A repeated triple lysine motif anchors complexes containing bone sialoprotein and the type XI collagen A1 chain involved in bone mineralization.

While details remain unclear, initiation of woven bone mineralization is believed to be mediated by collagen and potentially nucleated by bone sialoprotein (BSP). Interestingly, our recent publication showed that BSP and type XI collagen form complexes in mineralizing osteoblastic cultures. To learn more, we examined the protein composition of extracellular sites of de novo hydroxyapatite deposition which were enriched in BSP and Col11a1 containing an alternatively spliced "6b" exonal sequence. An alternate splice variant "6a" sequence was not similarly co-localized. BSP and Col11a1 co-purify upon ion-exchange chromatography or immunoprecipitation. Binding of the Col11a1 "6b" exonal sequence to bone sialoprotein was demonstrated with overlapping peptides. Peptide 3, containing three unique lysine-triplet sequences, displayed the greatest binding to osteoblastic cultures; peptides containing fewer lysine triplet motifs or derived from the "6a" exon yielded dramatically lower binding. Similar results were obtained with 6-carboxyfluorescein (FAM)-conjugated peptides and western blots containing extracts from osteoblastic cultures. Mass spectroscopic mapping demonstrated that FAM-peptide 3 bound to 90 kDa BSP and its 18 to 60 kDa fragments, as well as to 110 kDa nucleolin. In osteoblastic cultures, FAM-peptide 3 localized to biomineralization foci (site of BSP) and to nucleoli (site of nucleolin). In bone sections, biotin-labeled peptide 3 bound to sites of new bone formation which were co-labeled with anti-BSP antibodies. These results establish the fluorescent peptide 3 conjugate as the first nonantibody-based method to identify BSP on western blots and in/on cells. Further examination of the "6b" splice variant interactions will likely reveal new insights into bone mineralization during development.

Deletion of Mbtps1 (Pcsk8, S1p, Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age.

Conditional deletion of Mbtps1 (cKO) protease in bone osteocytes leads to an age-related increase in mass (12%) and in contractile force (30%) in adult slow twitch soleus muscles (SOL) with no effect on fast twitch extensor digitorum longus muscles. Surprisingly, bone from 10-12-month-old cKO animals was indistinguishable from controls in size, density, and morphology except for a 25% increase in stiffness. cKO SOL exhibited increased expression of Pax7, Myog, Myod1, Notch, and Myh3 and 6-fold more centralized nuclei, characteristics of postnatal regenerating muscle, but only in type I myosin heavy chain-expressing cells. Increased expression of gene pathways mediating EGF receptor signaling, circadian exercise, striated muscle contraction, and lipid and carbohydrate oxidative metabolism were also observed in cKO SOL. This muscle phenotype was not observed in 3-month-old mice. Although Mbtps1 mRNA and protein expression was reduced in cKO bone osteocytes, no differences in Mbtps1 or cre recombinase expression were observed in cKO SOL, explaining this age-related phenotype. Understanding bone-muscle cross-talk may provide a fresh and novel approach to prevention and treatment of age-related muscle loss.

MBTPS1/SKI-1/S1P proprotein convertase is required for ECM signaling and axial elongation during somitogenesis and vertebral development†.

