Matrix Gla Protein Gene Expression Research Articles

Assessment of MGP gene expression in cancer and contribution to prognosis

Matrix Gla protein (MGP) was first identified as a calcification physiological inhibitor and the causal agent of the Keutel syndrome. MGP has been suggested to play a role in development, cell differentiation, and tumorigenesis. This study aimed to compare MGP expression and methylation status in different tumors and adjacent tissues, using The Cancer Genome Atlas (TCGA) data repository. We investigated if changes in MGP mRNA expression were correlated to cancer progression and whether the correlation coefficients could be used for prognosis. Strong correlations were observed between altered MGP levels and disease progression in breast, kidney, liver, and thyroid cancers, suggesting that it could be used to complement current clinical biomarker assays, for early cancer diagnosis.We have also analyzed MGP methylation and identified CpG sites in its promoter and first intron with clear differences in methylation status between healthy and tumoral tissue providing evidence for epigenetic regulation of MGP transcription. Furthermore, we demonstrate that these alterations correlate with the overall survival of the patients suggesting that its assessment can serve as an independent prognostic indicator of patients’ survival.

Relationship between matrix Gla protein and clinical features of ulcerative colitis

Objective: To analyze the relationship between matrix Gla protein(MGP) and clinical characteristics of patients with ulcerative colitis (UC). Methods: Fifty-one UC patients who were admitted to the gastroenterology department of Peking union medical college hospital from July 1, 2015 to May 31, 2017 were included. Twenty-seven healthy subjects in the same period were included as normal controls. The expression of MGP mRNA in the colonic mucosa was detected by real-time fluorescence quantitative PCR. Clinical data of the patients were collected during a 2-year follow-up. The public data set containing MGP gene expression profile of UC patients was downloaded from the GEO database, and was divided into four groups according to the microscopic Mayo score. The differential expression of MGP in each group was analyzed. Results: All the fifty-one UC patients were followed up. The expression of MGP mRNA in the colonic mucosa of UC patients treated with hormone and immunosuppressive agents or biological agents or surgery was higher than that of patients treated with mesalazine. MGP mRNA expression was positively correlated with C-reactive protein level. It was also higher in the colonic mucosa of UC patients with clostridium difficilis or cytomegalovirus infection than that of patients without opportunistic infection. The difference of MGP mRNA expression between groups in GEO public data set was statistically significant(P<0.01), showing an up-regulation trend with the aggravation of inflammation. The expression level of MGP was moderately correlated with the microscopic Mayo score. The relationship between MGP mRNA expression and lifestyle, lesion range, erythrocyte sedimentation rate, parenteral manifestations and recurrence frequency of UC patients was not obvious. Conclusions: MGP is associated with colonic inflammation and its abnormal expression can help to predict the disease activity of patients with UC.

623: Decreased myometrial expression of matrix-Gla protein (MGP) is associated with preterm and term laboring state

Uterine contractions are driven by complex calcium mediated mechanisms. Matrix Gla-protein (MGP) is an emerging vitamin K2-dependent protein with key roles in calcium homeostasis and inhibition of smooth muscle calcification. We sought to study MGP expression to provide new insight into the physiology of term and preterm contracting human myometrium. Myometrial expression of MGP was investigated in the following groups: 1) term labor (TL, n=5, GA: 39); 2) term non-labor (TNL, n=5, GA: 39); 3) preterm labor (PTL, n=5, GA: 28); 4) preterm non-labor (PTNL, n=13, GA: 29). Serum levels of MGP were measured by ELISA. Myometrial expression was assessed using immunohistochemistry and RNAseq with qRT-PCR validation. MGP and calcium-related proteins were localized with immunofluorescence in pregnant (PHM1) and non-pregnant (hTERT) human myometrial cell lines. 1) Pregnant myometrial cell lines had greater expression of MGP compared to non-pregnant state (Figure A). 2) At term, maternal serum MGP levels were unaffected by labor. 3) Cytoplasm of human myocytes display MGP immunostaining constitutively. 4) By qRT-PCR, we identified significantly higher levels of MGP gene expression in non-laboring myometrium compared to laboring preterm (p=0.002) and term (p=0.0004) tissues (Figure B). 5) This expression pattern was paralleled in mRNAseq of term (p=0.003) and preterm (p=0.002) uterus. 6) Immunocytochemistry showed that MGP colocalized with smooth-muscle actin monofilaments, but not calcium exchangers (SERCA, PMCA1) and Golgi marker. Our study presents the novel findings that MGP is present in human myometrium, and its tissue withdrawal is associated with a laboring state.

