an amphibian (African clawed toad) were identified and characterized. As expected, the toad IBSP genes were transcribed only in calcified tissue (jaw and tibia), as also seen in mammals. The caiman, toad, avian, and mammalian IBSPs share several unique features specific for IBSP and apparently have similar properties. Furthermore, analysis of the sequences suggested that the IBSP molecule might have gradually intensified its functions related to calcification during its evolutionary process through tetrapods.

It is the goal of the current research to develop serum measures of a family of proteins that we have termed SIBLINGs (for small integrin binding ligand N-linked glycoproteins) as markers for use in prostate cancer detection and progression by studying a large population of prostate cancer patients, a large normal (cancer-free) population, a population of men with noncancerous prostate disease, and individuals with prostate cancer before, during and after treatment. Blood levels of SIBLING gene family members in normal (n=200) and subjects with diagnosed prostate cancer (n=200) have been measured for most of the five SIBLING proteins. The sensitivity, specificity and predictive value of these markers will be completed once all assays have been finished and the data "secured." Similarly, determining how the levels of SIBLINGs in blood correlate with clinical stage and progression, although underway, requires the completion of each SIBLING assay for all samples. This research will determine whether SIBLING levels in blood (a) have high sensitivity and high specificity for prostate cancer detection, (b) can be measured in a general laboratory setting, and (c) provide useful prognostic information on response to treatment and the likelihood of disease progression.

Various combinations of the SIBLING family of proteins have been found to be up-regulated in many human cancers and have been linked to different stages of tumor progression, including metastasis. Bone sialoprotein (BSP), osteopontin (OPN) and dentin matrix protein 1 (DMP1) specifically bind and activate MMP-2, MMP-3, and MMP-9, respectively. These proteases have also been shown to play important roles in oral squamous cell carcinoma (OSCC) invasion and metastasis. However, with the exception of OPN, there are no reports on the expression of the family of five SIBLING proteins in OSCC. This study examines the expression patterns of the SIBLING family (and MMP partners when known) in OSCC, correlating expression to outcome variables. Archived paraffin sections of 87 cases of primary OSCC were screened by immunohistochemistry for the SIBLINGs and their MMP partners. Three SIBLINGs (BSP, DSPP, and OPN), were expressed in OSCC, while DMP1 and MEPE expression were never observed. Furthermore, BSP and OPN were always expressed with their known MMP partners, MMP-2 and MMP-3, respectively. Poorly differentiated tumors exhibited reduced or no immunoreactivity for BSP and OPN but increased immunoreactivity for DSPP. Seventy eight (90%) cases were positive for BSP and DSPP, while 79 cases (91%) were positive for OPN. Overall, 91% of the cases were positive for at least one SIBLING. There were no correlations between SIBLING expression and tumor size ("T"; of the Union Internationale Contre le Cancer [UICC]-TNM classification for OSCC), and between SIBLING expression and lymph node spread for the T1/T2 tumors. The levels of DSPP expression for floor of mouth and retromolar region tumors were higher than for tongue tumors. Statistically significant correlations were, however, found between the expression levels of BSP and MMP-2 (p

Small integrin-binding ligand, N-linked glycoproteins (SIBLING) are believed to play key roles in the process of biomineralization. Reactionary dentin (RD), formed by odontoblasts in response to external stimuli, differs morphologically from primary dentin (PD). To test our hypothesis that the microscopic changes reflect variations in molecular mechanisms involved in formation of the two forms of dentin, and to characterize RD further, we compared the distributions of four SIBLING proteins [bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein 1 (DMP-1) and dentin sialophosphoprotein (DSPP)] in naturally occurring RD with those in PD. Molars of rats aged 12, 18, 24 and 36 wk were analyzed using immunohistochemistry with antibodies against BSP, OPN, DMP-1, and dentin sialoprotein (a fragment of DSPP). Differences in the distribution of the four SIBLING proteins were evident. Bone sialoprotein, not seen in PD, was consistently observed in RD. Osteopontin, almost absent from PD, was clearly observed in RD. The expression levels of DMP-1 and DSP in RD were lower than in PD. Elevated expression of BSP and OPN, along with a marked decrease of dentin sialoprotein and DMP-1 in RD, suggests a difference in the mechanism of formation of the two forms of dentin.

Renal expression of SIBLING proteins and their partner matrix metalloproteinases (MMP). Background Three members of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family of proteins have recently been shown to bind and activate specific promatrix metalloproteinases (MMPs) and to overcome the inhibition of tissue inhibitors of MMPs (TIMPs). Although usually associated with mineralized tissues, we have shown that the SIBLINGs and their MMP partners, when known, are coexpressed in salivary gland ductal cells. The present study examined the expression patterns of both the SIBLINGs and their MMP partners in adult kidney. Methods The expression patterns of all five SIBLINGs known to date, and their MMP partners were determined in monkey kidney using immunohistochemistry and in situ hybridization techniques. Results Bone sialoprotein (BSP) and its partner, MMP-2, were coexpressed in both the proximal and distal tubules. Osteopontin, as previously shown, was expressed in the distal tubules while its partner MMP-3 was expressed in both the proximal tubule and distal tubles. Dentin matrix protein-1 (DMP1) and MMP-9 were coexpressed throughout the nephron, including both parietal cells of Bowman's capsule and the thin limb of the loop of Henle. Dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE) were expressed in the proximal tubule and distal tubule, and proximal tubule, respectively. Conclusion In contrast to salivary gland in which all SIBLINGs and their MMP partners were coexpressed throughout the length of the ducts, these proteins were differentially expressed within the normal adult nephron. We hypothesize that the cells use the SIBLING/MMP pairs in the normal turnover of cell surface proteins and/or pericellular matrix proteins such as those in basement membranes.

