Noncollagenous Matrix Proteins Research Articles

A single nucleotide polymorphism in osteonectin 3' untranslated region regulates bone volume and is targeted by miR-433.

Osteonectin/SPARC is one of the most abundant noncollagenous extracellular matrix proteins in bone, regulating collagen fiber assembly and promoting osteoblast differentiation. Osteonectin-null and haploinsufficient mice have low-turnover osteopenia, indicating that osteonectin contributes to normal bone formation. In male idiopathic osteoporosis patients, osteonectin 3' untranslated region (UTR) single-nucleotide polymorphism (SNP) haplotypes that differed only at SNP1599 (rs1054204) were previously associated with bone mass. Haplotype A (containing SNP1599G) was more frequent in severely affected patients, whereas haplotype B (containing SNP1599C) was more frequent in less affected patients and healthy controls. We hypothesized that SNP1599 contributes to variability in bone mass by modulating osteonectin levels. Osteonectin 3' UTR reporter constructs demonstrated that haplotype A has a repressive effect on gene expression compared with B. We found that SNP1599G contributed to an miR-433 binding site, and miR-433 inhibitor relieved repression of the haplotype A, but not B, 3' UTR reporter construct. We tested our hypothesis in vivo, using a knock-in approach to replace the mouse osteonectin 3' UTR with human haplotype A or B 3' UTR. Compared with haplotype A mice, bone osteonectin levels were higher in haplotype B mice. B mice displayed higher bone formation rate and gained more trabecular bone with age. When parathyroid hormone was administered intermittently, haplotype B mice gained more cortical bone area than A mice. Cultured marrow stromal cells from B mice deposited more mineralized matrix and had higher osteocalcin mRNA compared with A mice, demonstrating a cell-autonomous effect on differentiation. Altogether, SNP1599 differentially regulates osteonectin expression and contributes to variability in bone mass, by a mechanism that may involve differential targeting by miR-433. This work validates the findings of the previous candidate gene study, and it assigns a physiological function to a common osteonectin allele, providing support for its role in the complex trait of skeletal phenotype. © 2014 American Society for Bone and Mineral Research.

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.

Early B-cell factor 1 is an essential transcription factor for postnatal glomerular maturation

The coordination of multiple cytokines and transcription factors with their downstream signaling pathways have been shown to be integral to nephron maturation. Here we present a completely novel role for the helix-loop-helix transcription factor Early B cell Factor 1 (Ebf1), originally identified for B cell maturation, for the proper maturation of glomerular cells from mesenchymal progenitors. The expression of Ebf1 was both spatially and temporally regulated within the developing cortex and glomeruli. Using Ebf1-null mice we then identified biochemical, metabolic, and histological abnormalities in renal development that arose in the absence of this transcription factor. In the Ebf1 knockout mice the developed kidneys show thinned cortices and reduced glomerular maturation. The glomeruli showed abnormal vascularization and severely effaced podocytes. The mice exhibited early albuminuria and elevated blood urea nitrogen levels. Moreover, the GFR was reduced over 66 percent and the expression of podocyte-derived VEGF-A was decreased compared to wild type control mice. Thus, Ebf1 has a significant and novel role in glomerular development, podocyte maturation, and the maintenance of kidney integrity and function.

Age‐related differences in the plasma proteome in healthy adults: modulatory effect of regular aerobic exercise (LB178)

Aging causes physiological dysfunction, some of which can be prevented or reduced by habitual aerobic exercise (AE). However, the molecular mechanisms underlying these effects remain largely unknown, particularly in humans. We used a novel high‐throughput molecular assay (SOMAscan v3.0, SomaLogic Inc., Boulder, CO) to characterize the effects of age and regular AE on the plasma proteome of healthy adult humans. From 1129 analytes, the expression of 48 proteins was greater and 37 lower in older (O; 64±6 yr; n=16) vs. young (Y; 24±4 yr; n=16) healthy men (false discovery rate [FDR] < 15%, p<0.01). Expression of the non‐collagenous extracellular matrix (ECM) protein dermatopontin was greater in O vs. Y (P=0.007), but was lower in older AE‐trained men (63±7 yr; n=8; VO2max = 49.6±3.7 mL/kg⋅min) compared with their sedentary counterparts (VO2max = 27.4±2.4 mL/kg⋅min) (FDR < 1.28%, p<0.001). Within O, no other group differences in plasma protein expression associated with exercise status met our FDR threshold (>35%). These preliminary results suggest that normal human aging is associated with several changes to the plasma proteome that may serve as molecular biomarkers of physiological dysfunction or disease risk. Regular AE status influences age‐related changes in circulating dermatopontin, a possible mediator of age‐related ECM remodeling.Grant Funding Source: Supported by NIH AG013038 and RR025780