Caudal regression syndrome (sacral agenesis), which impairs development of the caudal region of the body, occurs with a frequency of about 2 live births per 100 000 newborns although this incidence rises to 1 in 350 infants born to mothers with gestational diabetes. The lower back and limbs can be affected as well as the genitourinary and gastrointestinal tracts. The axial skeleton is formed during embryogenesis through the process of somitogenesis in which the paraxial mesoderm periodically segments into bilateral tissue blocks, called somites. Somites are the precursors of vertebrae and associated muscle, tendons and dorsal dermis. Vertebral anomalies in caudal regression syndrome may arise through perturbation of somitogenesis or, alternatively, could result from defective bone formation and patterning. We discovered that MBTPS1/SKI-1/S1P, which proteolytically activates a class of transmembrane transcription factors, plays a critical role in somitogenesis and the pathogenesis of lumbar/sacral vertebral anomalies. Conditional deletion of Mbtps1 yields a viable mouse with misshapen, fused and reduced number of lumbar and sacral vertebrae, under-developed hind limb bones and a kinky, shortened tail. We show that Mbtps1 is required to (i) maintain the Fgf8 'wavefront' in the presomitic mesoderm that underpins axial elongation, (ii) sustain the Lfng oscillatory 'clock' activity that governs the periodicity of somite formation and (iii) preserve the composition and character of the somitic extracellular matrix containing fibronectin, fibrillin2 and laminin. Based on this spinal phenotype and known functions of MBTPS1, we reason that loss-of-function mutations in Mbtps1 may cause the etiology of caudal regression syndrome.

Enamel organic matrix: potential structural role in enamel and relationship to residual basement membrane constituents at the dentin enamel junction.

Although mature enamel is predominantly composed of mineral, a previously uncharacterized organic matrix layer remains in the post-eruptive tissue that begins at the dentin enamel junction and extends 200-300 µm towards the outer tooth surface. Identification of the composition of this layer has been hampered by its insolubility; however, we have developed a single step method to isolate the organic enamel matrix relatively intact. After dissociative dissolution of the matrix with SDS and urea, initial characterization by Western blotting and gel zymography indicates the presence of type IV and type VII basement membrane collagens and active matrix metalloproteinase-20. When combined with data from transgenic knockout mice and from human mutations, these data suggest that the enamel organic matrix (EOM) and dentin enamel junction may have a structural and functional relationship with basement membranes, e.g. skin. To clarify this relationship, we hypothesize a "foundation" model which proposes that components of the EOM form a support structure that stabilizes the crystalline enamel layer, and bonds it to the underlying dentin along the dentin enamel junction. Since we have also co-localized an active matrix metalloproteinase to this layer, our hypothesis suggests that, under pathologic conditions, MMP-mediated degradation of the EOM could destabilize the enamel-dentin interface.

Type VII collagen is enriched in the enamel organic matrix associated with the dentin-enamel junction of mature human teeth.

The inner enamel region of erupted teeth is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the residual enamel organic matrix. Since enamel-forming ameloblasts are known to express type VII collagen and type VII collagen null mice display abnormal amelogenesis, the aim of this study was to determine whether type VII collagen is a component of the enamel organic matrix at the dentin-enamel junction (DEJ) of mature human teeth. Immunofluorescent confocal microscopy of demineralized tooth sections localized type VII collagen to the organic matrix surrounding individual enamel rods near the DEJ. Morphologically, immunoreactive type VII collagen helical-bundles resembled the gnarled-pattern of enamel rods detected by Coomassie Blue staining. Western blotting of whole crown or enamel matrix extracts also identified characteristic Mr=280 and 230 kDa type VII dimeric forms, which resolved into 75 and 25 kDa bands upon reduction. As expected, the collagenous domain of type VII collagen was resistant to pepsin digestion, but was susceptible to purified bacterial collagenase. These results demonstrate the inner enamel organic matrix in mature teeth contains macromolecular type VII collagen. Based on its physical association with the DEJ and its well-appreciated capacity to complex with other collagens, we hypothesize that enamel embedded type VII collagen fibrils may contribute not only to the structural resilience of enamel, but may also play a role in bonding enamel to dentin.

Identification of a protein-containing enamel matrix layer which bridges with the dentine-enamel junction of adult human teeth.

