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PubMed1995

Quantitation of matrix Gla protein mRNA by competitive polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase as an internal control.

Matrix Gla (gamma-carboxyglutamic acid) protein (MGP) is a vitamin-K-dependent extracellular matrix protein. A method was developed to quantitate MGP mRNA based on competitive polymerase chain reaction following reverse transcription (competitive RT-PCR). The MGP cDNA was coamplified with a mutant MGP cDNA (competitor). The ratio of MGP to competitor after the PCR reaction was compared to standards to determine the amount of MGP mRNA in RT samples. MGP mRNA in as little as 3.125 ng total RNA was accurately quantitated and was far more sensitive than RNA hybridization methods. To control for variations due to sample preparation, a second competitive RT-PCR was developed to measure the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA from the same sample as an internal control. Thus, the amount of MGP is normalized to the amount of the housekeeping gene GAPDH. The accuracy, sensitivity and ease of this new method enables rapid mRNA quantitation without blotting, hybridization or autoradiography. The method is particularly advantageous for MGP mRNA measurement from a small amount of sample. Using this assay, we established that MGP mRNA increases approx. fivefold with co-treatment of retinoic and ascorbic acids.

Animals↗
PubMed2004

Matrix GLA protein modulates branching morphogenesis in fetal rat lung.

The regulation of matrix gamma-carboxyglutamic acid protein (MGP) expression during the process of lung branching morphogenesis and development was investigated. MGP mRNA expression was determined over an embryonic and postnatal time course and shown to be developmentally regulated. Immunohistochemical analysis revealed increased staining for MGP in peripheral mesenchyme surrounding distal epithelial tubules. Fetal lung explants were used as an in vitro growth model to examine expression and regulation of MGP during branching morphogenesis. MGP mRNA expression over the culture interval mimicked the in vivo time course. Explants cultured in the presence of antibodies against MGP showed gross dilation and reduced terminal lung bud counts, accompanied by changes in MGP, sonic hedgehog, and patched mRNA expression. Similarly, antifibronectin antibody treatment resulted in explant dilation and reduced MGP expression, providing evidence for an interaction with MGP and fibronectin. Conversely, intraluminal microinjection of anti-MGP antibodies had no effect either on explant growth or MGP expression, supporting the hypothesis that MGP exerts its effects through the mesenchyme. Taken together, the results suggest that MGP plays a role in lung growth and development, likely via temporally and spatially specific interactions with other branching morphogenesis-related proteins to influence growth processes.

Animals↗
PubMed1999

Mutations in the gene encoding the human matrix Gla protein cause Keutel syndrome.

Keutel syndrome (KS, MIM 245150) is an autosomal recessive disorder characterized by abnormal cartilage calcification, peripheral pulmonary stenosis and midfacial hypoplasia. A genome search using homozygosity mapping provided evidence of linkage to chromosome 12p12.3-13.1 (maximum multipoint lod score, 4.06). MGP was a candidate on the basis of its localization to this chromosomal region and the known function of its protein. MGP maps to chromosome 12p near D12S363. Human MGP is a 10-kD skeletal extracellular matrix (ECM) protein that consists of an 84-aa mature protein and a 19-aa transmembrane signal peptide. It is a member of the Gla protein family, which includes osteocalcin, another skeletal ECM protein, and a number of coagulation factors (factors II, VII, IX, X and proteins S and C). All members of this family have glutamic acid residues modified to gamma-carboxyglutamic acids (Gla) by a specific gamma-carboxylase using vitamin K as a cofactor. The modified glutamic acid residues of Gla proteins confer a high affinity for mineral ions such as calcium, phosphate and hydroxyapatite crystals, the mineral components of the skeletal ECM. The pattern and tissue distribution of Mgp expression in mice suggest a role for Mgp in regulating ECM calcification. Mglap-deficient mice (Mglap-/-) have been reported to have inappropriate calcification of cartilage. Mutational analysis of MGP in three unrelated probands identified three different mutations: c.69delG, IVS1-2A-->G and c.113T-->A. All three mutations predict a non-functional MGP. Our data indicate that mutations in MGP are responsible for KS and confirm its role in the regulation of extracellular matrix calcification.

Abnormalities, Multiple↗
PubMed2006

Matrix GLA protein function in human trabecular meshwork cells: inhibition of BMP2-induced calcification process.

