Noncollagenous Bone Matrix Proteins Research Articles

Identification of gravity-responsive proteins in the femur of spaceflight mice using a quantitative proteomic approach

Although the microgravity (μ-g) environment that astronauts encounter during spaceflight can cause severe acute bone loss, the molecular mechanism of this bone loss remains unclear. To investigate the gravity-response proteins involved in bone metabolism, it is important to comprehensively determine which proteins exhibit differential abundance associated with mechanical stimuli. However, comprehensive proteomic analysis using small bone samples is difficult because protein extraction in mineralized bone tissue is inefficient. Here, we established a high-sensitivity analysis system for mouse bone proteins using data-independent acquisition mass spectrometry. This system successfully detected 40 proteins in the femoral diaphysis showing differential abundance between mice raised in a μ-g environment, where the bone mass was reduced by gravity unloading, and mice raised in an artificial 1-gravity environment on the International Space Station. Additionally, 22 proteins, including noncollagenous bone matrix proteins, showed similar abundance between the two groups in the mandible, where bone mass was unaltered due to mastication stimuli, suggesting that these proteins are responsive to mechanical stimuli. One of these proteins, SPARCL1, is suggested to promote osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand. We expect these findings to lead to new insights into the mechanisms of bone metabolism induced by mechanical stimuli. SignificanceWe aimed to investigate the gravity-response proteins involved in bone metabolism. To this end, we established a comprehensive analysis system for mouse bone proteins using data-independent acquisition mass spectrometry, which is particularly useful in comprehensively analyzing the bone proteome using small sample volumes. In addition, a comprehensive proteomic analysis of the femoral diaphysis and mandible, which exhibit different degrees of bone loss in mice raised on the International Space Station, identified proteins that respond to mechanical stimuli. SPARCL1, a mechanical stimulus-responsive protein, was consequently suggested to be involved in osteoclast differentiation associated with bone remodeling. Our findings represent an important step toward elucidating the molecular mechanism of bone metabolism induced by mechanical stimuli.

Bone sialoprotein promotes lung cancer osteolytic bone metastasis via MMP14-dependent mechanisms

Bone metastases during lung cancer are common. Bone sialoprotein (BSP), a non-collagenous bone matrix protein, plays important functions in bone mineralization processes and in integrin-mediated cell-matrix interactions. Importantly, BSP induces bone metastasis in lung cancer, but the underlying mechanisms remain unclear. This study therefore sought to determine the intracellular signaling pathways responsible for BSP-induced migration and invasion of lung cancer cells to bone. Analyses of the Kaplan-Meier, TCGA, GEPIA and GENT2 databases revealed that high levels of BSP expression in lung tissue samples were associated with significantly decreased overall survival (hazard ratio = 1.17; p = 0.014) and with a more advanced clinical disease stage (F-value = 2.38, p < 0.05). We also observed that BSP-induced stimulation of matrix metalloproteinase (MMP)-14 promoted lung cancer cell migration and invasion via the PI3K/AKT/AP-1 signaling pathway. Notably, BSP promoted osteoclastogenesis in RAW 264.7 cells exposed to RANKL and BSP neutralizing antibody reduced osteoclast formation in conditioned medium (CM) from lung cancer cell lines. Finally, at 8 weeks after mice were injected with A549 cells or A549 BSP shRNA cells, the findings revealed that the knockdown of BSP expression significantly reduced metastasis to bone. These findings suggest that BSP signaling promotes lung bone metastasis via its direct downstream target gene MMP14, which reveals a novel potential therapeutic target for lung cancer bone metastases.

Structural role of osteocalcin and its modification in bone fracture.