OBJECTIVE: To investigate the ultrastructure and chemical composition of the dentine-enamel junction and adjacent enamel of minimally processed third molar tooth sections. DESIGN: Undecalcified human third molar erupted teeth were sectioned and etched with 4% EDTA or 37% phosphoric acid prior to visualization by scanning electron microscopy. Confocal Raman spectroscopy was carried out at 50 µm and more than 400 µm away from the dentine-enamel junction before and after mild etching. RESULTS: A novel organic protein-containing enamel matrix layer was identified for the first time using scanning electron microscopy of etched bucco-lingual sections of crowns. This layer resembles a three-dimensional fibrous meshwork that is visually distinct from enamel "tufts". Previous studies have generally used harsher solvent conditions which likely removed this layer and precluded its prior characterization. The shape of the organic enamel layer generally reflected that of sheath regions of enamel rods and extended from the dentine-enamel junction about 100-400 µm into the cuspal enamel. This layer exhibited a Raman CH stretching peak at ∼2931 cm(-1) characteristic of proteins and this signal correlated directly with the presence and location of the matrix layer as identified by scanning electron microscopy. CONCLUSIONS: The enamel protein layer was most prominent close to the dentine-enamel junction and was largely absent in cuspal enamel >400 µm away from the dentine enamel junction. We hypothesize that this protein containing matrix layer could provide an important biomechanical linkage between the enamel and the dentine-enamel junction and by extension, with the dentine, of the adult tooth (246 words).

Inhibition of proprotein convertase SKI-1 blocks transcription of key extracellular matrix genes regulating osteoblastic mineralization.

Mineralization, a characteristic phenotypic property of osteoblastic lineage cells, was blocked by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and decanoyl-Arg-Arg-Leu-Leu-chloromethyl ketone (dec-RRLL-cmk), inhibitors of SKI-1 (site 1; subtilisin kexin like-1) protease. Because SKI-1 is required for activation of SREBP and CREB (cAMP-response element-binding protein)/ATF family transcription factors, we tested the effect of these inhibitors on gene expression. AEBSF decreased expression of 140 genes by 1.5-3.0-fold including Phex, Dmp1, COL1A1, COL11A1, and fibronectin. Direct comparison of AEBSF and dec-RRLL-cmk, a more specific SKI-1 inhibitor, demonstrated that expression of Phex, Dmp1, COL11A1, and fibronectin was reduced by both, whereas COL1A2 and HMGCS1 were reduced only by AEBSF. AEBSF and dec-RRLL-cmk decreased the nuclear content of SKI-1-activated forms of transcription factors SREBP-1, SREBP-2, and OASIS. In contrast to AEBSF, the actions of dec-RRLL-cmk represent the sum of its direct actions on SKI-1 and indirect actions on caspase-3. Specifically, dec-RRLL-cmk reduced intracellular caspase-3 activity by blocking the formation of activated 19-kDa caspase-3. Conversely, overexpression of SKI-1-activated SREBP-1a and CREB-H in UMR106-01 osteoblastic cells increased the number of mineralized foci and altered their morphology to yield mineralization nodules, respectively. In summary, SKI-1 regulates the activation of transmembrane transcription factor precursors required for expression of key genes required for mineralization of osteoblastic cultures in vitro and bone formation in vivo. Our results indicate that the differentiated phenotype of osteoblastic cells and possibly osteocytes depends upon the non-apoptotic actions of SKI-1.

Confocal laser Raman microspectroscopy of biomineralization foci in UMR 106 osteoblastic cultures reveals temporally synchronized protein changes preceding and accompanying mineral crystal deposition.

