Functional involvement of PHOSPHO1 in matrix vesicle-mediated skeletal mineralization.

PHOSPHO1 is a phosphatase highly expressed in bone. We studied its functional involvement in mineralization through the use of novel small molecule inhibitors. PHOSPHO1 expression was present within matrix vesicles, and inhibition of enzyme action caused a decrease in the ability of matrix vesicles to calcify. INTRODUCTION: The novel phosphatase, PHOSPHO1, belongs to the haloacid dehalogenase superfamily of hydrolases and is capable of cleaving phosphoethanolamine (PEA) and phosphocholine to generate inorganic phosphate. Our aims in this study were to examine the expression of PHOSPHO1 in murine mineralizing cells and matrix vesicles (MV) and to screen a series of small-molecule PHOSPHO1-specific inhibitors for their ability to pharmacologically inhibit the first step of MV-mediated mineralization. MATERIALS AND METHODS: q-PCR and immunohistochemistry were used to study the expression and localization profiles of PHOSPHO1. Inhibitors of PHOSPHO1's PEA hydrolase activity were discovered using high-throughput screening of commercially available chemical libraries. To asses the efficacy of these inhibitors to inhibit MV mineralization, MVs were isolated from TNAP-deficient (Akp2(-/-)) osteoblasts and induced to calcify in their presence. RESULTS: q-PCR revealed a 120-fold higher level of PHOSPHO1 expression in bone compared with a range of soft tissues. The enzyme was immunolocalized to the early hypertrophic chondrocytes of the growth plate and to osteoblasts of trabecular surfaces and infilling primary osteons of cortical bone. Isolated MVs also contained PHOSPHO1. PEA hydrolase activity was observed in sonicated MVs from Akp2(-/-) osteoblasts but not intact MVs. Inhibitors to PHOSPHO1 were identified and characterized. Lansoprazole and SCH202676 inhibited the mineralization of MVs from Akp2(-/-) osteoblasts by 56.8% and 70.7%, respectively. CONCLUSIONS: The results show that PHOSPHO1 localization is restricted to mineralizing regions of bone and growth plate and that the enzyme present within MVs is in an active state, inhibition of which decreases the capacity of MVs to mineralize. These data further support our hypothesis that PHOSPHO1 plays a role in the initiation of matrix mineralization.     

PHOSPHO1 is essential for mechanically competent mineralization and the avoidance of spontaneous fractures

Phosphatases are essential for the mineralization of the extracellular matrix within the skeleton. Their precise identities and functions however remain unclear. PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase involved in the generation of inorganic phosphate for bone mineralization. It is highly expressed at sites of mineralization in bone and cartilage. The bones of Phospho1(-/-) mice are hypomineralized, bowed and present with spontaneous greenstick fractures at birth. In this study we show that PHOSPHO1 is essential for mechanically competent mineralization that is able to withstand habitual load. Long bones from Phospho1(-/-) mice did not fracture during 3-point bending but deformed plastically. With dynamic loading nanoindentation the elastic modulus and hardness of Phospho1(-/-) tibiae were significantly lower than wild-type tibia. Raman microscopy revealed significantly lower mineral:matrix ratios and lower carbonate substitutions in Phospho1(-/-) tibia. The altered dihydroxylysinonorleucine/hydroxylysinonorleucine and pyridinoline/deoxypyridinoline collagen crosslink ratios indicated possible changes in lysyl hydroxylase-1 activity and/or bone mineralization status. The bone formation and resorption markers, N-terminal propeptide and C-terminal telopeptide of Type I collagen, were both increased in Phospho1(-/-) mice and this we associated with increased bone remodeling during fracture repair or an attempt to remodel a mechanically competent bone capable of withstanding physiological load. In summary these data indicate that Phospho1(-/-) bones are hypomineralized and, consequently, are softer and more flexible. An inability to withstand physiological loading may explain the deformations noted. We hypothesize that this phenotype is due to the reduced availability of inorganic phosphate to form hydroxyapatite during mineralization, creating an undermineralized yet active bone.     

Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and alkaline phosphatase function: A unified model of the mechanisms of initiation of skeletal calcification

Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Alkaline phosphatase (TNAP) plays a crucial role promoting mineralization of the extracellular matrix by restricting the concentration of the calcification inhibitor inorganic pyrophosphate (PP_i). Mutations in the TNAP gene cause hypophosphatasia, a heritable form of rickets and osteomalacia. Here we show that PHOSPHO1, a phosphatase with specificity for phosphoethanolamine and phosphocholine, plays a functional role in the initiation of calcification and that ablation of PHOSPHO1 and TNAP function prevents skeletal mineralization. Phospho1^?/? mice display growth plate abnormalities, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis in early life. Primary cultures of Phospho1^?/? tibial growth plate chondrocytes and chondrocyte‐derived matrix vesicles (MVs) show reduced mineralizing ability, and plasma samples from Phospho1^?/? mice show reduced levels of TNAP and elevated plasma PP_i concentrations. However, transgenic overexpression of TNAP does not correct the bone phenotype in Phospho1^?/? mice despite normalization of their plasma PP_i levels. In contrast, double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality. We conclude that PHOSPHO1 has a nonredundant functional role during endochondral ossification, and based on these data and a review of the current literature, we propose an inclusive model of skeletal calcification that involves intravesicular PHOSPHO1 function and P_i influx into MVs in the initiation of mineralization and the functions of TNAP, nucleotide pyrophosphatase phosphodiesterase‐1, and collagen in the extravesicular progression of mineralization. © 2011 American Society for Bone and Mineral Research.     

Kinetic analysis of substrate utilization by native and TNAP‐, NPP1‐, or PHOSPHO1‐deficient matrix vesicles

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite seed crystals in the sheltered interior of membrane‐limited matrix vesicles (MVs). Here, we have studied phosphosubstrate catalysis by osteoblast‐derived MVs at physiologic pH, analyzing the hydrolysis of ATP, ADP, and PP_i by isolated wild‐type (WT) as well as TNAP‐, NPP1‐ and PHOSPHO1‐deficient MVs. Comparison of the catalytic efficiencies identified ATP as the main substrate hydrolyzed by WT MVs. The lack of TNAP had the most pronounced effect on the hydrolysis of all physiologic substrates. The lack of PHOSPHO1 affected ATP hydrolysis via a secondary reduction in the levels of TNAP in PHOSPHO1‐deficient MVs. The lack of NPP1 did not significantly affect the kinetic parameters of hydrolysis when compared with WT MVs for any of the substrates. We conclude that TNAP is the enzyme that hydrolyzes both ATP and PP_i in the MV compartment. NPP1 does not have a major role in PP_i generation from ATP at the level of MVs, in contrast to its accepted role on the surface of the osteoblasts and chondrocytes, but rather acts as a phosphatase in the absence of TNAP. © 2010 American Society for Bone and Mineral Research     

Primary mineralization and extracellular matrix vesicles in rat bone after administration of glass-ceramic implants

Summary Glass-ceramic implants were administered into cavities prepared in rat femoral shaft. Electron microscope examination revealed formation of collagenous rich matrix in the implant-bone interface. Features typical to primary mineralization as well as bone and implant resorption were present in the interface. Primary mineralization was characterized by the occurrence of active forming cells, extracellular matrix vesicles and calcifying calcospheritic structures. Intensive primary mineralization in association with the implants indicates that glass-ceramic may be stimulative to ossification, allowing favourable tissue-implant relationship.

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Zusammenfassung Glaskeramikimplantate wurden in passende Bohrlöcher des Rattenfemurschafts eingesetzt. Die elektronenmikroskopische Untersuchung ergab die Bildung einer kollagenreichen Matrix an der Grenze zwischen Implantat und Knochen. Typische Erscheinungen der primären Mineralisation und der Resorption von Implantat und Knochen wurden an der Implantat-Knochengrenze festgestellt. Die primäre Mineralisation war gekennzeichnet durch aktive knochenbildende Zellen, extrazelluläre Matrixvesikel und kalzifizierende globuläre Strukturen. Die intensive primare Mineralisation in der Umgebung der Implantate spricht dafür, daß die Glaskeramik die Verknöcherung fördern und eine günstige Beziehung zwischen Gewebe und Implantat zustande kommen kann.

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This study was supported in part by a grant from The Government of Israel, The Ministry of Health by grant MT 0250 Ministerium für Forschung und Technologie, Bonn, West Germany, and by E. Leitz Wetzlar GmbH, Wetzlar, West Germany     

Elevated calcium, phosphorus, and magnesium retention in pregnant rats prior to the onset of fetal skeletal mineralization

Intestinal absorption and renal excretion rates of Ca, P, and Mg were compared in pregnant and control virgin rats fed a purified diet containing 0.55% Ca, 0.54% P, and 0.067% Mg. Four consecutive balance intervals of 5 days each were examined, beginning on day 1 of gestation. During days 6-10 of gestation, pregnant rats had elevated fractional intestinal absorptions of Ca (29.6 +/- 1.7 versus 20.6 +/- 1.5%), P (65.4 +/- 2.0 versus 59.9 +/- 0.9%), and Mg (54.3 +/- 1.5 versus 42.4 +/- 2.6%). Since urinary excretions of these elements did not change, the pregnant rats retained more Ca, P, and Mg than virgin rats. Fractional intestinal absorptions of these elements during pregnancy were similar to control values during days 1-5 and 11-15 of gestation and then rose for each element during days 16-20 of gestation. Presumably because of an increase in glomerular filtration rate, urinary excretions of Ca and Mg were elevated by 40 and 26% during days 16-20 of gestation. In contrast, urinary P excretion was decreased by 30% during days 11-20 of gestation. Analyses of uterine mineral contents indicated the increased maternal Ca and Mg retentions during pregnancy were balanced by the transfers of these elements to the fetuses. In contrast, pregnancy was associated with a net maternal retention of P. These data are consistent with previous observations of increased maternal skeletal mineralization during early pregnancy before the onset of fetal osteogenesis and subsequent enhanced maternal Ca intestinal absorption concurrent with fetal skeletal mineralization.     