Osteocalcin (OC), an abundant non-collagenous protein in bone extracellular matrix, plays a vital role in both its biological and mechanical function. OC undergoes post-translational modification, such as glycation; however, it remains unknown whether glycation of OC affects bone's resistance to fracture. Here, for the first time, we demonstrate the formation of pentosidine, an advanced glycation end-product (AGE) cross-link on mouse OC analyzed by ultra-performance liquid chromatography. Next, we establish that the presence of OC in mouse bone matrix is associated with lower interlamellar separation (distance) and thicker bridges spanning the lamellae, both of which are critical for maintaining bone's structural integrity. Furthermore, to determine the impact of modification of OC by glycation on bone toughness, we glycated bone samples in vitro from wild-type (WT) and osteocalcin deficient (Oc-/-) mice, and compared the differences in total fluorescent AGEs and fracture toughness between the Oc -/- glycated and control mouse bones and the WT glycated and control mouse bones. We determined that glycation resulted in significantly higher AGEs in WT compared to Oc-/- mouse bones (delta-WT > delta-OC, p = 0.025). This observed change corresponded to a significant decrease in fracture toughness between WT and Oc-/- mice (delta-WT vs delta-OC, p = 0.018). Thus, we propose a molecular deformation and fracture mechanics model that corroborates our experimental findings and provides evidence to support a 37%-90% loss in energy dissipation of OC due to formation of pentosidine cross-link by glycation. We anticipate that our study will aid in elucidating the effects of a major non-collagenous bone matrix protein, osteocalcin, and its modifications on bone fragility and help identify potential therapeutic targets for maintaining skeletal health.

Nanoscale Imaging and Analysis of Bone Pathologies

Understanding the properties of bone is of both fundamental and clinical relevance. The basis of bone’s quality and mechanical resilience lies in its nanoscale building blocks (i.e., mineral, collagen, non-collagenous proteins, and water) and their complex interactions across length scales. Although the structure–mechanical property relationship in healthy bone tissue is relatively well characterized, not much is known about the molecular-level origin of impaired mechanics and higher fracture risks in skeletal disorders such as osteoporosis or Paget’s disease. Alterations in the ultrastructure, chemistry, and nano-/micromechanics of bone tissue in such a diverse group of diseased states have only been briefly explored. Recent research is uncovering the effects of several non-collagenous bone matrix proteins, whose deficiencies or mutations are, to some extent, implicated in bone diseases, on bone matrix quality and mechanics. Herein, we review existing studies on ultrastructural imaging—with a focus on electron microscopy—and chemical, mechanical analysis of pathological bone tissues. The nanometric details offered by these reports, from studying knockout mice models to characterizing exact disease phenotypes, can provide key insights into various bone pathologies and facilitate the development of new treatments.

Effect of Azithromycin on Mineralized Nodule Formation in MC3T3-E1 Cells.

Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.

Serum Osteocalcin Level is Negatively Associated with Vascular Reactivity Index by Digital Thermal Monitoring in Kidney Transplant Recipients.

Background and Objectives: Osteocalcin is the most abundant noncollagenous protein in bone matrix, which is considered a marker of bone formation. Previous studies indicate that circulating osteocalcin can be expressed by osteoblasts and even by osteoblast-like cells in vessel walls, and it is often associated with arterial stiffness. Our study aims to examine the potential association between osteocalcin levels and endothelial function among kidney transplant (KT) recipients. Materials and Methods: Fasting blood samples were obtained from 68 KT recipients. To measure the endothelial function and vascular reactivity index (VRI), a digital thermal monitoring test (VENDYS) was used. A commercial enzyme-linked immunosorbent assay kit was also utilized to measure serum total osteocalcin levels. In this study, a VRI of less than 1.0 indicated poor vascular reactivity; a VRI of 1.0–2.0 indicated intermediate vascular reactivity; and a VRI of 2.0 or higher indicated good vascular reactivity. Results: Our findings show that 8 KT recipients (11.8%) had poor vascular reactivity (VRI < 1.0), 26 (38.2%) had intermediate vascular reactivity (1.0 ≤ VRI < 2.0), and 34 (50%) had good vascular reactivity. Increased serum osteocalcin levels (p < 0.001) were found to be associated with poor vascular reactivity. Advanced age (r = −0.361, p = 0.002), serum alkaline phosphate level (r = −0.254, p = 0.037), and log-transformed osteocalcin levels (r = − 0.432, p < 0.001) were identified to be negatively correlated with VRI in KT recipients. Multivariable forward stepwise linear regression analysis revealed that the serum level of osteocalcin (β = −0.391, adjusted R2 change = 0.174; p < 0.001) and advanced age (β = −0.308, adjusted R2 change = 0.084; p = 0.005) were significantly and independently associated with VRI in KT recipients. Conclusions: Higher serum osteocalcin level was associated with lower VRI and poorer endothelial dysfunction among KT recipients.

