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.     

Lung, heart, and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla protein. Implications for the possible functions of matrix Gla protein and for the tissue distribution of the gamma-carboxylase

We have used cDNA probes for two small vitamin K-dependent bone matrix proteins, bone Gla protein (BGP) and matrix Gla protein (MGP), to evaluate the possibility that either of these proteins might be synthesized by the various soft tissues previously shown to have gamma-carboxylase activity. BGP mRNA was found in bone but not in any of the soft tissues tested, a result which reinforces the view that plasma BGP is a specific marker for bone metabolism. In contrast, MGP mRNA was found in all rat tissues examined. Lung and heart have 10-fold higher levels of MGP mRNA than bone, and kidney has a 5-fold higher level. Despite the high levels of MGP mRNA in heart and kidney, these tissues contain 40-500-fold lower concentrations of MGP protein than bone. Immunofluorescence was used to identify cells that contain MGP in kidney, lung, heart, and spleen. In each tissue, MGP was found in discrete tissue-specific cell types. In most of the soft tissues tested, MGP is the first well characterized substrate for the vitamin K-dependent carboxylase found to be synthesized. The exceptionally broad tissue distribution for MGP synthesis demonstrates that the function of MGP is not specific to connective tissues, and the low levels of MGP antigen in soft tissues with high MGP mRNA levels indicate that MGP is unlikely to act solely by virtue of its accumulation in an extracellular matrix.     

The identification of matrix Gla protein in cartilage

The vitamin K-dependent bone protein matrix gamma-carboxyglutamic acid (Gla) protein (MGP) has been identified by radioimmunoassay in the guanidine extract of rat cartilage. MGP was present in all cartilages tested at levels comparable to the MGP level in bone. Western blot analysis indicated that the molecular weight of cartilage MGP is the same as bone MGP, and Northern blot analysis revealed that MGP mRNA from cartilage is the same size as the MGP mRNA from bone. The structurally related vitamin K-dependent protein bone Gla protein could not be detected in cartilage by radioimmunoassay or by Northern blot analysis. The discovery that MGP is synthesized by growth plate cartilage could provide an explanation for the excessive growth plate mineralization disorder seen in rats treated with the vitamin K antagonist warfarin and the punctate mineralization of the growth plate seen in infants whose mothers received warfarin in the first trimester of pregnancy (the fetal warfarin syndrome). Both disorders appear to be caused by the inactivation of a vitamin K-dependent mineralization inhibitor in cartilage, an inhibitor which we suggest is MGP.     

1,25-Dihydroxyvitamin D3 stimulates the synthesis of matrix gamma-carboxyglutamic acid protein by osteosarcoma cells. Mutually exclusive expression of vitamin K-dependent bone proteins by clonal osteoblastic cell lines

Several clonal rat osteosarcoma cell lines were tested for the ability to express and secrete matrix Gla protein (MGP), a small vitamin K-dependent protein found in bone and cartilage. Two independently derived cell lines, UMR 106-01 and ROS 25/1, expressed MGP mRNA and secreted MGP antigen identical in size with that found in bone. No MGP message could be detected in ROS 17/2 and 2/3 cells, cell lines previously shown to synthesize the other known vitamin K-dependent bone protein, bone Gla protein (BGP), and no BGP mRNA could be detected in the cell lines which synthesize MGP. Since UMR 106-01 and ROS 17/2 are presently the best characterized clonal osteoblastic cell lines, the discovery of the mutually exclusive expression of MGP and BGP by these cell lines indicates that osteosarcoma cells can be fixed in different phenotypic states and that MGP and BGP should be useful markers for the analysis of phenotypic expression in bone. Treatment of UMR 106-01 cells with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) dramatically increased MGP mRNA within 4 h and, by 24 h, increased MGP secretion 15-fold. This is only the second example of a bone matrix protein whose synthesis is dramatically increased by vitamin D, the first being the 6-fold stimulation of BGP synthesis by 1,25(OH)2D3 in ROS 17/2 cells. The discovery that MGP and BGP are similarily regulated by 1,25(OH)2D3 was unexpected since the two proteins differ markedly in structure, physical properties, and tissue distribution. Since the synthesis of MGP is rapidly and dramatically increased by 1,25(OH)2D3, it is probable that MGP plays a role in the normal bone response to the hormone. MGP may also be the vitamin K-dependent protein whose abnormal synthesis in the Warfarin-treated animal modifies the bone response to 1,25(OH)2D3.     