Three members of the SIBLING family of integrin-binding phosphoglycoproteins (bone sialoprotein, BSP; osteopontin, OPN; and dentin matrix protein-1, DMP1) were recently shown to bind with high affinity (nM) and to activate 3 different matrix metalloproteinases (MMP-2, MMP-3, and MMP-9, respectively) in vitro. The current study was designed to document the possible biological relevance of the SIBLING-MMP activation pathway in vivo by showing that these 3 SIBLINGs and their known MMP partners are co-expressed in normal adult tissue. BSP, OPN, and DMP1 were invariably co-expressed with their partner MMPs in salivary glands of humans and mice. The 2 SIBLING proteins without known MMP partners, dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE), were also expressed in salivary glands. Expression of all SIBLINGs in this normal, non-mineralizing epithelial tissue suggests that they serve at least one function in vivo other than directly promoting matrix mineralization—a function we hypothesize involves local activation of MMPs.

Matrix metalloproteinases (MMPs) are critical for development, wound healing, and for the progression of cancer. It is generally accepted that MMPs are secreted in a latent form (proMMP) and are activated only upon removal of their inhibitory propeptides. This report shows that three members of the SIBLING (Small, Integrin-Binding LIgand, N-linked Glycoprotein) family can specifically bind (Kd approximately equal nM) and activate three different MMPs. Binding of SIBLING to their corresponding proMMPs is associated with structural changes as indicated by quenching of intrinsic tryptophan fluorescence, increased susceptibility to plasmin cleavage, and decreased inhibition by specific natural and synthetic inhibitors. Activation includes both making the proMMPs enzymatically active and the reactivation of the TIMP (tissue inhibitors of MMP) inhibited MMPs. Bone sialoprotein specifically binds proMMP-2 and active MMP-2, while osteopontin binds proMMP-3 and active MMP-3, and dentin matrix protein-1 binds proMMP-9 and active MMP-9. Both pro and active MMP-SIBLING complexes are disrupted by the abundant serum protein, complement Factor H, thereby probably limiting SIBLING-mediated activation to regions immediately adjacent to sites of secretion in vivo. These data suggest that the SIBLING family offers an alternative method of controlling the activity of at least three MMPs.

We have previously shown that breast and prostate cancers express bone matrix proteins. DMP1 expression was evaluated in 59 human lung cancer samples at the protein and mRNA levels. It was detectable in 80% of the cases, suggesting a potential role for DMP1 in tumor progression and bone metastasis. INTRODUCTION: Previously, we and others have shown that bone extracellular matrix proteins such as bone sialoprotein (BSP) and osteopontin (OPN) are expressed in various types of cancer that are characterized by a high affinity for bone including breast, prostate, and lung adenocarcinoma. Based on biochemical and genetic features, BSP, OPN, dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP) have been recently classified in a unique family named SIBLING (small integrin-binding ligand, N-linked glycoprotein). Therefore, we investigated whether DMP1 could also be detected in osteotropic cancers. MATERIALS AND METHODS: We first used a cancer array for evaluating the relative abundance of DMP1 transcript in a broad spectrum of human cancer tissues. This screening showed that DMP1 was strongly detectable in lung tumors compared with normal corresponding tissue. In a second step, we used an immunophosphatase technique and a specific polyclonal antibody directed against DMP1 to examine the expression of DMP1 in 59 human non-small cell lung cancer samples, including 29 squamous carcinoma, 20 adenocarcinoma, and 10 bronchioloalveolar carcinoma. Student's t-test was used to determine the statistical significance of immunostaining scores between the lung cancer histological groups studied and between cancer and normal lung tissues. RESULTS: Our results show that DMP1 is detectable in 90% of the adenocarcinoma and squamous carcinoma analyzed while 8 of 10 bronchioloalveolar specimens were negative. DMP1 immunostaining intensity and extent scores were significantly higher in adenocarcinoma (p = 0.0004) and squamous carcinoma (p < 0.0001) samples compared with adjacent normal lung tissue. In situ hybridization experiments confirmed that DMP1 mRNA is localized in lung cancer cells. CONCLUSION: In this study, we show that a third SIBLING protein is ectopically expressed in lung cancer. The role of DMP1 in lung cancer is largely unknown. Further studies are required to determine the implication of this protein, next to its sisters SIBLING proteins, in tumor progression and bone metastasis development.