Functional Role of Matrix gla Protein in Glioma Cell Migration.

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor subtype. Despite that metastasis of GBM beyond the central nervous system (CNS) is rare, its malignancy is attributed to the highly infiltration trait, leading to the difficulty of complete surgical excision. Matrix gla protein (MGP) is a vitamin K-dependent small secretory protein, and functions as a calcification inhibitor. The involvement of MGP function in glioma cell dynamics remains to be clarified. The study showed that a low proliferative rat C6 glioma cell line named as C6-2 exhibited faster migratory and invasive capability compared to that observed in a high tumorigenic rat C6 glioma cell line (called as C6-1). Interestingly, C6-2 cells expressed higher levels of MGP molecules than C6-1 cells did. Lentivirus-mediated short hairpin RNA (shRNA) against MGP gene expression (MGP-KD) in C6-2 cells or lentivirus-mediated overexpression of MGP transcripts in C6-1 cells resulted in the morphological alteration of the two cell lines. Moreover, MGP-KD caused a decline in cell migration and invasion ability of C6-2 cells. In contrast, increased expression of MGP in C6-1 cells promoted their cell migration and invasion. The observations were further verified by the results from the implantation of C6-1 and C6-2 cells into ex vivo brain slice and in vivo rat brain. Thus, our results demonstrate that the manipulation of MGP expression in C6 glioma cells can mediate glioma cell migratory activity. Moreover, our findings indicate the possibility that high proliferative glioma cells expressing a high level of MGP may exist and contribute to tumor infiltration and recurrence.

Selenium levels in early weaning diets for gilthead seabream larvae

Abstract The inclusion of complementary antioxidative factors, such as selenium (Se), could counteract the high oxidation risk in early weaning diets high in polyunsaturated fatty acids (PUFA). The present study investigated the effects of graded levels of Se derived yeast with krill phospholipids (KPL) on skeletal development, survival, stress resistance, oxidative status and biochemical composition of seabream larvae. Seabream larvae were completely weaned at 16 dph and fed five microdiets for 30 days with different levels of Se: 2SE, 4SE, 6SE, 8SE and 12SE (1.73, 3.91, 6.41, 8.47, 11.65 mg kg− 1 dietary dry weight, respectively). Increases in Se up to 11.65 mg kg− 1 dietary dry weight significantly improved survival rate (54%) and stress resistance, but did not affect larval growth. Seabream larvae fed diets supplemented with 12SE (11.65 mg kg− 1) showed a gradual increase in this mineral according to dietary Se levels, denoting the progressive absorption of this nutrient. The degree of larval lipid oxidation, as indicated by malondialdehyde (MDA) content and antioxidant enzyme (AOE) gene expression, was significantly lower in larvae fed 8SE and 12SE diets compared to those fed 2SE and 4SE diets. Furthermore, a reactive response as a result of Se inclusion was observed by the increase in osteocalcin, osteonectin, osteopontin, alkaline phosphatase and matrix gla protein gene expression in larval tissues, suggesting a well skeletal development. These results denoted the high efficiency of Se as an antioxidant factor and the importance of the inclusion of adequate levels (11.65 mg Se kg− 1 diet) in early weaning diets.

Molecular characterization and methylation study of matrix gla protein in articular cartilage from pig with osteochondrosis