Mineralization in UMR 106-01 osteoblastic cultures occurs within extracellular biomineralization foci (BMF) within 12 h after addition of beta-glycerol phosphate to cells at 64 h after plating. BMF are identified by their enrichment with an 85-kDa glycoprotein reactive with Maackia amurensis lectin. Laser Raman microspectroscopic scans were made on individual BMF at times preceding (64-76 h) and following the appearance of mineral crystals (76-88 h). The range of variation between spectra for different BMF in the same culture was rather small. In contrast, significant differences were observed for spectral bands at 957-960, 1004, and 1660 cm(-1) when normalized BMF spectra at different times were compared. Protein-dependent spectral bands at 1004 and 1660 cm(-1) increased and then decreased preceding the detection of hydroxyapatite crystals via the phosphate stretching peak at 959-960 cm(-1). When sodium phosphate was substituted for beta-glycerol phosphate, mineralization occurred 3-6 h earlier. Irrespective of phosphate source, the Raman full peak width at half-maximum ratio for 88 h cultures was similar to that for 10-day-old marrow ablation primary bone. However, if mineralization was blocked with serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 64-88-h BMF spectra remained largely invariant. In summary, Raman spectral data demonstrate for the first time that formation of hydroxyapatite crystals within individual BMF is a multistep process. Second, changes in protein-derived signals at 1004 and 1660 cm(-1) reflect events within BMFs that precede or accompany mineral crystal production because they are blocked by mineralization inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride. Finally, the low extent of spectral variability detected among different BMF at the same time point indicates that mineralization of individual BMF within a culture is synchronized.

Isolation of calcospherulites from the mineralization front of bone.

Calcium-containing spherical bodies (calcospherulites) exist along the mineralization front of bone and are thought to play a role in bone formation. Existing methods to isolate calcospherulites involve harsh treatments that remove much of their organic matter. This study sought to isolate them using a less destructive approach to better preserve their organic components. Juvenile rats were injected with a low dose of calcein to label the newly formed mineral at the mineralization front of bone in vivo. Periosteum was completely dissected from the tibial diaphysis and unmineralized osteoid matrix was removed by collagenase in order to expose calcospherulites. Calcein-labeled calcospherulites of approximately 0.5 mum average diameter were observed all along the mineralization front and they exhibited a Ca/P ratio of 1.3 in situ. Calcospherulites were released from the mineralization front by a short dispase digestion and isolated via fluorescence flow sorting. X-ray diffraction revealed they contained apatite crystals (c-axis length of 17.5 +/- 0.2 nm) and their Ca/P ratio was preserved during isolation. Calcospherulites treated with ice-cold ethanol exhibited a Ca/P ratio of 1.6, suggesting the presence of some extractable phospholipids. Proteins extracted from isolated calcospherulites were resolved by SDS-PAGE into more than 20 distinct bands. Western blot analyses showed the presence of matrix proteins in these preparations. These results indicate that calcospherulites can be isolated from the mineralization front of bone in a form that can be used to study their proteome and lipid composition.

Potential role of proprotein convertase SKI-1 in the mineralization of primary bone.

The biochemical mechanism controlling nucleation of mineral crystals in developing bone, along with the growth and propagation of these crystals once formed, remains poorly understood. To define the nucleation mechanism, a proteomics analysis was begun on isolated biomineralization foci (BMF), sites of initial crystal nucleation in osteoblastic cell cultures and in primary bone. Comparative analyses of the protein profile for mineralized BMF with that for total osteoblast cultures revealed the latter were enriched in several proteins including BAG-75 and BSP, as well as fragments of each. When 12 protease inhibitors were added separately to UMR 106-01 osteoblastic cultures, only the serine protease inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) blocked cleavage of BAG-75 and BSP, and prevented mineral crystal nucleation within BMF. Consideration of the specificities of the inhibitors tested and the fact that AEBSF inhibition was not dependent upon inclusion of FBS in the culture media indicated that mineral nucleation does not require serine protease plasmin, thrombin, kallikrein, urokinase, C1s or furin. In contrast, SKI-1 (S1P or site-1) is a membrane-bound serine protease inhibitable by AEBSF. We show here for the first time that mineralizing UMR 106 cells express a 98-kDa active, soluble form of SKI-1 within BMF. In contrast, nonmineralizing UMR cells appear to differentially process SKI-1 into smaller immunoreactive fragments (<35 kDa). These findings suggest that SKI-1 plays a direct or indirect role in assembly of functional nucleation complexes containing BAG-75 and BSP and their fragments, thus facilitating initial mineral nucleation within these biomineralization foci.

Calcospherulites isolated from the mineralization front of bone induce the mineralization of type I collagen.