Caveolin-1 in extracellular matrix vesicles secreted from osteoblasts

Caveolin-1 is an essential and signature protein of caveolae, which are small invaginations of the plasma membrane enriched in cholesterol and sphingolipids. Although high levels of expression of caveolin-1 have been demonstrated in osteoblasts as well as endothelial cells, fibroblasts, and muscular cells, the role of caveolin-1 in osteoblasts has not been clarified. Here, we show that caveolin-1 is secreted from osteoblasts in the form of matrix vesicles; extracellular vesicles released from the plasma membrane of osteoblasts. In this study, caveolae and matrix vesicles were similarly enriched in cholesterol and sphingomyelin in fractions isolated from mineralizing MC3T3-E1 cells. Interestingly, in the MC3T3-E1 cells caveolin-1 was enriched in the matrix vesicle fraction as well as the caveolar membrane fraction, and the amount of caveolin-1 in the matrix vesicle fraction increased as differentiation progressed. Localization of caveolin-1 in matrix vesicles was also confirmed in murine tibia. Furthermore, overexpression of caveolin-1 enhanced matrix calcification in MC3T3-E1 cells, whereas knockdown of caveolin-1 diminished it. These results suggest that secreted caveolin-1 as a component of matrix vesicles may play an important role in osteoblast calcification.     

Differential involvement of matrix vesicles during the initial and appositional mineralization processes in bone, dentin, and cementum

The distribution of matrix vesicles and its role in biological mineralization were examined in bone and dental hard tissues of the rat after daily administrations of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP), a potent inhibitor of mineralization, for 7 or 14 days. Newly formed, nonmineralized matrices of the HEBP-affected bone and mesodermal dental hard tissues other than circumpulpal dentin contained numerous mineral-filled matrix vesicles (MV), randomly distributed throughout the collagenous matrix. The distribution density of the mineral-filled MV in the HEBP-affected matrices of calvaria, metaphyseal trabecular bone, alveolar bone, and cellular cementum ranged from 60 to 70 per 100 microm(2), and no statistically significant differences were noted among the values. In the HEBP-affected dentin, however, MV were located only in the nonmineralized matrix of mantle dentin and totally absent in the circumpulpal dentin layers. Instead, the HEBP-affected circumpulpal dentin contained a dense meshwork of noncollagenous matrix enriched with calcium and phosphorus. Comparable meshwork structures were undetectable in nonmineralized matrices of the other hard tissues affected by HEBP. These observations suggest that a certain population of MV (60-70 per 100 microm(2)) is involved in the process of appositional mineralization in most of the mesodermal hard tissues, in addition to their well-known role in initial mineral induction in these tissues. Circumpulpal dentin appears to be an exception, where MV are not required for the appositional mineralization process. Exclusive localization of dentin phosphoproteins in circumpulpal dentin layers must take place to facilitate appositional mineralization at the calcification front, in the absence of MV.     

The amount of proteoglycans in cartilage matrix and the onset of mineralization

An electron microscopic investigation of the relationship between proteoglycans and cartilage mineralization has been carried out. On the basis of the number of matrix granules and the affinity of the matrix for colloidal ThO₂, we found that in embryonic mouse radii:

• 1.1. The amount of proteoglycans does not decline before the onset of mineralization.
• 2.2. In the calcified portions of the cartilage matrix the concentration of proteoglycans remains constant during and after mineralization.
• 3.3. There is a degradation of proteoglycans in the uncalcified portions of the matrix near the marrow cavity.

This last finding explains why chemical analyses of epiphyseal disks so often show a decline in the amount of proteoglycans in the mineralizing zones. It was concluded that degradation of proteoglycans is not a first, necessary step in cartilage mineralization. The loss of proteoglycans in the uncalcified matrix in the lower zones of the epiphyseal disks is probably devoid of any particular significance for calcification but is rather a preparation for the formation of the marrow cavity.     