Osteochondrosis (OC) or leg weakness is an economically important disease of young fast growing pigs and is a concern of animal welfare. The etiology and pathogenesis of osteochondrosis is not fully understood yet, but any abnormalities in the formation of hypertrophic chondrocytes and disrupted blood supply to the growth cartilage are very important predisposing factors. Matrix gla protein (MGP) as a potential calcification inhibitor of extracellular matrix might contribute to the development of OC. Molecular characterization, polymorphisms analysis, methylation at promoter region and expression of MGP gene and protein were performed in both healthy and OC cartilage collected from a Duroc × Pietrain resource population. The porcine MGP gene consists of 4 exons and 3 introns. The full-length MGP cDNA isolated from articular cartilage consists of 606 bp with a 69-bp 5′ UTR, a 312-bp open reading frame with a start codon, a 225-bp 3′ UTR. Three single-nucleotide polymorphisms (SNP) were detected in the intron 1 (A-115G, C-1073T and C-1135A) and one in the 3′UTR (C-3767T). The relative abundance of MGP mRNA was lower ( P < 0.05) in OC compared with healthy cartilage. Moreover, the intensity of MGP band was lower ( P < 0.05) in OC group when quantified by western blot. Furthermore, one CpG region was identified in MGP promoter and DNA methylation of three CG sites were higher in OC compared with normal cartilage. This suggested that the high DNA methylation at specific CG sites in the MGP promoter might be involved in the down regulation of MGP in OC. Immunofluorescence of normal cartilage collected from pigs of different ages revealed that MGP signals were higher in younger pigs and decreased in the older pigs. The MGP protein was expressed more near to the cartilage canals. These results suggest that the MGP gene might be a potential candidate gene for the development of OC in pigs.

Upregulation of cardiac matrix Gla protein expression in response to hypertrophic stimuli

Matrix Gla protein (MGP) expression is increased in cardiac hypertrophy, but the precise mechanisms regulating its expression are unknown. Here we characterized the effect of pressure overload and myocardial infarction in vivo as well as mechanical stretch and hypertrophic agonists in vitro on MGP expression. When angiotensin II (Ang II) was administered by osmotic minipumps, left ventricular (LV) MGP mRNA levels increased significantly from 6 h to 2weeks, whereas intravenous arginine8-vasopressin increased LV MGP mRNA levels within 4 h. During post-infarction remodeling process, MGP mRNA levels were elevated at 24 h (1.3-fold, p<0.05) and the maximal increase was observed at 4 weeks (2.8-fold, p<0.01). Ang II increased MGP mRNA levels 20% (p<0.05) in neonatal rat cardiac myocytes and 40% (p<0.05) in cardiac fibroblasts, whereas endothelin-1 decreased MGP mRNA levels 30% (p<0.01) in myocytes and had no effect in fibroblasts. Cyclic mechanical stretch resulted in reduction of MGP gene expression in both cardiac myocytes and fibroblasts. These results demonstrate that MGP is rapidly upregulated in response to cardiac overload well before the development of LV hypertrophy and post-infarction remodeling process. Our results also suggest that Ang II may be involved in mediating load-induced activation of MGP expression.

Retinoic acid is a negative regulator of matrix Gla protein gene expression in teleost fish Sparus aurata

Matrix Gla protein (MGP) is an extracellular mineral-binding protein expressed in several tissues while accumulated only in bone and cartilage under physiological conditions. Although the precise molecular mechanism of action of MGP remains unknown, all available evidence indicates that it acts as a physiological inhibitor of mineralization. This work presents the cloning of gilthead seabream MGP gene (SaMGP) and the functional analysis of its promoter. SaMGP gene was found to be organized in five exons and to be under control of a distal and a proximal promoter, both, capable of activating SaMGP transcription in transient transfections. Furthermore, we present strong evidence that retinoic acid down-regulates SaMGP gene transcription by interacting, through binding of its receptor, with a specific region within distal promoter. Interestingly, the presence of repetitive motifs in the proximity of SaMGP gene regulatory regions suggests that they may modulate promoter accessibility to transcription machinery, as already seen for other genes. This work provides additional evidence of the usefulness of non-mammalian model systems to elucidate the complex regulation of MGP gene transcription.

Cloning of matrix Gla protein in a marine cartilaginous fish, Prionace glauca: preferential protein accumulation in skeletal and vascular systems

Matrix Gla protein (MGP) belongs to the family of vitamin K dependent, Gla containing proteins and, in mammals, birds and Xenopus, its mRNA has been previously detected in bone, cartilage and soft tissue extracts, while the accumulation of the protein was found mainly in calcified tissues. More recently, the MGP gene expression was also studied in marine teleost fish where it was found to be associated with chondrocytes, smooth muscle and endothelial cells. To date no information is available on the sites of MGP expression or accumulation in cartilaginous fishes that diverged from osteichthyans, a group that includes mammals, over 400 million years ago. The main objectives of this work were to study the sites of MGP gene expression and protein accumulation by means of in situ hybridization and immunohistochemistry. MGP mRNA and protein were localized as expected not only in cartilage from branchial arches and vertebra but also in the endothelia of the vascular system as well as in the tubular renal endothelium. The accumulation of MGP in non mineralized soft tissues was unexpected and suggests differences in localization or regulation of this protein in shark soft tissues compared to tetrapods and teleosts. Our results also corroborate the hypothesis that in Prionace glauca, as previously shown in mammals, the MGP protein probably also acts as a calcification inhibitor, protecting soft tissues from abnormal and ectopic calcification.

