Contributions of increased osteopontin and hypophosphatemia to dentoalveolar defects in osteomalacic Hyp mice.

X-linked hypophosphatemia (XLH) is an inherited disorder caused by inactivating mutations in the PHEX gene leading to renal phosphate wasting, rickets and osteomalacia. XLH is also associated with dentoalveolar mineralization defects in tooth enamel, dentin and cementum, and in alveolar bone, which lead to an increased prevalence of dental abscesses, periodontal disease and tooth loss. Genetic mouse experiments, and deficiencies in XLH patient therapies where treatments do not fully ameliorate mineralization defects, suggest that other pathogenic mechanisms may exist in XLH. The mineralization-inhibiting, secreted extracellular matrix phosphoprotein osteopontin (OPN, gene Spp1) is a substrate for the PHEX enzyme whereby extensive and inactivating degradation of inhibitory OPN by PHEX facilitates mineralization. Conversely, excess OPN accumulation in skeletal and dental tissues - for example in XLH where inactivating mutations in the PHEX gene limit degradation of inhibitory OPN, or as occurs in Fgf23-null mice - contributes to mineralization defects. We hypothesized that Spp1/OPN ablation in Hyp mice (a mouse model for XLH) would reduce dentoalveolar mineralization defects. Immunostaining revealed increased OPN in Hyp vs. wild-type (WT) alveolar bone, particularly in osteocyte lacunocanalicular networks where Hyp mice have characteristic hypomineralized peri-osteocytic lesions (POLs). Micro-computed tomography and histology showed that ablation of Spp1 in Hyp mice (Hyp;Spp1-/-) on a normal diet did not ameliorate bulk defects in enamel, dentin, or alveolar bone. On a high-phosphate diet, both Hyp and Hyp;Spp1-/- mice showed improved mineralization of enamel, dentin, and alveolar bone. Silver staining indicated Spp1 ablation did not improve alveolar or mandibular bone osteocyte POLs in Hyp mice; however, they were normalized by a high-phosphate diet in both Hyp and Hyp;Spp1-/- mice, although inducing increased OPN. Collectively, these data indicate that despite changes in OPN content in the dentoalveolar mineralized tissues, there exist other compensatory mineralization mechanisms that arise from knockout of Spp1/OPN in the Hyp background.

The role of extracellular matrix phosphorylation on energy dissipation in bone.

Protein phosphorylation, critical for cellular regulatory mechanisms, is implicated in various diseases. However, it remains unknown whether heterogeneity in phosphorylation of key structural proteins alters tissue integrity and organ function. Here, osteopontin phosphorylation level declined in hypo- and hyper- phosphatemia mouse models exhibiting skeletal deformities. Phosphorylation increased cohesion between osteopontin polymers, and adhesion of osteopontin to hydroxyapatite, enhancing energy dissipation. Fracture toughness, a measure of bone's mechanical competence, increased with ex-vivo phosphorylation of wildtype mouse bones and declined with ex-vivo dephosphorylation. In osteopontin-deficient mice, global matrix phosphorylation level was not associated with toughness. Our findings suggest that phosphorylated osteopontin promotes fracture toughness in a dose-dependent manner through increased interfacial bond formation. In the absence of osteopontin, phosphorylation increases electrostatic repulsion, and likely protein alignment and interfilament distance leading to decreased fracture resistance. These mechanisms may be of importance in other connective tissues, and the key to unraveling cell-matrix interactions in diseases.

Genetic Ablation of Osteopontin in Osteomalacic Hyp Mice Partially Rescues the Deficient Mineralization Without Correcting Hypophosphatemia.

PHEX is predominantly expressed by bone and tooth-forming cells, and its inactivating mutations in X-linked hypophosphatemia (XLH) lead to renal phosphate wasting and severe hypomineralization of bones and teeth. Also present in XLH are hallmark hypomineralized periosteocytic lesions (POLs, halos) that persist despite stable correction of serum phosphate (Pi ) that improves bulk bone mineralization. In XLH, mineralization-inhibiting osteopontin (OPN, a substrate for PHEX) accumulates in the extracellular matrix of bone. To investigate how OPN functions in Hyp mice (a model for XLH), double-null (Hyp;Opn-/- ) mice were generated. Undecalcified histomorphometry performed on lumbar vertebrae revealed that Hyp;Opn-/- mice had significantly reduced osteoid area/bone area (OV/BV) and osteoid thickness of trabecular bone as compared to Hyp mice, despite being as hypophosphatemic as Hyp littermate controls. However, tibias examined by synchrotron radiation micro-CT showed that mineral lacunar volumes remained abnormally enlarged in these double-null mice. When Hyp;Opn-/- mice were fed a high-Pi diet, serum Pi concentration increased, and OV/BV and osteoid thickness normalized, yet mineral lacunar area remained abnormally enlarged. Enpp1 and Ankh gene expression were increased in double-null mice fed a high-Pi diet, potentially indicating a role for elevated inhibitory pyrophosphate (PPi ) in the absence of OPN. To further investigate the persistence of POLs in Hyp mice despite stable correction of serum Pi , immunohistochemistry for OPN on Hyp mice fed a high-Pi diet showed elevated OPN in the osteocyte pericellular lacunar matrix as compared to Hyp mice fed a control diet. This suggests that POLs persisting in Hyp mice despite correction of serum Pi may be attributable to the well-known upregulation of mineralization-inhibiting OPN by Pi , and its accumulation in the osteocyte pericellular matrix. This study shows that OPN contributes to osteomalacia in Hyp mice, and that genetic ablation of OPN in Hyp mice improves the mineralization phenotype independent of systemic Pi -regulating factors. © 2020 American Society for Bone and Mineral Research.