PURPOSE: The matrix GLA (MGP) gene has been found to be among the 10 most highly expressed genes in the human trabecular meshwork (TM), and its expression is affected by conditions associated with glaucoma. Because MGP protein has been shown to play a key role in inhibiting calcification in cartilage and arterial vessels, MGP's function in human TM was investigated. METHODS: Perfused TM tissue and primary human TM (HTM) cells originated from donors of nonglaucomatous eyes. MGP mRNA was assayed by relative quantitative and real-time PCR. AdhMGP recombinant adenovirus was generated by bacterial transposition. Western blot analyses were cross-reacted with MGP N-terminal- and conformational-specific antibodies. MGP/BMP2 colocalization was analyzed by confocal microscopy. gamma-Carboxylation activity was measured by incorporation of 14CO2 into FLEEL synthetic peptide. Alkaline phosphatase (ALP) activity was used as a marker of osteogenic differentiation and a calcification precursor. Calcification was assessed by measuring direct calcium (o-cresolphthalein). Normalization was conducted with a telomerase probe (genomic DNA). RESULTS: HTM cells contained high levels of gamma-carboxylase activity and were able to convert MGP to its active conformation. Overexpression of MGP in HTM cells reduced ALP activity in a model of BMP2-induced osteogenesis. MGP colocalized intracellularly with BMP2. HTM cells aged in culture exhibited increased calcium content, increased ALP, decreased normalized MGP expression and lower gamma-carboxylase activity. CONCLUSIONS: MGP protein is active and functions as an inhibitor of BMP2-induced ALP activity in the HTM cells. The human TM may undergo a calcification process with age. Inhibition of the calcification mechanism mediated by MGP could be used to regulate resistance and elevated IOP.

Adenoviridae↗
PubMed1992

Retinoic acid induces matrix Gla protein gene expression in human cells.

The objective of this study was to investigate the possible regulation of the vitamin K-dependent matrix Gla (gamma-carboxyglutamic acid) protein (MGP) by retinoic acid, a regulation suggested by the recent observation that the human MGP promoter has a perfect direct repeat which is nearly identical to the retinoic acid-responsive element in the retinoic acid receptor-beta gene. We report that retinoic acid strongly increases MGP mRNA levels in all human cells tested, including osteoblasts, articular cartilage chondrocytes, and fibroblasts. In osteoblastic cells, MGP mRNA levels are increased by 25-fold at 1 microM retinoic acid and achieve half-maximal levels at 0.1 microM hormone. MGP is a small secreted protein of unknown function that is synthesized in a wide variety of vertebrate tissues. The present results suggest that part of the known actions of retinoic acid on skin, bone, cartilage, and other tissues in the human may be mediated by the stimulation of MGP synthesis and the consequent effect of increased MGP secretion on nearby target cells.

Calcitriol↗
PubMed2006

Expression of osteonectin and matrix Gla protein in scleroderma patients with and without calcinosis.

OBJECTIVES: Our aim was to evaluate (i) whether the bone matrix proteins osteonectin and matrix gamma-carboxyglutamic acid protein (MGP) are up-regulated in skin biopsies from patients with systemic sclerosis (SSc) and (ii) whether there is differential expression between patients with and without dermal calcinosis, a distressing and debilitating complication of SSc. METHODS: Skin punch biopsies were taken from the forearms of 38 SSc patients with the limited cutaneous subtype of SSc [17 without calcinosis (lcSSc) and 21 with calcinosis (lcSScCal)] and from 11 healthy control subjects. Immunohistochemistry was performed with antibodies to osteonectin and MGP. Staining was assessed semiquantitatively in the microvascular endothelium and in dermal fibroblasts. The Kruskal-Wallis one-way ANOVA was used to compare the data between patient groups. RESULTS: Both lcSSc and lcSScCal groups showed a statistically significant increase in the percentage of microvessels with osteonectin-positive endothelial cells (EC) (especially the lcSScCal group), whereas lcSScCal alone showed an increase in the percentage of microvessels with MGP-positive EC when compared with controls. In both SSc groups, the percentage of osteonectin and MGP-stained fibroblasts was increased in the reticular dermis (for osteonectin this was more marked in the lcSScCal group). In the papillary dermis, the percentage of osteonectin-stained fibroblasts was increased in both SSc groups but the lcSScCal group alone had a higher percentage of MGP-stained fibroblasts. CONCLUSIONS: When compared with controls, protein expression of osteonectin and MGP was greater in SSc patients generally, and osteonectin expression was significantly higher in EC and fibroblasts of the lcSScCal patients than the lcSSc patients without calcinosis.