Previous work has suggested that "calcospherulites" actively participate in the mineralization of developing and healing bone. This study sought to directly test this hypothesis by developing a method to isolate calcospherulites and analyzing their capacity to seed mineralization of fibrillar collagen. The periosteal surface of juvenile rat tibial diaphysis was enriched in spherulites of approximately 0.5-mum diameter exhibiting a Ca/P ratio of 1.3. Their identity as calcospherulites was confirmed by their uptake of calcein at the tibial mineralization front 24 h following in vivo injection. Periosteum was dissected and unmineralized osteoid removed by collagenase in order to expose calcospherulites. Calcein-labeled calcospherulites were then released from the mineralization front by dispase digestion and isolated via fluorescence flow sorting. X-ray diffraction analysis revealed they contained apatite crystals (c-axis length of 17.5+/-0.2 nm), though their Ca/P ratio of 1.3 is lower than that of hydroxyapatite. Much of their non-mineral phosphorous content was removed by ice-cold ethanol, elevating their Ca/P ratio to 1.6, suggesting the presence of phospholipids. Western blot analyses showed the presence of bone matrix proteins and type I collagen in these preparations. Incubating isolated calcospherulites in collagen hydrogels demonstrated that they could seed a mineralization reaction on type I collagen fibers in vitro. Ultrastructural analyses revealed crystals on the collagen fibers that were distributed rather uniformly along the fiber lengths. Furthermore, crystals were observed at distances well away from the observed calcospherulites. Our results directly support an active role for calcospherulites in inducing the mineralization of type I collagen fibers at the mineralization front of bone.

Association of specific proteolytic processing of bone sialoprotein and bone acidic glycoprotein-75 with mineralization within biomineralization foci.

Mineral crystal nucleation in UMR 106-01 osteoblastic cultures occurs within 15-25-microm extracellular vesicle-containing biomineralization foci (BMF) structures. We show here that BAG-75 and BSP, biomarkers for these foci, are specifically enriched in laser capture microscope-isolated mineralized BMF as compared with the total cell layer. Unexpectedly, fragments of each protein (45-50 kDa in apparent size) were also enriched within captured BMF. When a series of inhibitors against different protease classes were screened, serine protease inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride HCl (AEBSF) was the only one that completely blocked mineral nucleation within BMF in UMR cultures. AEBSF appeared to act on an osteoblast-derived protease at a late differentiation stage in this culture model just prior to mineral deposition. Similarly, mineralization of bone nodules in primary mouse calvarial osteoblastic cultures was completely blocked by AEBSF. Cleavage of BAG-75 and BSP was also inhibited at the minimum dosage of AEBSF sufficient to completely block mineralization of BMF. Two-dimensional SDS-PAGE comparisons of AEBSF-treated and untreated UMR cultures showed that fragmentation/activation of a limited number of other mineralization-related proteins was also blocked. Taken together, our results indicate for the first time that cleavage of BAG-75 and BSP by an AEBSF-sensitive, osteoblast-derived serine protease is associated with mineral crystal nucleation in BMF and suggest that such proteolytic events are a permissive step for mineralization to proceed.

Extracellular bone acidic glycoprotein-75 defines condensed mesenchyme regions to be mineralized and localizes with bone sialoprotein during intramembranous bone formation.

Bone acidic glycoprotein-75 is expressed very early during in vivo models of intramembranous bone formation, highly enriched in condensing osteogenic mesenchyme after marrow ablation and the osteoprogenitor layer of tibial periosteum. Bone sialoprotein accumulates within bone acidic glycoprotein-75-enriched matrix areas at a later stage in both models. Decalcification of initial sites of mineralization consistently revealed focal immunostaining for bone acidic glycoprotein-75 underneath these sites suggesting that mineralization occurs within bone acidic glycoprotein-75-enriched matrix areas. Ultrastructural immunolocalization of bone acidic glycoprotein-75 does not support a direct association with banded collagen fibrils, but rather suggests it is a component of a separate, amorphous scaffold occupying interfibrillar spaces. Double immunogold labeling demonstrated that a sizeable proportion of bone sialoprotein particles were located within a 50-nm radius of bone acidic glycoprotein-75. These results define bone acidic glycoprotein-75 as the earliest bone-restricted, extracellular marker of osteogenic mesenchyme. Based on this early bone-restricted expression pattern and a previously documented propensity of bone acidic glycoprotein-75 to form supramolecular complexes through self-association, bone acidic glycoprotein-75 may serve a key structural role in setting boundary limits of condensing osteogenic mesenchyme.