Osteopontin as a novel substrate for the proprotein convertase 5/6 (PCSK5) in bone.

Seven proprotein convertases cleave the basic amino acid consensus sequence K/R-Xn-K/R↓ (where n=0, 2, 4 or 6 variable amino acids) to activate precursor proteins. Despite similarities in substrate specificity, basic amino acid-specific proprotein convertases have a distinct tissue distribution allowing for enzymatic actions on tissue-resident substrates. Proprotein convertase 5/6 (PC5/6) has two splice variants - soluble PC5/6A and membrane-bound PC5/6B - and is expressed during mouse development in many tissues including bone and tooth, but little is known about the substrates for PC5/6 therein. Osteopontin (OPN) is an abundant bone extracellular matrix protein with roles in mineralization, cell adhesion and cell migration, and it has putative consensus sequence sites for cleavage by PC5/6, which may modify its function in bone. Since PC5/6-knockout mouse embryos show developmental abnormalities, and reduced overall mineralization, we designed this study to determine whether OPN is a substrate of PC5/6. In silico analysis of OPN protein sequences identified four potential PC5/6 consensus cleavage sites in human OPN, and three sites - including a noncanonical sequence - in mouse OPN. Ex vivo co-transfections with human OPN revealed complete OPN cleavage reducing full-length OPN (~70kDa) to an N-terminal fragment migrating at ~50kDa and two C-terminal fragments at ~18kDa and ~16kDa. Direct cleavage of OPN by PC5/6A - the predominant isoform expressed in human osteoblast cells - was confirmed by cell-free enzyme-substrate assays and by mass spectrometry. The latter was also used to investigate potential cleavage sites. Co-transfections of PC5/6 and mouse OPN showed partial cleavage of OPN into a C-terminal OPN fragment migrating at ~30kDa and an N-terminal fragment migrating at ~29kDa. Micro-computed tomography of PC5/6-knockout embryos at E18.5 confirmed a reduction in mineralized bone, and in situ hybridization performed on cryo-sections of normal mouse bone using Pcsk5 and Opn anti-sense and control-sense cRNA probes indicated the co-localization of the expression of these genes in bone cells. This mRNA expression profile was supported by semi-quantitative RT-PCR using osteoblast primary cultures, and cultured MC3T3-E1 osteoblast and MLO-Y4 osteocyte cell lines. Immunoblotting for OPN from mouse bone extracts showed altered OPN processing in PC5/6-knockout mice compared to wildtype mice. OPN fragments migrated at ~25kDa and ~16kDa in wildtype bone and were not present in PC5/6-deficient bone. In conclusion, this study demonstrates that Pcsk5 is expressed in bone-forming cells, and that OPN is a novel substrate for PC5/6. Cleavage of OPN by PC5/6 may modify the function of OPN in bone and/or modulate other enzymatic cleavages of OPN, leading to alterations in the bone phenotype.

Mineralization-inhibiting effects of transglutaminase-crosslinked polymeric osteopontin.

Osteopontin (OPN) belongs to the SIBLING family (Small, Integrin-Binding LIgand N-linked Glycoproteins) of mineral-binding matrix proteins found in bones and teeth. OPN is a well-known inhibitor of matrix mineralization, and enzymatic modification of OPN can affect this inhibitory function. In bone, OPN exists both as a monomer and as a high-molecular-weight polymer - the latter is formed by transglutaminase-mediated crosslinking of glutamine and lysine residues in OPN to create homotypic protein assemblies. OPN can be covalently crosslinked by transglutaminase 2 (TG2) and Factor XIII-A. Polymeric OPN has increased binding to collagen and promotes osteoblast adhesion, but despite these initial observations, its role in mineralization is not clear. In this study, we investigated the effect of polymerized OPN on mineralization using a hydroxyapatite crystal growth assay and mineralizing MC3T3-E1 osteoblast cultures. In the cultures, endogenous polymeric OPN was detected after mineralization occurred. In cell-free conditions, TG2 was used to crosslink bovine OPN into its polymeric form, and atomic force microscopy and dynamic light scattering revealed variably-sized, large branched aggregates ranging across hundreds of nanometers. These OPN polymers inhibited the growth of hydroxyapatite crystals in solution at concentrations similar to monomeric OPN, although the crosslinking slightly reduced its inhibitory potency. When added to MC3T3-E1 osteoblast cultures, this exogenous polymeric OPN essentially did not inhibit mineralization when given during the later mineralization stages of culture; however, cultures treated early and then continuously with polymeric OPN throughout both the matrix assembly and mineral deposition stages showed reduced mineralization. Immunoblotting of protein extracts from these continuously treated cultures revealed exogenous OPN polymers incorporated into mature matrix that had not yet mineralized. These results suggest that in bone, the increased size and branched structure of crosslinked inhibitory polymeric OPN near the mineralization front could hinder it from accessing focal mineralization sites in the dense collagen-rich matrix, suggesting that OPN-crosslinking into polymers may represent a way to fine-tune the inhibitory potency of OPN on bone mineralization.