Dentin matrix proteins (DMPs) enhance differentiation of BMMSCs via ERK and P38 MAPK pathways

Dentin, the predominant mineralized tissue of the tooth, comprises an extracellular matrix of collagen and a heterogeneous mixture of non-collagenous components, many of which have cellular signaling properties. These properties may be important in signaling stem cell involvement in tissue regeneration following injury and the present study investigates their morphogenic effects on differentiation of Bone Marrow Stromal Stem Cells (BMMSCs) in vitro. Non-collagenous dentin matrix proteins (DMPs) were isolated from healthy human teeth and their effects on BMMSCs behavior examined during in vitro culture. In vitro, DMPs enhanced alkaline phosphatase activity and mineralization in BMMSCs cultures as well as increasing the expression of dentinogenic and osteogenic differentiation markers (including runt-related transcription factor 2, osterix, bone sialoprotein, dentin sialophosphoprotein and osteocalcin) at both transcript and protein levels, with 10 μg/mL DMPs being the optimal stimulatory concentration. Expression of phosphor-ERK/phosphor-P38 in BMMSCs was up-regulated by DMPs and, in the presence of the ERK1/2- and p38-specific inhibitors, the differentiation of BMMSCs was inhibited. These data indicate that DMPs promote the dentinogenic/osteogenic differentiation of BMMSCs via the ERK/p38 MAPK pathways.

Cartilage Oligomeric Matrix Protein in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5–3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes (Fold change 13, p-value <0.05) in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-β1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-β1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-β1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-β1 signaling should be persuaded.

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.

Implications of combined ovariectomy and glucocorticoid (dexamethasone) treatment on mineral, microarchitectural, biomechanical and matrix properties of rat bone.

Osteoporosis is one of the deleterious side effects of long-term glucocorticoid therapy. Since the condition is particularly aggressive in postmenopausal women who are on steroid therapy, in this study we have attempted to analyse the combined effect of glucocorticoid (dexamethasone) treatment and cessation of oestrogen on rat bone. The dual aim was to generate osteoporotic bone status in a short time scale and to characterise the combination of glucocorticoid-postmenopausal osteoporotic conditions. Sprague Dawley rats (N = 42) were grouped randomly into three groups: untreated control, sham-operated and ovariectomized-steroid (OVX-Steroid) rats. Control animals were euthanized with no treatment [Month 0 (M0)], while sham and OVX-Steroid rats were monitored up to 1 month (M1) and 3 months (M3) post laparotomy/post OVX-Steroid treatment. Histology, dual-energy X-ray absorptiometry (DXA), micro-computed tomography (micro-CT), and biomechanical and mRNA expression analysis of collagenous, non-collagenous matrix proteins and osteoclast markers were examined. The study indicated enhanced osteoclastogenesis and significantly lower bone mineral density (BMD) in the OVX-Steroid rats with Z-scores below -2.5, reduced torsional strength, reduced bone volume (BV/TV%), significantly enhanced trabecular separation (Tb.S), and less trabecular number (Tb.N) compared with sham rats. Osteoclast markers, cathepsin K and MMP 9 were upregulated along with Col1α1 and biglycan with no significant expression variation in fibronectin, MMP 14, LRP-5, Car II and TNC. These results show higher bone turnover with enhanced bone resorption accompanied with reduced torsional strength in OVX-Steroid rats; and these changes were attained within a short timeframe. This could be a useful model which mimics human postmenopausal osteoporosis that is associated with steroid therapy and could prove of value both in disease diagnosis and for testing generating and testing biological agents which could be used in treatment.

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.