Impact of Glutamate Carboxylation in the Adsorption of the α-1 Domain of Osteocalcin to Hydroxyapatite and Titania.

One proposed mechanism of implant fouling is attributed to the nonspecific adsorption of non-collagenous bone matrix proteins (NCPs) onto a newly implanted interface. With the goal of capturing the fundamental mechanistic and thermodynamic forces that govern changes in these NCP recognition domains as a function of γ-carboxyglutamic acid (Gla) post-translational modification and surface chemistry, we probe the adsorption process of the most commonly occurring NCP, osteocalcin, onto a mineral and metal oxide surface. Here, we apply two enhanced sampling methods to independently probe the effects of post-translational modification and peptide structure on adsorption. First, well-tempered metadynamics was used to capture the binding of acetyl and N-methylamide capped glutamic acid and Gla single amino acids onto crystalline hydroxyapatite and titania model surfaces at physiological pH. Following this, parallel tempering metadynamics in the well-tempered ensemble (PTMetaD-WTE) was used to study adsorption of the α-1 domain of osteocalcin onto hydroxyapatite and titania. Simulations were performed for the α-1 domain of osteocalcin in both its fully decarboxylated (dOC) and fully carboxylated (OC) form. Our simulations find that increased charge density due to carboxylation results in increased interactions at the interface, and stronger adsorption of the single amino acids to both surfaces. Interestingly, the role of Gla in promoting compact and helical structure in the α-1 domain resulted in disparate binding modes at the two surfaces, which is attributed to differences in interfacial water behavior. Overall, this work provides a benchmark for understanding the mechanisms that drive adsorption of Gla-containing mineralizing proteins onto different surface chemistries.

Synergistic effect of extracellularly supplemented osteopontin and osteocalcin on stem cell proliferation, osteogenic differentiation, and angiogenic properties.

A high demand for functional bone grafts is being observed worldwide, especially due to the increased life expectancy. Osteoinductive components should be incorporated into functional bone grafts, accelerating cell recruitment, cell proliferation, angiogenesis, and new bone formation at a defect site. Noncollagenous bone matrix proteins, especially osteopontin (OPN) and osteocalcin (OC), have been reported to regulate some physiological process, such as cell migration and bone mineralization. However, the effects of OPN and OC on cell proliferation, osteogenic differentiation, mineralization, and angiogenesis are still undefined. Therefore, we assessed the exogenous effect of OPN and OC supplementation on human bone marrow mesenchymal stem/stromal cells (hBM MSC) proliferation and osteogenic differentiation. OPN dose-dependently increased the proliferation of hBM MSC, as well as improved the angiogenic properties of human umbilical vein endothelial cells (HUVEC) by increasing the capillary-like tube formation in vitro. On the other hand, OC enhanced the differentiation of hBM MSC into osteoblasts and demonstrated an increase in extracellular calcium levels and alkaline phosphatase activity, as well as higher messenger RNA levels of mature osteogenic markers osteopontin and osteocalcin. In vivo assessment of OC/OPN-enhanced scaffolds in a critical-sized defect rabbit long-bone model revealed no infection, while new bone was being formed. Taken together, these results suggest that OC and OPN stimulate bone regeneration by inducing stem cell proliferation, osteogenesis and by enhancing angiogenic properties. The synergistic effect of OC and OPN observed in this study can be applied as an attractive strategy for bone regeneration therapeutics by targeting different vital cellular processes.

Evidence of Osteogenic Regulation in Calcific Porcine Aortic Valves.