Osteopontin and the dento-osseous pathobiology of X-linked hypophosphatemia.

Seven young patients with X-linked hypophosphatemia (XLH, having inactivating PHEX mutations) were discovered to accumulate osteopontin (OPN) at the sites of defective bone mineralization near osteocytes - the so-called hallmark periosteocytic (lacunar) "halos" of XLH. OPN was also localized in the pericanalicular matrix extending beyond the osteocyte lacunae, as well as in the hypomineralized matrix of tooth dentin. OPN, a potent inhibitor of mineralization normally degraded by PHEX, is a member of a family of acidic, phosphorylated, calcium-binding, extracellular matrix proteins known to regulate dental, skeletal, and pathologic mineralization. Associated with the increased amount of OPN (along with inhibitory OPN peptide fragments) in XLH bone matrix, we found an enlarged, hypomineralized, lacuno-canalicular network - a defective pattern of skeletal mineralization that decreases stiffness locally at: i) the cell-matrix interface in the pericellular environment of the mechanosensing osteocyte, and ii) the osteocyte's dendritic network of cell processes extending throughout the bone. Our findings of an excess of inhibitory OPN near osteocytes and their cell processes, and in dentin, spatially correlates with the defective mineralization observed at these sites in the skeleton and teeth of XLH patients. These changes likely contribute to the dento-osseous pathobiology of XLH, and participate in the aberrant bone adaptation and remodeling seen in XLH.

Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins, enzymes, and relationship to hypophosphatasia and X-linked hypophosphatemia.

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.

Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures.

Studies on various compounds of inorganic phosphate, as well as on organic phosphate added by post-translational phosphorylation of proteins, all demonstrate a central role for phosphate in biomineralization processes. Inorganic polyphosphates are chains of orthophosphates linked by phosphoanhydride bonds that can be up to hundreds of orthophosphates in length. The role of polyphosphates in mammalian systems, where they are ubiquitous in cells, tissues and bodily fluids, and are at particularly high levels in osteoblasts, is not well understood. In cell-free systems, polyphosphates inhibit hydroxyapatite nucleation, crystal formation and growth, and solubility. In animal studies, polyphosphate injections inhibit induced ectopic calcification. While recent work has proposed an integrated view of polyphosphate function in bone, little experimental data for bone are available. Here we demonstrate in osteoblast cultures producing an abundant collagenous matrix that normally show robust mineralization, that two polyphosphates (PolyP5 and PolyP65, polyphosphates of 5 and 65 phosphate residues in length) are potent mineralization inhibitors. Twelve-day MC3T3-E1 osteoblast cultures with added ascorbic acid (for collagen matrix assembly) and ß-glycerophosphate (a source of phosphate for mineralization) were treated with either PolyP5 or PolyP65. Von Kossa staining and calcium quantification revealed that mineralization was inhibited in a dose-dependent manner by both polyphosphates, with complete mineralization inhibition at 10µM. Cell proliferation and collagen assembly were unaffected by polyphosphate treatment, indicating that polyphosphate inhibition of mineralization results not from cell and matrix effects but from direct inhibition of mineralization. This was confirmed by showing that PolyP5 and PolyP65 bound to synthetic hydroxyapatite in a concentration-dependent manner. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) efficiently hydrolyzed the two PolyPs as measured by Pi release. Importantly, at the concentrations of polyphosphates used in this study which inhibited bone cell culture mineralization, the polyphosphates competitively saturated TNAP, thus potentially interfering with its ability to hydrolyze mineralization-inhibiting pyrophosphate (PPi) and mineralizing-promoting ß-glycerophosphate (in cell culture). In the biological setting, polyphosphates may regulate mineralization by shielding the essential inhibitory substrate pyrophosphate from TNAP degradation, and in the same process, delay the release of phosphate from this source. In conclusion, the inhibition of mineralization by polyphosphates is shown to occur via direct binding to apatitic mineral and by mixed inhibition of TNAP.

Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine model of X-linked hypophosphatemia.

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.