Dentine matrix protein 1 (DMP-1) is a marker of bone-forming tumours

Dentin matrix protein 1 (DMP-1) is highly expressed by osteocytes and is a non-collagenous matrix protein found in dentin and bone. In this study, we determined the expression of DMP-1 in mature and immature human bone and examined whether DMP-1 is useful in distinguishing osteoid/bone-forming tumours from other primary and secondary bone tumours. DMP-1 expression was immunohistochemically determined in paraffin sections of a wide range of benign and malignant primary bone tumours and tumour-like lesions (n = 353). DMP-1 mRNA expression was also examined in osteosarcoma and fibrosarcoma cell lines as well as bone tumour specimens (n = 5) using real-time PCR. In lamellar and woven bone, DMP-1 was expressed in the matrix around osteocyte lacunae and canaliculi; osteoblasts and other cell types in the bone were negative. Matrix staining of the osteoid and bone was seen in bone-forming tumours including osteoma, osteoid osteoma, osteoblastoma and osteosarcoma. DMP-1 staining was also seen in fibrous dysplasia, osteofibrous dysplasia and chondroblastoma and in reactive bone in solitary bone cysts and aneurysmal bone cysts. DMP-1 was not expressed in the tumour component of other bone neoplasms including Ewing sarcoma, chondrosarcoma, leiomyosarcoma, fibrosarcoma, giant cell tumour of bone and metastatic carcinoma. DMP-1 mRNA was expressed in osteosarcoma cell lines and tumour samples. DMP-1 is a matrix marker expressed around osteocytes in human woven and lamellar bone and is useful in identifying osteosarcoma and other bone-forming tumours.

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.

Osteopontin expression is an independent factor for poor survival in oral squamous cell carcinoma: a computer‐assisted analysis on TMA sections

Osteopontin (OPN) is non-collagenous extracellular matrix protein involved in various physiological and pathological events, including tumor progression. The aim of this study was to analyze the expression of OPN in normal oral mucosa and oral squamous cell carcinoma (OSCC) and to assess its prognostic significance. The expression of OPN was immunohistochemicaly analyzed in 86 OSCC and compared with clinicopathological variable such as tumor size, nodal stage, WHO clinical stage, Ki-67 proliferation index, and patients' outcome. OPN mRNA was analyzed using quantitative real-time PCR and compared with protein OPN expression and clinical outcome in 18 OSCC samples. The expression of OPN protein was found in OSCC tumor cells (t-OPN) and various stromal cells (s-OPN). High level of t-OPN expression was associated with higher nodal stage (P=0.045), higher WHO clinical stage (P=0.033), and poor clinical outcome (P=0.022). In multivariate analysis, t-OPN emerged as an adverse independent factor for survival (P=0.049). Although correlated with t-OPN (P=0.005), s-OPN was not significantly associated with clinical parameters, including patients' outcome. Also, there was no association between OPN and clinical parameters at the mRNA level. OPN is upregulated in tumor and stromal OSCC cells. Tumor cell-derived OPN is involved in tumor progression and can independently predict the clinical outcome. Stromal-derived OPN probably has a different function compared with OPN secreted from tumor cells.

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.

The Expression of Alkaline Phosphatase, Osteopontin, Osteocalcin, and Chondroitin Sulfate during Pectoral Fin Regeneration inCarassius auratus gibelio: A Combined Histochemical and Immunohistochemical Study

Dermal bone is an important component of the teleost fins, and its ability to regenerate after fin amputation appears to be unlimited. The organic bone matrix contain type I collagen fibers, proteoglycans enriched in chondroitin sulfate, and noncollagenous matrix protein such as osteocalcin, osteopontin, and osteonectin. These molecules are synthesized by fin osteoblasts. Inorganic components chiefly consist of calcium and phosphate that form crystals of hydroxyapatite. Fin rays are described as models to study ossification. Due to this, the identification of the components involved in the synthesis of the organic and inorganic components of lepidotrichial bone are of great interest for the analysis of skeletal disorders in fish ossification. The present study investigates expression of alkaline phosphatase, osteopontin, osteocalcin, and chondroitin sulfate during pectoral fin regeneration in Carassius auratus gibelio. Alkaline phosphatase reaction has been found in the epidermis covering the wound, proximal blastema, near the cells that surround newly-formed lepidotrichia matrix and the tips of regenerating fin rays. Osteopontin has been observed throughout the regeneration blastema but excluded from the scleroblasts lining the inner side of the lepidotrichia. Osteocalcin and chondroitin sulfate expression coincides with the onset of mineralization of lepidotrichial matrix, suggesting its involvement in bone mineralization.