Primary structure of bovine matrix Gla protein, a new vitamin K-dependent bone protein

The complete amino acid sequence of bovine bone matrix Gla protein (MGP) was determined by automatic sequence analysis of the intact protein and of peptides isolated from tryptic and BNPS-skatole digests. This 79-residue, vitamin K-dependent protein contains a single disulfide bond and 4.8 gamma-carboxyglutamate (Gla) residues, one each at positions 37, 41, 48, and 52, and 0.8 Gla and 0.2 Glu at position 2. There is sufficient sequence homology between MGP and bone Gla protein (BGP) to indicate that these two bovine bone proteins arose by gene duplication and subsequent divergent evolution. Although MGP has a very low solubility in water compared to BGP, there is no hydrophobic domain in MGP which could account for its insolubility, and the overall fraction of hydrophobic residues is 32% for MGP compared to 43% for BGP. MGP is the first vitamin K-dependent protein to be discovered which has several non-gamma-carboxylated residues to the NH2-terminal side of its Gla residues. The presence of NH2-terminal Glu residues between the putative targeting domain for the gamma-carboxylase in the MGP leader sequence and the mid-molecule Gla residues suggests that the gamma-carboxylase may have additional, as yet unrecognized, specificity requirements which determine the susceptibility of Glu residues for gamma-carboxylation.     

Osteocalcin and matrix GLA protein in developing teleost teeth: identification of sites of mRNA and protein accumulation at single cell resolution

In this study, the tissue distribution and accumulation of osteocalcin or bone Gla protein (BGP) and matrix Gla protein (MGP) were determined during tooth development in a teleost fish, Argyrosomus regius. In this species, the presence of BGP and MGP mRNA in teeth was revealed by in situ hybridization. mRNA for BGP was detected in the odontoblasts as well as in its cytoplasmic processes emerging through dentinal tubules, while mRNA for MGP was expressed in the enamel portion within the apical portion of the elongated cell bodies of enameloblasts, adjacent to the root of the teeth as well as in cells within the pulpal space. Immunolocalization of BGP and MGP demonstrated that these proteins accumulate mainly in the mineralized dentin or in enameloblastic processes, confirming in situ hybridization results. In this study, we examined for the first time the localization of both BGP and MGP gene expression and protein accumulation within the different regions of the vertebrate tooth. We clearly demonstrated that although the overall pattern of BGP and MGP gene expression and protein accumulation in A. regius teeth was in general agreement to what is known for other vertebrates such as rats or rodents, our study provided novel information and highlighted some species-differences between fish and higher vertebrates.     

Evolution of matrix and bone gamma-carboxyglutamic acid proteins in vertebrates

The evolution of calcified tissues is a defining feature in vertebrate evolution. Investigating the evolution of proteins involved in tissue calcification should help elucidate how calcified tissues have evolved. The purpose of this study was to collect and compare sequences of matrix and bone gamma-carboxyglutamic acid proteins (MGP and BGP, respectively) to identify common features and determine the evolutionary relationship between MGP and BGP. Thirteen cDNAs and genes were cloned using standard methods or reconstructed through the use of comparative genomics and data mining. These sequences were compared with available annotated sequences (a total of 48 complete or nearly complete sequences, 28 BGPs and 20 MGPs) have been identified across 32 different species (representing most classes of vertebrates), and evolutionarily conserved features in both MGP and BGP were analyzed using bioinformatic tools and the Tree-Puzzle software. We propose that: 1) MGP and BGP genes originated from two genome duplications that occurred around 500 and 400 million years ago before jawless and jawed fish evolved, respectively; 2) MGP appeared first concomitantly with the emergence of cartilaginous structures, and BGP appeared thereafter along with bony structures; and 3) BGP derives from MGP. We also propose a highly specific pattern definition for the Gla domain of BGP and MGP.     

Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins,osteocalcin, and matrix Gla protein in vitro

The role of the vitamin K dependent proteins, osteocalcin which is bone specific and matrix Gla protein (MGP) found in many tissues, has been studied by inhibition of synthesis of their characteristic amino acid, γ-carboxyglutamic acid (Gla) with the anticoagulant sodium warfarin. The effect of sodium warfarin on expression of these proteins, and other phenotypic markers of bone and cartilage during cellular differentiation and development of tissue extracellular matrix, was examined in several model systems. Parameters assayed include cell growth (reflected by histone gene expression) and collagen types I and II, osteopontin, alkaline phosphatase, and mineralization. Studies were carried out in calvarial bone organ cultures, normal diploid rat osteoblast and chondrocyte cultures, and rat osteosarcoma cell lines ROS 17/2.8 and 25/1. In normal diploid cells, warfarin consistently stimulated cell proliferation (twofold). In osteoblast cultures, MGP mRNA levels were generally increased (three to tenfold). Notably, MGP mRNA levels were not affected in chondrocyte cultures, either with chronic or acute warfarin treatments. Osteocalcin mRNA levels and synthesis were decreased up to 50% in ROS 17/2.8 cells and in chronically treated (1 and 5 μg/ml sodium warfarin) rat osteoblast cultures after 22 days. Early stages of osteoblast phenotype development from the proliferation period to initial tissue formation (nodules) appeared unaffected; while after day 14, further growth and mineralization of the nodule areas were significantly decreased in warfarin-treated cultures. In summary, warfarin has opposing effects on the expression of two vitamin K dependent proteins, MGP and osteocalcin, in osteoblast cultures and MGP is regulated differently between cartilage and bone as reflected by cellular mRNA levels. Additionally, warfarin effects expression of nonvitamin K dependent proteins which may reflect the influence of warfarin on endoplasmic reticulum associated enzymes. © 1994 Wiley-Liss, Inc.     

Discovery of bone gamma-carboxyglutamic acid protein in mineralized scales. The abundance and structure of Lepomis macrochirus bone gamma-carboxyglutamic acid protein.

The mineralized scale of the freshwater sunfish Lepomis macrochirus (bluegill) contains a Gla protein. The protein was identified in extracts of scale by a new colorimetric assay for Gla-containing proteins. The protein was purified by gel filtration chromatography followed by reversed phase high performance liquid chromatography (HPLC). Several tests establish the identity of scale Gla protein and bone Gla protein (BGP). First, the proteins exhibit identical mobilities on electrophoresis and by reversed phase HPLC. Second, they have identical amino-terminal amino acid sequences. Finally, identical peptides are generated by proteolytic digestion. The 45-residue amino acid sequence of the bone Gla protein from L. macrochirus has a high sequence homology with swordfish, as well as homology to mammalian bone Gla protein. The BGP of bluegill shares with swordfish BGP a truncated NH2 terminus and an extended COOH terminus. These features may be unique to fish, as they have not been observed in terrestrial vertebrates. The bluegill BGP is the first vitamin K-dependent protein to contain a non-gamma-carboxylated residue to the NH2-terminal side of all of its Gla residues. In all other vitamin K-dependent proteins, Gla always appears to the NH2-terminal side of the first Glu. The implications of this result are discussed. The bluegill rib bone is curiously enriched in BGP, as are other mineralized tissues of this species. One hypothesis is that this may be due to the acellular nature of the bone in this species. The abundance of BGP in the bones of this fish may provide clues to the unknown function of this bone protein.     

Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone γ-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat

New aspects of the distribution and developmental appearance of the 44-kDa bone phosphoprotein (44K BPP, also called sialoprotein I or osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP, also called osteocalcin) during osteogenesis and dentinogenesis were investigated with immunocytochemical techniques using monospecific, affinity-purified polyclonal antibodies. Sections from newborn rat incisors and from various bone anlagen of newborn animals and fetuses were processed for detection of 44K BPP or BGP antigenicity. In addition, histochemical reactions for detection of alkaline phosphatase or calcium salts were performed on a number of the sections. The 44K BPP appears to be synthesized and secreted by chondrocytes only in the areas of cartilage-to-bone transition; these cells could participate indirectly in the process of bone formation by providing a suitable scaffold onto which primary marrow osteoblasts attach and spread. During osteogenesis, 44K BPP is found in bone-forming cells almost concomitantly with the appearance of alkaline phosphatase and before osteoid deposition, whereas BGP is still absent during early stages of mineralization. We hypothesize that this dramatic difference between the developmental appearance of 44K BPP and BGP reflects the delayed expression of the BGP gene relative to that of 44K BPP. In long-term cultures of bone marrow from adult mice, some fibroblastic cells expressed the 44K BPP phenotype; these cells could represent early osteogenic progenitor cells. Some experiments also suggested that, as with BGP, 44K BPP or an immunologically related protein is synthesized by some odontoblasts and secreted into predentin, prior to the onset of mineralization.     

Matrix Gla protein accumulates at the border of regions of calcification and normal tissue in the media of the arterial vessel wall.

Vitamin K-dependent matrix Gla protein (MGP) has been suggested to play a role in the inhibition of soft-tissue calcification. Here we report the expression of recombinant prokaryotic MGP as part of a fusion protein and the preparation of two antibodies that specifically recognize MGP. Monoclonal antibodies were raised against synthetic peptides homologous to the sequences 3-15 and 63-75 of human MGP. Both antibodies recognize recombinant and synthetic human MGP. Immunohistochemical analysis showed that MGP was associated with the extracellular matrix of noncalcified bone and with chondrocytes in cartilage. In the healthy human arterial vessel wall, MGP antigen was demonstrated in association with smooth muscle cells and elastic laminae of the tunica media and with the extracellular matrix of the adventitia. Colocalization with the elastic laminae was lost at sites of medial calcification; in both human and rat arteries, high amounts of MGP were found in the extracellular matrix at borders of intimal and medial calcification. Our data demonstrate the close association between MGP and calcification. It is suggested that undercarboxylated MGP is biologically inactive and that poor vascular vitamin K status may form a risk factor for vascular calcification.     

Carboxyl-terminal proteolytic processing of matrix Gla protein

The present study was undertaken to determine the extent of COOH-terminal proteolytic processing in matrix Gla protein (MGP), a 10-kDa protein which contains 5 residues of the vitamin K-dependent Ca2+ binding amino acid, gamma-carboxyglutamic acid (Gla). Two forms of MGP were isolated from demineralization and urea extracts of bovine cortical bone, one 79 residues in length with the COOH terminus Phe-Arg-Gln and the other 83 residues in length with the COOH terminus Phe-Arg-Gln-Arg-Arg-Gly-Ala. The 84-residue form of bovine MGP predicted from the message structure could not be detected in the bone extracellular matrix extracts, and it therefore seems probable that the lysine at position 84 was removed by the action of a carboxypeptidase B-like enzyme prior to secretion. A plausible sequence of proteolytic cleavages that could generate the 79-residue form of MGP would be a trypsin-like cleavage at Arg80-Arg81 or Arg81-Gly82 followed by carboxypeptidase B-like cleavage to remove COOH-terminal arginine(s). Since essentially equal amounts of the 79- and 83-residue forms of MGP were also detected in bovine articular cartilage and plasma, it seems likely that the COOH-terminal processing events identified in bone apply to many of the other tissues which synthesize this protein. Only one form of MGP was detected in human bone extracts, a 77-residue protein that lacks the COOH-terminal residues Arg-Lys-Arg-Arg-Gly-Thr-Lys. This shortened version of human MGP is consistent with the proposed model for COOH-terminal processing, since the amino acid substitution in the COOH terminus of the human protein, Lys79 for Gln79, would allow removal of the additional basic residues from the human MGP COOH terminus by the action of the carboxypeptidase B-like enzymic activity. Recent studies have shown that MGP is strongly induced by retinoic acid in fibroblasts, chondrocytes, and osteoblasts, a response which suggests that MGP mediates an action of retinoic acid on an aspect of cell growth or differentiation. If this hypothesis is true, the present evidence for complex COOH-terminal processing events could provide a means to regulate the as yet unknown activity of MGP in the extracellular environment in a mechanism similar to the activation of hormones such as anaphlotoxins and kinins.     