Bone acidic glycoprotein-75 delineates the extracellular sites of future bone sialoprotein accumulation and apatite nucleation in osteoblastic cultures.

Addition of an organophosphate source to UMR osteoblastic cultures activates a mineralization program in which BSP localizes to extracellular matrix sites where hydroxyapatite crystals are subsequently nucleated. This study identifies for the first time novel extracellular spherical structures, termed biomineralization foci (BMF), containing bone acidic glycoprotein-75 (BAG-75), bone sialoprotein (BSP), and alkaline phosphatase that are the exclusive sites of initial nucleation of hydroxyapatite crystals in the UMR model. Importantly, in the absence of added phosphate, UMR cultures after reaching confluency contain two size populations of morphologically identifiable BMF precursors enriched in BAG-75 (15-25 and 150-250 microm in diameter). The shape and size of the smaller population are similar to structures assembled in vitro through self-association of purified BAG-75 protein. After organophosphate addition, BSP accumulates within these BAG-75-containing BMF precursors, with hydroxyapatite crystal nucleation occurring subsequently. In summary, BAG-75 is the earliest detectable biomarker that accurately predicts the extracellular sites of de novo biomineralization in UMR cultures. We hypothesize that BAG-75 may perform a key structural role in the assembly of BMF precursors and the recruitment of other proteins such as alkaline phosphatase and BSP. Furthermore, we propose a hypothetical mechanism in which BAG-75 and BSP function actively in nucleation of apatite within BMF.

New alternatively spliced form of galectin-3, a member of the beta-galactoside-binding animal lectin family, contains a predicted transmembrane-spanning domain and a leucine zipper motif.

Osteoclasts or their precursors interact with the glycoprotein-enriched matrix of bone during extravasation from the vasculature, and upon attachment prior to resorption. Reverse transcriptase-PCR studies showed that two new alternatively spliced forms of chicken galectin-3, termed Gal-3TM1 and Gal-3TR1, were enriched and preferentially expressed in highly purified chicken osteoclast-like cells. Gal-3TM1 and Gal-3TR1 mRNA were also detected in chicken intestinal tissue, but not in kidney, liver, or lung. Gal-3TM1 and Gal-3TR1 messages both contain an open reading frame encoding a predicted 70-amino acid TM1 sequence inserted between the N-terminal Gly/Pro repeat domain and the carbohydrate recognition domain (exons 3 and 4). Gal-3TR1 mRNA contains an additional 241-bp sequence, which encodes a truncated open reading frame between the 4th and 5th exons, and, whose translation is expected to terminate within the carbohydrate recognition domain encompassing exons 4, 5, and 6. Immunoblotting and affinity chromatography showed that purified osteoclast preparations and intestinal homogenates contained a 36-kDa lactose-binding galectin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analyses on chymotryptic peptides from the 36-kDa lectin confirmed its identity as Gal-3TM1. The TM1 insert contains a single transmembrane-spanning region and a leucine zipper-like stalk domain that is predicted to position the intact carbohydrate recognition domain of Gal-3TM1 on the exterior surface of the plasma membrane. Immunofluorescent staining of chicken osteoclasts confirmed the expression of Gal-3TM1 at the plasma membrane. Gal-3TM1 is the first example of a galectin superfamily member capable of being expressed as a soluble protein and as a transmembrane protein.