High Serum Osteopontin Levels Are Associated with Low Bone Mineral Density in Postmenopausal Women

Osteopontin (OPN) is an acidic, noncollagenous matrix protein produced by the bone and kidneys. It is reportedly involved in bone resorption and formation. We examined the association between serum OPN levels and bone mineral density in postmenopausal women. Premenopausal women (n=32) and postmenopausal women (n=409) participated in the study. We measured serum osteopontin levels and their relationships with bone mineral density and previous total fragility fractures. The postmenopausal women had higher mean serum OPN levels compared to the premenopausal women (43.6±25.9 vs 26.3±18.6 ng/mL; P<0.001). In the postmenopausal women, high serum OPN levels were negatively correlated with mean lumbar bone mineral density (BMD) (r=-0.113, P=0.023). In a stepwise multiple linear regression model, serum OPN levels were associated with BMD of the spine, femoral neck, and total hip after adjustment for age, body mass index, smoking, and physical activity in postmenopausal women. However, serum OPN levels did not differ between postmenopausal women with and without fractures. Postmenopausal women exhibit higher serum OPN levels than premenopausal women and higher serum OPN levels were associated with low BMD in postmenopausal women.

In vitro and clinical studies examining the expression of osteopontin in cigarette smoke-exposed endothelial cells and cigarette smokers

BackgroundCigarette smoking is a leading cause of mortality and morbidity and is associated with cardiovascular disease via contributory processes such as endothelial dysfunction, inflammation and thrombosis. Cigarette smoke both contains and stimulates the production of cellular oxidants and it may also promote vascular inflammation. Osteopontin is a non-collagenous matrix protein first identified in bone and there is increasing evidence for its role in inflammation and cardiovascular disease via its action as a soluble cytokine.MethodsIn this study we have examined the mechanisms underlying the expression of osteopontin in human vascular endothelial cells in vitro following exposure to cigarette smoke particulate matter (PM), using PCR, electrochemiluminescence, immunostaining and Western blotting. We further determined if serum osteopontin levels changed in humans who quit smoking.ResultsNon-cytotoxic concentrations of PM increased osteopontin levels in cultured human endothelial cells and this effect was reduced in the presence of ascorbate, suggesting a role for oxidants in the response to PM. However, oxidant production played no role in the PM-evoked induction MMP-3, an enzyme which cleaves osteopontin. In smokers who quit smoking for 5 days, serum osteopontin levels were significantly lowered compared to those measured prior to smoking cessation.ConclusionsIn vitro cigarette smoke extract exposure induced osteopontin expression in human endothelial cells in an oxidative stress-dependent manner, which may involve MMP-3 cleavage. In humans, serum osteopontin was decreased with short-term smoking cessation. Endothelial-derived osteopontin may contribute to inflammation in smokers, and may also contribute to atherosclerosis and cardiovascular disease-related processes.

Relationship of Sympathetic Activity to Bone Microstructure, Turnover, and Plasma Osteopontin Levels in Women

Studies in rodents have demonstrated that sympathetic activity reduces bone formation and bone mass; these effects are mediated by the noncollagenous matrix protein, osteopontin. The objective of the study was to relate sympathetic activity (measured using microneurography at the peroneal nerve) to bone microstructure (assessed by high resolution peripheral quantitative computed tomography), bone turnover, and plasma osteopontin levels. Twenty-three women aged 20-72 yr (10 premenopausal and 13 postmenopausal) were studied in the Clinical Research Unit. Sympathetic activity (bursts per 100 heart beats) was 2.4-fold higher in postmenopausal as compared with premenopausal women (P < 0.001). In the two groups combined and after age adjustment, sympathetic activity was inversely correlated with trabecular bone volume fraction (r = -0.55, P < 0.01) and thickness (r = -0.59, P < 0.01) and positively correlated with trabecular separation (r = 0.45, P < 0.05). Sympathetic activity was negatively correlated with serum amino-terminal propeptide of type I collagen in postmenopausal women (r = -0.65, P = 0.015), with a similar trend in premenopausal women (r = -0.58, P = 0.082). Sympathetic activity was also negatively correlated with plasma osteopontin levels (r = -0.43, P = 0.045), driven mainly by the correlation in postmenopausal women (r = -0.76, P = 0.002). These findings represent the first demonstration in humans of a relationship between sympathetic activity and bone microstructure and circulating levels of amino-terminal propeptide of type I collagen and osteopontin. Given the critical role of osteopontin in mediating the effects of β-adrenergic signaling on bone, the inverse association between sympathetic activity and plasma osteopontin levels may reflect a negative feedback loop to limit the deleterious effects of sympathetic activity on bone metabolism. Based on the higher sympathetic activity observed in postmenopausal women, additional human studies are needed to define the role of increased sympathetic activity in mediating postmenopausal bone loss.