Chemically cross-linked animal tissues, such as porcine aortic valves (PAVs) have many documented advantages over mechanical valves. However, calcification is the major underlying pathologic process that results in bioprosthetic valve failure. Recently, several reports described the expression of noncollagenous bone matrix proteins in bioprosthetic valves and suggested an actively regulated process of tissue repair. Thirty-one explanted PAVs with evidence of calcification were collected and examined for the protein expression implicated in myofibroblast activation, osteoblast differentiation, and bone matrix deposition by using immunohistochemistry. The mean duration that PAVs were implanted was 11.5 ± 5.6 years, ranging from 12 months to 28 years. Pearson correlation analysis showed a significant relationship between the duration and valvular calcification (r = 0.3818, P = .034). The number of vimentin-positive mesenchymal cells in explanted PAVs was significantly lower than that of unused PAVs (P < .01). However, increased expression of α-smooth muscle actin (α-SMA) (P < .01), proliferating cell nuclear antigen (PCNA, P < .01), Cbfa1/Runx2 (P < .01), osterix (P = .0126), bone sialoprotein (BSP, P < .01), osteocalcin (P < .01), and osteopontin (P < .01) was found in explanted PAVs. Immunohistochemical staining of alkaline phosphatase (ALP) and osteocalcin was negative in the unused PAVs. In explanted PAVs, the expression level of these 2 proteins was also significantly increased. Our results support the view that PAV calcification is an actively regulated process with osteogenic signaling activation.

Expression of Non-collagenous Bone Matrix Proteins in Osteoblasts Stimulated by Mechanical Stretching in the Cranial Suture of Neonatal Mice.

We investigated the influence of mechanical stretching on the genetic expression pattern of non-collagenous bone matrix proteins in osteoblasts. The cranial sutures of neonatal mice were subjected to ex vivo mechanical stretching. In the non-stretched control group, as osteoblast differentiation progressed, the successive genetic expression of bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) was detected using in situ hybridization, in that order. In the stretched group, the sutures were widened, and after 24 hr of cultivation, a large number of osteoblasts and abundant new osteoid were observed on the borders of the parietal bones. All new osteoblasts expressed BSP and some of them expressed OPN, but very few of them expressed OCN. After 48 hr, more extensive presence of osteoid was noted on the borders of the parietal bones, and this osteoid was partially mineralized; all osteoblasts on the osteoid surface expressed BSP, and more osteoblasts expressed OPN than those after 24 hr cultivation. Surprisingly, many of the osteoblasts that did not express OPN, expressed OCN. This suggests that when osteoblast differentiation is stimulated by mechanical stress, the genetic expression pattern of non-collagenous proteins in the newly differentiated osteoblasts is affected.

Bioactive Glass Particles in Two-Dimensional and Three-Dimensional Osteogenic Cell Cultures.

This study aimed to investigate the influence of a three-dimensional cell culture model and bioactive glass (BG) particles on the expression of osteoblastic phenotypes in rat calvaria osteogenic cells culture. Cells were seeded on two-dimensional (2D) and three-dimensional (3D) collagen with BG particles for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity was performed. Cell morphology and immunolabeling of noncollagenous bone matrix proteins were assessed by epifluorescence and confocal microscopy. The expressions of osteogenic markers were analyzed using RT-PCR. Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay. Experimental cultures produced a growing cell viability rate up to 14 days. Although ALP activity at 7 days was higher on BG cultures, cells on 3D and 3D+BG had an activity decrease of ALP at 14 days. Three-dimensional conditions favored the immunolabeling for OPN and BSP and the expression of ALP and COL I mRNAs. BG particles influenced positively the OC and OPN mRNAs expression and calcified nodule formation in vitro. The results indicated that the 3D cultures and BG particles contribute to the expression of osteoblastic phenotype and to differentiated and mineralized matrix formation.

Evaluation of Bone Mineral Density in Children with Type I Diabetes Mellitus and Relationship to Serum Levels of Osteopontin.

Type I diabetes mellitus (T1DM) patients are at risk of osteoporosis and fracture due to the osteoblast and osteoclast malfunction. Osteopontin (OPN) as the major non-collagenous bone matrix protein is produced by osteoblasts and osteoclasts and involve in bone resorption, formation and remodeling. To evaluate the serum levels of OPN, bone mineral density (BMD) and correlation in patients with T1DM this study was designed. In this case-control study, 87 children with T1DM and 87 age/sex-matched healthy subjects were enrolled. Blood samples were tested for OPN levels by ELISA. Dual energy X-ray absorptiometry was used to measure BMD. The mean levels of BMD in patients was significantly lower than control group (p<0.0001). There was no significant difference between patients and healthy subjects regarding the levels of OPN. However, in patients with high levels of OPN (mean+1.5 standard deviation) the BMD was significantly lower than other patients (p<0.0001). Totally there was a negative correlation between serum levels of OPN and BMD in patients with T1DM (p<0.016). These results indicated that the BMD in T1DM is less than healthy children and elevated level of OPN in patients could be associated with low BMD. A linear negative correlation between serum OPN and total BMD of T1DM patients compared to control group was noticed in this study indicating that the amount of serum OPN could be effective on BMD and a good predicting factor for osteoporosis in patients.