瘢痕中缺氧诱导因子-1α的表达及其与凋亡的关系

目的 探讨瘢痕内缺氧环境减轻的机制。方法 采用免疫组化法检测烧伤后肉芽、不同时期瘢痕和正常皮肤中缺氧诱导因子-1α（hypoxia-inducible factor 1α，HIF-1α）、p53的表达，采用权重方法分别对各组瘢痕的HIF-1α、p53表达结果进行量化，分析各自规律及相互间关系。结果 随着瘢痕的成熟，HIF-1α表达强度逐渐减弱，p53表达强度在1年内逐渐增强，相互间具有负相关性（P〈0．01）。结论 p53在瘢痕成熟过程中起重要作用，HIF-1α可能通过协同p53诱导细胞凋亡，减少耗氧来减轻缺氧环境。     

Comparative calcification of native articular cartilage matrix vesicles and nitroprusside-generated vesicles

OBJECTIVE: Articular cartilage matrix vesicles generate calcium pyrophosphate- and basic calcium phosphate-like mineral in vitro. We sought to determine the morphologic features and calcifying capacity of sodium nitroprusside (SNP)-induced vesicles for comparison to those of controls. METHODS: Porcine articular cartilage was exposed to 1 mM SNP for 24 h and vesicles were isolated by enzymatic digestion and serial ultracentrifugation. Control vesicles were derived from an equal weight of untreated articular cartilage. Vesicles-containing fractions pelleted at 2 x 10(5) g x min (pellet I), 3 x 10(6) g x min (pellet II, the heavy vesicle fraction) and 8 x 10(6) g x min (pellet III, the light vesicle fraction) were analysed for Lowry protein content, nucleoside triphosphate pyrophosphohydrolase specific activity (NTPPPH) and ATP-dependent calcifying capacity. RESULTS: Electron micrographs (EM) revealed two populations of vesicular structures in both SNP and control pellets. In most experiments, there were no significant differences between the protein contents or ATP-dependent calcium accumulation of SNP vesicles compared to control vesicles. SNP vesicles in pellets I and II had lower NTPPPH activities than their respective controls, P < or = 0.01. CONCLUSIONS: Our data confirmed that 24-hour treatment with the apoptosis-inducing agent did not increase matrix vesicle protein or alter the calcifying activity of vesicles compared to those from control cartilage. SNP did generate vesicles with lower NTPPPH specific activity in most experiments. SNP vesicles, although morphologically similar to controls, are not biochemically identical to them.     

Enhanced expression of the inorganic phosphate transporter Pit-1 is involved in BMP-2-induced matrix mineralization in osteoblast-like cells.

Pi handling by osteogenic cells is important for bone mineralization. The role of Pi transport in BMP-2-induced matrix calcification was studied. BMP-2 enhances Pit-1 Pi transporters in osteogenic cells. Experimental analysis suggest that this response is required for bone matrix calcification. INTRODUCTION: Bone morphogenetic proteins (BMPs) are produced by osteogenic cells and play an important role in bone formation. Inorganic phosphate (Pi) is a fundamental constituent of hydroxyapatite, and its transport by osteogenic cells is an important function for primary calcification of the bone matrix. In this study, we investigated the role of Pi transport in BMP-2-induced matrix mineralization. MATERIALS AND METHODS: Confluent MC3T3-E1 osteoblast-like cells were exposed to BMP-2 for various time periods. Pi and alanine transport was determined using radiolabeled substrate, Pit-1 and Pit-2 expression by Northern blot analysis, cell differentiation by alkaline phosphatase activity, matrix mineralization by alizarin red staining, and the characteristics of mineral deposited in the matrix by transmission electron microscopy, electron diffraction analysis, and Fourier transformed infrared resolution (FTIR). RESULTS: BMP-2 time- and dose-dependently stimulated Na-dependent Pi transport in MC3T3-E1 cells by increasing the V(max) of the transport system. This effect was preceded by an increase in mRNA encoding Pit-1 but not Pit-2. BMP-2 also dose-dependently enhanced extracellular matrix mineralization, an effect blunted by either phosphonoformic acid or expression of antisense Pit-1. Enhanced Pi transport and matrix mineralization induced by BMP-2 were blunted by a specific inhibitor of the c-Jun-N-terminal kinase (JNK) pathway. CONCLUSIONS: Results presented in this study indicate that, in addition to its well-known effect on several markers of the differentiation of osteoblastic cells, BMP-2 also stimulates Pi transport activity through a selective increase in expression of type III Pi transporters Pit-1. In MC3T3-E1 cells, this effect is mediated by the JNK pathway and plays an essential role in bone matrix calcification induced by BMP-2.     

The role of the collagen matrix in skeletal fragility

The collagen network in bone provides resistance against fracture and may be susceptible to changes with aging and disease. This review identifies the changes in quality of collagen matrix as contributors to bone fragility. With aging and in diabetes, cross-links accumulate in bone collagen as a result of nonenzymatic glycation and consequently impair matrix properties, increasing bone fragility. Cell-culture and animal studies suggest that the accumulation of cross-links induced by nonenzymatic glycation may be related to a reduction in bone turnover resulting from the altered responses of osteoblasts and osteoclasts to advanced glycation end products.