Osteocalcin and selected biochemical parameters of bone turnover in healthy subjects

Osteocalcin (OC) is non-collagenous bone matrix protein produced by osteoblasts. OC binds to hydroxyapatite and is deposited in the bone matrix. Concentration of OC in the blood seems to reflect both osteogenesis and increased bone metabolism and depends on age, sex, menopausal status. The aim of our study was to evaluate changes in the concentration of osteocalcin and selected biochemical parameters of bone turnover in different stages of life in healthy people. We found that the mean serum levels of OC were significantly greater in women than in men. The highest OC levels were found in men under the age of 30 with a decrease in the fourth decade of life. In the group of women of up to 40 years of age, OC concentrations were comparable with the equivalent group of men. The lowest concentration of OC was found in the age group 40-49, with the significant increase in the subsequent decades of life. In both men and women, we found a significant correlation of OC with the calcium concentrations and alkaline phosphatase activity. Osteocalcin determinationa, particulary in women, may be useful laboratory parameters of osteoporosis process.

Osteocyte and DMP1

Dentin matrix protein (DMP1) is a non-collagenous bone matrix protein produced specifically by osteocytes. Because of its highly acidic nature, DMP1 can participate in bone matrix mineralization through Ca(2 + ) binding capacity. Inactivating mutations in DMP1 have recently been shown to cause autosomal recessive hypophosphatemic rickets (ARHR) . In a murine model of ARHR, DMP1-null mice had increased fibroblast growth factor23 (FGF23) expression in osteocytes, indicating that DMP1 participates in the systemic phosphate regulation by restraining osteocytic FGF23 production. In addition, the DMP1 promoter has been used frequently as an osteocyte-specific promoter. However, DMP1 is not expressed in all osteocytes. DMP1 expression is seen in osteoblastic-, osteoid-, young osteocytes of the superficial bone layer, but is reduced remarkably in old osteocytes distributed over the bone inside. This pattern of DMP1 expression may influence the interpretation of DMP1 function and the studies that use the DMP1 promoter.

Transcriptional repression of the Dspp gene leads to dentinogenesis imperfecta phenotype in Col1a1-Trps1 transgenic mice

Dentinogenesis imperfecta (DGI) is a hereditary defect of dentin, a calcified tissue that is the most abundant component of teeth. Most commonly, DGI is manifested as a part of osteogenesis imperfecta (OI) or the phenotype is restricted to dental findings only. In the latter case, DGI is caused by mutations in the DSPP gene, which codes for dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Although these two proteins together constitute the majority of noncollagenous proteins of the dentin, little is known about their transcriptional regulation. Here we demonstrate that mice overexpressing the Trps1 transcription factor (Col1a1-Trps1 mice) in dentin-producing cells, odontoblasts, present with severe defects of dentin formation that resemble DGI. Combined micro–computed tomography (µCT) and histological analyses revealed tooth fragility due to severe hypomineralization of dentin and a diminished dentin layer with irregular mineralization in Col1a1-Trps1 mice. Biochemical analyses of noncollagenous dentin matrix proteins demonstrated decreased levels of both DSP and DPP proteins in Col1a1-Trps1 mice. On the molecular level, we demonstrated that sustained high levels of Trps1 in odontoblasts lead to dramatic decrease of Dspp expression as a result of direct inhibition of the Dspp promoter by Trps1. During tooth development Trps1 is highly expressed in preodontoblasts, but in mature odontoblasts secreting matrix its expression significantly decreases, which suggests a Trps1 role in odontoblast development. In these studies we identified Trps1 as a potent inhibitor of Dspp expression and the subsequent mineralization of dentin. Thus, we provide novel insights into mechanisms of transcriptional dysregulation that leads to DGI. © 2012 American Society for Bone and Mineral Research.