Osteocalcin Mediates Biomineralization during Osteogenic Maturation in Human Mesenchymal Stromal Cells.

There is a growing interest in cell therapies using mesenchymal stromal cells (MSCs) for repairing bone defects. MSCs have the ability to differentiate into osteoprogenitors and osteoblasts as well as to form calcified bone matrix. However, the molecular mechanisms governing mineralization during osteogenic differentiation remain unclear. Non-collagenous proteins in the extracellular matrix are believed to control different aspects of the mineralization. Since osteocalcin is the most abundant non-collagenous bone matrix protein, the purpose of this study is to investigate the roles of osteocalcin in mineral species production during osteogenesis of MSCs. Using Raman spectroscopy, we found that the maturation of mineral species was affected by osteocalcin expression level. After osteocalcin was knocked down, the mineral species maturation was delayed and total hydroxyapatite was lower than the control group. In addition, the expression of osteogenic marker genes, including RUNX2, alkaline phosphatase, type I collagen, and osteonectin, was downregulated during osteogenic differentiation compared to the control group; whereas gene expression of osterix was upregulated after the knockdown. Together, osteocalcin plays an essential role for the maturation of mineral species and modulates osteogenic differentiation of MSCs. The results offer new insights into the enhancement of new bone formation, such as for the treatments of osteoporosis and fracture healing.

Osteonectin (SPARC) Expression in Vascular Calcification: In Vitro and Ex Vivo Studies

Osteonectin, also termed SPARC, is a noncollagenous protein of bone matrix. Since there are controversial results regarding its role during the process of vascular calcification, we investigated osteonectin expression in our in vitro calcification model. Rat vascular smooth muscle cells (VSMCs) were challenged with high phosphate (5mmol/L Pi) and analyzed quantifying calcium levels, through immunohistochemical studies, and studying gene expression. We detected a peak of osteonectin expression at day 7 in cell treated with high phosphate. The time course of calcium deposition, reflected the expression of osteonectin, resulting extensively present at day 7. On the contrary, the expression of the mitotic marker Ki-67 had a peak at day 4, showing no correlation between osteonectin and cell proliferation. Moreover, 7days was the time point in which Cbfα1/RUNX-2 had its maximal expression. Furthermore, ascorbic acid increased osteonectin expression, supporting a procalcifying role for this protein. Next we decided to study osteonectin expression ex vivo in fetal, adult not calcified, and adult calcific vessels. Immunohistochemical studies demonstrated a spread and strong reactivity in VSMCs of a 20-week fetus, confirming that osteonectin may have a potential role in regulation of mitosis and in cell differentiation. In adult not calcified arteries, osteonectin was constitutively expressed and its levels increased in atherosclerotic and in calcified plaques, where it could have a regulatory role in the calcification process. Our in vitro and ex vivo data show osteonectin expression during the calcification process and suggest its potential role as procalcifying factor.

Understanding the influence of phosphorylation and polysialylation of gelatin on mineralization and osteogenic differentiation

Post-translational modifications such as phosphorylation and sialylation impart crucial functions such as mineral deposition and osteogenic differentiation to non-collagenous bone matrix proteins. In this work, the influence of phosphorylation and polysialylation of gelatin on mineralization in simulated body fluid (SBF) and on osteogenic differentiation of mesenchymal stem cells (MSC) was studied. It was observed that increase in phosphorylation could be directly correlated with the mineralization ability of phosphorylated gelatin in SBF. The total calcium and phosphate deposited increased with increase in degree of phosphorylation and was >3 fold higher on the highest degree of phosphorylation. Whereas, polysialylation did not have any significant influence on mineral deposition in SBF. On the other hand, when MSCs were cultured on polysialylated surfaces they showed relatively higher cell elongation with 1.5 fold higher cell aspect ratio, higher alkaline phosphatase activity and 3 fold higher mineral deposition when compared to control and phosphorylated gelatin surfaces. In conclusion, phosphorylation and polysialylation of gelatin show a significant influence on mineralization and osteogenic differentiation respectively which can be advantageously used for bone tissue engineering.