Cell adhesion to matrix Gla protein and its inhibition by an Arg-Gly-Asp-containing peptide.

Matrix Gla protein (MGP) is a 14-kDa protein found in bone and cartilage which contains the unusual amino acid gamma-carboxyglutamic acid (Gla). The biological function of this protein has not been elucidated. Here we have demonstrated the adherence of chondrocytes, fibroblasts, osteosarcoma cells, and kidney mesangial cells to MGP purified from bovine bone. Maximum adherence occurred at MGP concentrations of 0.5-1.0 micrograms/ml. Removal of the calcium-binding Gla residues by thermal decarboxylation of MGP destroyed the proteins' cell adherence properties. Cell adherence to MGP was not affected by the presence of antibodies directed against the C-terminal (non-Gla) portion of the protein or the presence of cycloheximide during the adherence assay. However, the Arg-Gly-Asp-containing synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro significantly inhibited cell attachment to MGP, whereas the control peptide Gly-Arg-Gly-Glu-Ser-Pro had minimal effect. These data indicate that MGP may function in mediating cell attachment to the extracellular matrix via a receptor that requires intact Gla residues and that can be inhibited by Arg-Gly-Asp-containing peptides.     

Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival

Matrix Gla protein (MGP) is a 14-kD extracellular matrix protein of the mineral-binding Gla protein family. Studies of MGP-deficient mice suggest that MGP is an inhibitor of extracellular matrix calcification in arteries and the epiphyseal growth plate. In the mammalian growth plate, MGP is expressed by proliferative and late hypertrophic chondrocytes, but not by the intervening chondrocytes. To investigate the functional significance of this biphasic expression pattern, we used the ATDC5 mouse chondrogenic cell line. We found that after induction of the cell line with insulin, the differentiating chondrocytes express MGP in a stage-specific biphasic manner as in vivo. Treatment of the ATDC5 cultures with MGP antiserum during the proliferative phase leads to their apoptosis before maturation, whereas treatment during the hypertrophic phase has no effect on chondrocyte viability or mineralization. After stable transfection of ATDC5 cells with inducible sense or antisense MGP cDNA constructs, we found that overexpression of MGP in maturing chondrocytes and underexpression of MGP in proliferative and hypertrophic chondrocytes induced apoptosis. However, overexpression of MGP during the hypertrophic phase has no effect on chondrocyte viability, but it does reduce mineralization. This work suggests that coordinated levels of MGP are required for chondrocyte differentiation and matrix mineralization.     

Bone Gla protein messenger ribonucleic acid is regulated by both 1,25-dihydroxyvitamin D3 and 3',5'-cyclic adenosine monophosphate in rat osteosarcoma cells