Adult↗
PubMed2005

Characteristics and performance of an immunosorbent assay for human matrix Gla-protein.

BACKGROUND: Matrix gammacarboxyglutamate (Gla)-protein (MGP) is a strong inhibitor of soft tissue calcification and is mainly produced by chondrocytes and vascular smooth muscle cells (VSMCs). MGP-deficient mice have extensive calcifications of cartilage and arteries leading to osteopenia, fractures and blood vessel ruptures. Promotor polymorphisms resulting in decreased expression levels were found to be associated with an increased risk for cardiovascular disease in humans. METHODS: Recently, an ELISA-based assay has become available with which MGP may be detected in the circulation. The principle of the test kit is that of a competitive immunoassay using a monoclonal antibody against MGP bound to the microtiter plate. RESULTS: Here, we report on a critical evaluation of this assay and its potential diagnostic utility in diseases associated with the degeneration of the arterial vessel wall and cartilage. The biochemical performance of the kit is satisfactory, and significant differences were found between a number of patient cohorts and the reference population. Serum MGP concentrations were significantly decreased in patients with angina pectoris and in various cartilage diseases. CONCLUSIONS: The assay allows comparison of groups and may become a suitable marker for risk assessment or diagnosis in cardiovascular disease and osteoarthritis.

Aging↗
PubMed2003

Characterization of osteocalcin (BGP) and matrix Gla protein (MGP) fish specific antibodies: validation for immunodetection studies in lower vertebrates.

In fish species the basic mechanisms of bone development and bone remodeling are not fully understood. The classification of bone tissue in teleosts as cellular or acellular and the presence of transitional states between bone and cartilage and the finding of different types of cartilage in teleosts not previously recognized in higher vertebrates emphasizes the need for a study on the accumulation of the Gla-containing proteins MGP and BGP at the cellular level. In the present study, polyclonal antibodies developed against BGP and MGP from A. regius (a local marine teleost fish) and against MGP from G. galeus (a Pacific Ocean shark), were tested by Western blot for their specificity against BGP and MGP from several other species of teleost fish and shark. For this purpose we extracted and purified both proteins from various marine and freshwater teleosts, identified them by N-terminal amino acid sequence analysis and confirmed the presence of gamma-carboxylation in the proteins with the use of a stain specific for Gla residues. Each antibody recognized either BGP or MGP with no cross-reaction between proteins detected. All purified fish BGPs and MGPs tested were shown to be specifically recognized, thus validating the use of these antibodies for further studies.

Amino Acid Sequence↗
PubMed1996

Vertebrate mineralized matrix proteins: structure and function.

The mineralized matrices of enamel, cementum, dentin, calcified cartilage and bone are similar in their ability to form a microenvironment that facilitates deposition of hydroxyapatite. However, they are not identical, as witnessed by the nature of apatite crystals that are formed. Enamel is devoid of collagen; and is composed of enamelins, amelogenins, tuftelin and ameloblastin, first described at this meeting. Cementum, dentin and bone matrices are composed primarily of type I collagen, however, each matrix may also contain unique moieties. The exact composition of cementum is not fully known, but in dentin there are unique matrix proteins, phosphophoryn (dentin phosphoprotein, DPP), a distinctive dentin matrix protein (DMP-1), and dentin sialoprotein (DSP). In bone, dentin and cementum, the matrix proteins include proteoglycans (versican, decorin, biglycan) and hyaluronan, glycoproteins which are often phosphorylated and sulfated (osteonectin, RGD-containing proteins) and gla-containing proteins (matrix gla protein, protein S, osteocalcin). The exact nature of all the non-collagenous proteins of calcified cartilage is not yet fully known. While there are no definitive functions for any of the mineralized matrix proteins to date, they most likely participate in regulation of cell metabolism, matrix deposition and mineralization, and bone turnover.

Animals↗
PubMed2006

Osteopontin upregulation and polymerization by transglutaminase 2 in calcified arteries of Matrix Gla protein-deficient mice.