First titanium dental implants with white surfaces: Preparation and in vitro tests

ObjectivesTo evaluate first white titanium surfaces developed for improvement of existing clinically used titanium-based implants Ticer. MethodsThe anodic plasma-electrochemical oxidation in aqueous solutions of sodium hydroxide and calcium dihydrogen phospate was used to prepare three novel anodic conversion layers with white titanium oxide surfaces. The surfaces have been characterized by the means of scanning electron microscopy, surface microanalysis and X-ray diffraction. In vitro studies were conducted on primary human osteoblast cells using novel surfaces (M1–M3) as well as commercially pure titanium (Ti cp), Ticer and SS (subtracted surface). An indirect toxicity test using MTT and SRB assays has been carried out. Furthermore, immunohistochemical analysis of cell proliferation, morphology, and expression of non-collagenous bone matrix proteins (sialoprotein, BSP, and osteocalcin, OC) were performed. ResultsThe basic morphology of the surfaces shows clusters in a size of 100μm of knob-like structures. The coatings are composed of rutile and monoclinic sodium titanates. Novel white surfaces (M1–M3) induced proliferation rates, morphological changes and influenced the expression of OC and BSP similarly to Ticer. On the other hand, Ti cp and SS exhibited different in vitro behavior. SignificanceThe novel surfaces expressed similar in vitro behavior as Ticer, successfully used in clinical practice. Furthermore, due to their white color they are also promising from the esthetic point of view. The results described herein open the door toward a new generation of white titanium dental implants.

Novel sandwich ELISAs for rat DMP1: Age-related decrease of circulatory DMP1 levels in male rats

Dentin matrix protein 1 (DMP1), a noncollagenous bone matrix protein produced by osteocytes, regulates matrix mineralization and phosphate homeostasis. The lack of a precise assay for circulating DMP1 levels impairs further investigation of the protein's biological significance. Because full-length precursor DMP1 is cleaved into NH2- and COOH-terminal fragments during the secretory process, we developed two new sandwich ELISAs for the NH2- and COOH-terminal fragments of rat DMP1. One of these ELISAs, ELISA 1–2, is based on two affinity-purified polyclonal antibodies against the DMP1-1 and DMP1-2 peptides of the NH2-terminal fragment, whereas the other, ELISA 4–3, is based on two affinity-purified polyclonal antibodies against the DMP1-3 and DMP1-4 peptides of the COOH-terminal fragment. The polyclonal antibodies were characterized in immunohistochemical and liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) studies. Immunohistochemical analyses of rat bone using these polyclonal antibodies revealed DMP1 immunoreactivity in osteocytes and pericanalicular matrix, consistent with the previously reported osteocyte-specific expression of DMP1. LC–MS/MS analyses of rat plasma-derived immunoreactive products affinity-extracted with these antibodies revealed the presence of DMP1 in circulating blood. The ELISAs established with these antibodies met accepted standards for reproducibility, repeatability, precision, and accuracy. Circulating DMP1 and levels of other biochemical markers (osteocalcin, Trap5b, Dkk-1, and SOST) were measured in 2-, 4-, 8-, 12-, 18-, 24-, 72-, and 96-week-old Wistar male rats. Circulating DMP1 levels determined by ELISAs 1–2 and 4–3 significantly decreased with age. During rapid skeletal growth (2–12weeks), DMP1 levels measured by ELISA 4–3 were over three times higher than those measured by ELISA 1–2; however, DMP1 levels in old animals (72 and 96weeks) were almost the same when measured by either ELISA. DMP1 levels determined by both ELISAs were most highly positively correlated with the level of Dkk-1, second most highly correlated with the level of osteocalcin, and less highly correlated with the levels of Trap5b and SOST. These novel sandwich ELISAs for rat DMP1 are highly specific and allow precise measurements of circulating DMP1, which may be a new biochemical marker for osteocyte-mediated bone turnover.