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] regulates the synthesis of bone gamma-carboxyglutamic acid (Gla) protein (BGP) by osteoblastic cells. In this study we examined the effect of cAMP, alone and in combination with 1,25-(OH)2D3, on the regulation of BGP mRNA levels in ROS 17/2 rat osteosarcoma cells. Elevation of intracellular cAMP levels by cAMP analogs or by isobutylmethylxanthine (IBMX), forskolin, or PTH, resulted in increased BGP mRNA levels and BGP secretion after 1 day of treatment. The effects of these agents were additive with 1,25-(OH)2D3 in stimulating BGP gene expression. After 4 days of treatment, pertussis toxin (PT) and 1,25-(OH)2D3 were synergistic in stimulating BGP mRNA, and the effect of PT could be mimicked by (Bu)2cAMP, IBMX, forskolin, cholera toxin, and to a lesser extent by PTH. The effect of 1-day treatment with cAMP alone and the synergistic effect with 1,25-(OH)2D3 on the stimulation of BGP mRNA were dependent on cell density, while basal and 1,25-(OH)2D3-stimulated synthesis were not. Cyclic AMP inhibited ROS 17/2 cell growth after 1 day of treatment, an effect that was also dependent on initial cell density. After 4 days of treatment, 1,25-(OH)2D3, cAMP, and PT all demonstrated inhibition of cell growth. When cells were treated with actinomycin D, both 1,25-(OH)2D3 and cAMP stimulation of BGP mRNA were blocked. In addition, neither agent was effective in enhancing BGP mRNA stability when prestimulated cells were exposed to actinomycin D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucocorticoid effects on vitamin K-dependent carboxylase activity and matrix Gla protein expression in rat lung

The role of glucocorticoids in the regulation of vitamin K-dependent carboxylase activity was investigated in fetal and adult lung. Glucocorticoid deficiency induced by adrenalectomy (ADX) stimulated adult lung growth and reduced carboxylation in a tissue-specific manner. Type II epithelial cells were enriched in carboxylase activity, where ADX-induced downregulation was retained in freshly isolated cells. Carboxylase activity in fetal type II cells was one-half that found in fetal fibroblasts isolated from the same lungs, and both populations increased activity with time in culture. Both carboxylase activity and formation of gamma-carboxyglutamate (Gla)-containing proteins were stimulated by dexamethasone (Dex) in fetal type II cells. Matrix Gla protein (MGP), a vitamin K-dependent protein known to be synthesized in type II cells, was also found in fetal fibroblasts, where its expression was stimulated by Dex. These combined results suggested an important role for glucocorticoids and MGP in the developing lung, where both epithelial and mesenchymal cells coordinate precise control of branching morphogenesis. We investigated MGP expression and its regulation by Dex in the fetal lung explant model. MGP mRNA and protein were increased in parallel with the formation of highly branched lungs, and this increase was stimulated twofold by Dex at each day of culture. Dex-treated explants were characterized by large, dilated, conducting airways and a peripheral rim of highly branched saccules compared with uniformly branched controls. We propose that glucocorticoids are important regulators of vitamin K function in the developing and adult lung.     

Discovery of a high molecular weight complex of calcium, phosphate, fetuin, and matrix gamma-carboxyglutamic acid protein in the serum of etidronate-treated rats.

In the present study we report the discovery of a novel protein-mineral complex in the serum of rats treated with doses of the bone-active bisphosphonate etidronate that inhibit normal bone mineralization. The composition of this high molecular mass protein-mineral complex consists of about 18% mineral, 80% fetuin, and 2% matrix Gla protein (MGP) by weight, and the presence of the complex in serum after an injection of 8 mg etidronate/100 g of body weight elevates calcium by 1.8-fold (to 4.3 mm), phosphate by 1.6-fold (to 5.6 mm), and MGP by 25-fold (to 12 microg/ml). The serum mineral complex reaches maximal levels at 6 h after subcutaneous injection of etidronate and is subsequently cleared from serum by 24 h. This highly specific complex of fetuin, MGP, and mineral prevents the growth, aggregation, and precipitation of the mineral component, which indicates that the previously reported calcification inhibitory activities of fetuin and MGP may be related to their ability to form stable complexes with nascent mineral nuclei. Treatment with the vitamin K-antagonist warfarin prevents the increase in serum MGP after etidronate injection, which shows that the increase in serum MGP is due to new synthesis and that the gamma-carboxylation of MGP is necessary for its binding to the serum mineral complex.