Matrix Gla protein (MGP) is a potent inhibitor of soft tissue calcification, and Mgp gene deletion in mice results in arterial calcification. Our aim was to examine osteopontin (OPN) expression and localization, and posttranslational processing of OPN by the crosslinking enzyme transglutaminase 2 (TG2), in the calcified aorta of Mgp-deficient (Mgp(-/-)) mice. Using immunohistochemistry and light and electron microscopy, we report that following mineralization occurring in the arterial media of Mgp(-/-) aortas, OPN is upregulated and accumulates at the surface of the calcified elastic lamellae. Macrophages were observed in direct contact with this OPN-rich layer. Western blot analysis of extracted Mgp(-/-) aortas revealed that the majority of the OPN was in high molecular mass protein complexes, indicating modification by a crosslinking enzyme. Consistent with this observation, TG2 expression and gamma-glutamyl-epsilon-lysyl crosslink levels were also increased in Mgp(-/-) aortas. In addition to the mineral-inhibiting actions of OPN, and based on data linking OPN and TG2 with cell adhesion in various cell types including monocytes and macrophages, we propose that TG2 interactions with OPN lead to protein polymerization that facilitates macrophage adhesion to the calcified elastic lamellae to promote clearance of the ectopic mineral deposits.

Animals↗
PubMed1991

Vitamin K-dependent carboxylation in articular chondrocytes.

A nonhepatic vitamin K-dependent protein, matrix Gla protein, has recently been identified in cartilage where it may play an important role in the control of mineralization or matrix development. We have investigated the vitamin K cycle in chondrocytes isolated from bovine and rabbit articular cartilage and examined these cells for their ability to synthesize vitamin K-dependent proteins. Chondrocytes were found to have an active vitamin K-dependent carboxylation system. Preincubation of the cells with warfarin resulted in a significant increase in the measured carboxylase activity. Both vitamin K epoxide reductase and DT-diaphorase (EC 1.6.99.2) activity were present indicating that chondrocytes are capable of producing reduced vitamin K1H2, the cofactor for the vitamin K-dependent carboxylase. Specific 14C-labeling of microsomal vitamin K-dependent protein precursors demonstrated synthesis of several vitamin K-dependent proteins by chondrocytes. 35S-labeling of chondrocyte proteins provided evidence that matrix Gla protein is synthesized by these cells.

Animals↗
PubMed1992

In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections.

We have developed a method for in situ hybridization of adult bone tissue utilizing undecalcified sections and have used it to histologically examine the mRNA expression of non-collagenous bone matrix proteins such as osteocalcin (bone Gla protein, BGP), matrix Gla protein (MGP), and osteopontin in adult rats. Expression was compared with that in bone tissues of newborn rats. In the adult bone tissue, osteocalcin mRNA was strongly expressed in periosteal and endosteal cuboidal osteoblasts but not in primary spongiosa near the growth plate. Osteopontin mRNA was strongly expressed in cells present on the bone resorption surface, osteocytes, and hypertrophic chondrocytes, but not in cuboidal osteoblasts on the formation surface. Osteopontin and osteocalcin mRNAs were expressed independently and the distribution of cells expressing osteopontin mRNA corresponded with acid phosphatase-positive mononuclear cells and osteoclasts. Expression of MGP mRNA was noted only in hypertrophic chondrocytes. In newborn rat bone tissues, expression of osteocalcin mRNA was much weaker than in adult rat bone tissues. These results clearly indicate the differential expression of mRNAs of non-collagenous bone matrix proteins in adult rat bone tissues.

Animals↗
PubMed1994

Vitamin K--its essential role in craniofacial development. A review of the literature regarding vitamin K and craniofacial development.

The normal vitamin K status of the human embryo appears to be close to deficiency. Maternal dietary deficiency or use of a number of therapeutic drugs during pregnancy, may result in frank vitamin K deficiency in the embryo. First trimester deficiency results in maxillonasal hypoplasia in the neonate with subsequent facial and orthodontic implications. A rat model of the vitamin K deficiency embryopathy shows that the facial dysmorphology is preceded by uncontrolled calcification in the normally uncalcified nasal septal cartilage, and decreased longitudinal growth of the cartilage, resulting in maxillonasal hypoplasia. The developing septal cartilage is normally rich in the vitamin K-dependent protein matrix gla protein (MGP). It is proposed that functional MGP is necessary to maintain growing cartilage in a non-calcified state. Developing teeth contain both MGP and a second vitamin K-dependent protein, bone gla protein (BGP). It has been postulated that these proteins have a functional role in tooth mineralization. As yet this function has not been established and abnormalities in tooth formation have not been observed under conditions where BGP and MGP should be formed in a non-functional form.