Ameloblastin Inhibits Cranial Suture Closure by Modulating Msx2 Expression and Proliferation

Deformities of cranial sutures such as craniosynostosis and enlarged parietal foramina greatly impact human development and quality of life. Here we have examined the role of the extracellular matrix protein ameloblastin (Ambn), a recent addition to the family of non-collagenous extracellular bone matrix proteins, in craniofacial bone development and suture formation. Using RT-PCR, western blot and immunohistochemistry, Ambn was localized in mouse calvarial bone and adjacent condensed mesenchyme. Five-fold Ambn overexpression in a K14-driven transgenic mouse model resulted in delayed posterior frontal suture fusion and incomplete suture closure. Moreover, Ambn overexpressor skulls weighed 13.2% less, their interfrontal bones were 35.3% thinner, and the width between frontal bones plus interfrontal suture was 14.3% wider. Ambn overexpressing mice also featured reduced cell proliferation in suture blastemas and in mesenchymal cells from posterior frontal sutures. There was a more than 2-fold reduction of Msx2 in Ambn overexpressing calvariae and suture mesenchymal cells, and this effect was inversely proportionate to the level of Ambn overexpression in different cell lines. The reduction of Msx2 expression as a result of Ambn overexpression was further enhanced in the presence of the MEK/ERK pathway inhibitor O126. Finally, Ambn overexpression significantly reduced Msx2 down-stream target gene expression levels, including osteogenic transcription factors Runx2 and Osx, the bone matrix proteins Ibsp, ColI, Ocn and Opn, and the cell cycle-related gene CcnD1. Together, these data suggest that Ambn plays a crucial role in the regulation of cranial bone growth and suture closure via Msx 2 suppression and proliferation inhibition.

Platelets express and release osteocalcin and co‐localize in human calcified atherosclerotic plaques

Although vascular-calcification mechanisms are only partially understood, the role of circulating calcifying cells and non-collagenous bone matrix proteins in the bone-vascular axis is emerging. In spite of the fact that platelets represent a cellular interface between hemostasis, inflammation and atherosclerosis, and have a myeloid precursor, a possible involvement in the modulation of vascular calcification has rarely been investigated. We investigated if osteocalcin (OC) is released by platelets and described OC expression in patients with carotid artery occlusive disease. Expression and release of OC were determined by Western blot, immunofluorescence, fluorescence-activated cell sorting (FACS) and ELISA in human resting and activated platelets and megakaryocytes. Co-localization of platelet aggregates, macrophages, OC and calcifications was studied in carotid endarterectomy specimens and normal tissues. Human platelets expressed OC and co-localized with CD63 in δ-granules. Upon activation with an endogenous mechanism, platelets released OC in the extracellular medium. Expression of OC in megakaryocytes suggested lineage specificity. The OC count in circulating platelets and the released amount were significantly higher in patients with carotid artery occlusive disease than in healthy controls (P < 0.0001) in spite of similar serum levels. In atherosclerotic plaques, OC strongly overlapped with CD41+ platelets in the early stage of calcification, but this was not seen in normal tissues. CD68+OC+ cells were present at the periphery of the calcified zone. Given the active role played by platelets in the atherosclerotic process, the involvement of OC release from platelets in atherosclerotic lesions and the impact of genetic and cardiovascular risk factors in mediating bone-marrow preconditioning should be investigated further.

Endocrine Actions of Osteocalcin

Osteocalcin is the most abundant noncollagenous protein of bone matrix. Once transcribed, this protein undergoes posttranslational modifications within osteoblastic cells before its secretion, including the carboxylation of three glutamic residues in glutamic acid, which is essential for hydroxyapatite binding and deposition in the extracellular matrix of bone. Recent provocative data from experimental observations in mice showed that the circulating undercarboxylated fraction of osteocalcin increases insulin secretion and sensitivity, lowers blood glucose, and decreases visceral fat in both genders, while it enhances testosterone production by the testes in males. Moreover, both total and undercarboxylated osteocalcins increase following physical activity with potential positive effects on glucose tolerance. Despite that these evidences have been only in part confirmed in humans, further prospective investigations are needed to definitively establish the endocrine role of osteocalcin both in the general population and cohorts of patients with diabetes or other metabolic disorders.