1-Carboxyglutamic Acid↗
PubMed2005

Differential effects of 17beta-estradiol and raloxifene on VSMC phenotype and expression of osteoblast-associated proteins.

Several studies demonstrate an association between osteoporosis and arterial calcific disease, both of which being common in elderly women. Estradiol and raloxifene, a selective estrogen receptor modulator, prevent bone loss in postmenopausal women. Little is known regarding how these agents affect arterial calcification. The aim of this study was to determine whether or not 17beta-estradiol and raloxifene reduced vascular smooth muscle cell (VSMC) differentiation and expression of bone-associated proteins during phosphate-induced calcification in vitro. Aortic VSMC were cultured from adult, gonadally intact, and ovariectomized (OVX) female pigs. Calcifying medium was added, and cells were treated with solvent (control), 17beta-estradiol (E(2)), or raloxifene. Extent of calcification and phenotypic expression of bone-associated proteins [matrix gla protein (MGP), osteoprotegerin (OPG), and bone sialoprotein (BSP)] were examined at 3-day intervals over 2 wk. Calcium content increased in all groups but was greater in VSMC derived from intact compared with OVX animals. E(2) reduced calcification and preserved a contractile phenotype. Expression of OPG significantly decreased with time; this decrease was significantly greater in VSMC derived from OVX compared with gonadally intact pigs. E(2) and raloxifene preserved expression of OPG only in VSMC from intact pigs. Expression of MGP increased significantly with time and was not affected by E(2) or raloxifene treatments. E(2) treatment significantly inhibited synthesis of BSP in cells from both groups. In conclusion, E(2) slows differentiation of VSMC induced by excess phosphate. Effectiveness of raloxifene to preserve expression of bone cell-associated proteins depends on the hormonal status of the tissue donor.

Animals↗
PubMed2001

Cloning of the bone Gla protein gene from the teleost fish Sparus aurata. Evidence for overall conservation in gene organization and bone-specific expression from fish to man.

Bone Gla protein (BGP, Osteocalcin) is a bone-specific vitamin K-dependent protein which has been intensively studied in mammals. Although BGP is the most abundant non-collagenous protein of bone, its mode of action at the molecular level remains unclear. From an evolutionary point of view, the appearance of BGP seems to parallel the appearance of hydroxyapatite-containing bone structures since it has never been found in elasmobranchs, whose skeleton is composed of calcified cartilage. Accordingly, recent work indicates that, in mammalian bone, BGP is required for adequate maturation of the hydroxyapatite crystal. Taken together, these data suggest that teleost fishes, presumably the first vertebrates to develop a BGP-containing skeleton, may be a useful model to further investigate BGP function. In addition, fish offer several advantages over mammalian models, due to a large progeny, external embryonic development and transparency of larvae. In the present work, the BGP cDNA and gene were cloned from a teleost fish, Sparus aurata, and its tissue distribution, pattern of developmental expression and evolutionary pathways analyzed. The molecular organization of the Sparus BGP (spBGP) gene is similar to mammalian BGP genes, and its expression throughout development follows the onset of calcification. The spBGP gene encodes a pre-propeptide of 97 amino acid residues, expressed only in bone and showing extensive homology to its mammalian homologs. Phylogenetic analysis of the available BGP sequences supports the hypothesis that all BGPs have a single origin and share a common ancestor with a related vitamin K-dependent protein (Matrix Gla protein).

Amino Acid Sequence↗
PubMed2000

Warfarin exposure and calcification of the arterial system in the rat.

There is evidence from knock-out mice that the extrahepatic vitamin K-dependent protein, matrix gla protein, is necessary to prevent arterial calcification. The aim of this study was to determine if a warfarin treatment regimen in rats, designed to cause extra-hepatic vitamin K deficiency, would also cause arterial calcification. Sprague-Dawley rats were treated from birth for 5-12 weeks with daily doses of warfarin and concurrent vitamin K1. This treatment causes an extrahepatic vitamin K deficiency without affecting the vitamin K-dependent blood clotting factors. At the end of treatment the rats were killed and the vascular system was examined for evidence of calcification. All treated animals showed extensive arterial calcification. The cerebral arteries and the veins and capillaries did not appear to be affected. It is likely that humans on long-term warfarin treatment have extrahepatic vitamin K deficiency and hence they are potentially at increased risk of developing arterial calcification.

Animals↗