Thyroid Hormone Targets Matrix Gla Protein Gene Associated With Vascular Smooth Muscle Calcification

Thyroid hormones have marked cardiovascular effects in vivo. However, their direct effects on vascular smooth muscle cells have been unclear. Because thyroid hormones play critical roles in bone remodeling, we hypothesized that they are also associated with vascular smooth muscle calcification, one of the pathological features of vascular sclerosis. To test this hypothesis, we examined the effects of 3',3,5-triiodo-L-thyronine (T3) on the expression of calcification-associated genes in rat aortic smooth muscle cells (RAOSMCs). Quantitative RT-PCRs revealed that a physiological concentration of T3 (15 pmol/L free T3) increased mRNA level of matrix Gla protein (MGP), which acts as a potent inhibitor of vascular calcification in vivo, by 3-fold in RAOSMCs, as well as in cultured human coronary artery smooth muscle cells. In RAOSMCs transiently transfected with a luciferase reporter gene driven by the MGP promoter, T3 significantly stimulated luciferase activity. In addition, RNA interference against thyroid hormone receptor-alpha gene diminished the effect of T3 on MGP expression. Aortic smooth muscle tissues from methimazole-induced hypothyroid rats (400 mg/L drinking water; 4 weeks) also showed a 68% decrease in the MGP mRNA level, as well as a 33% increase in calcium content compared with that from the control euthyroid animals, whereas hyperthyroidism (0.2 mg T3/kg IP; 10 days) upregulated MGP mRNA by 4.5-fold and reduced calcium content by 11%. Our findings suggest that a physiological concentration of thyroid hormone directly facilitates MGP gene expression in smooth muscle cells via thyroid hormone nuclear receptors, leading to prevention of vascular calcification in vivo.

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.

Matrix Gla protein gene expression and protein accumulation colocalize with cartilage distribution during development of the teleost fish Sparus aurata

Matrix Gla protein (MGP) is a member of the family of extracellular mineral-binding Gla proteins, expressed in several tissues with high accumulation in bone and cartilage. Although the precise molecular mechanism of action of this protein remains unknown, all available evidence indicates that MGP plays a role as an inhibitor of mineralization. We investigated the sites of gene expression and protein accumulation of MGP throughout development of the bony fish Sparus aurata, by in situ hybridization, Northern and RT-PCR Southern hybridization, and immunohistochemistry. The results obtained were compared with the patterns of developmental appearance of cartilaginous and mineralized structures in this species, identified by histological techniques and by detection of mRNA presence and protein accumulation of osteocalcin (Bone Gla protein), a marker for osteoblasts known to accumulate in bone mineralized extracellular matrix. The expression of MGP mRNA was first detected at 2 days posthatching (dph) by Northern analysis, RT-PCR amplification, and in situ hybridization, and thereafter continuously detected at various levels of intensity, until 130 dph. In situ hybridization analysis performed in parallel with immunohistochemistry indicated that until ca. 45 dph, the MGP gene was highly expressed in a number of different tissues including skull, jaw, neural and hemal arches, and heart and the protein accumulated in cartilaginous tissues. At 85 dph, a stage when most skeletal structures are mineralized, MGP gene expression and protein accumulation were restricted to the remaining cartilaginous structures, whereas osteocalcin gene expression and protein accumulation were localized in most mineralized structures. MGP gene expression was also detected in heart and kidney, although in situ hybridization only detected MGP mRNA in heart, located in the arterial bulbus and not in the cardiac muscle. Our results are in agreement with those recently described for MGP localization in adult tissues of another teleost fish, as well as available data from higher vertebrates, strengthening the hypothesis of a conserved function for MGP from teleost fish to human, a period of more than 200 million years of evolution. In addition, Sparus aurata, a marine teleost fish routinely grown in captivity, appears to be a good model to further analyze MGP gene expression and regulation.

Purification of matrix Gla protein from a marine teleost fish, Argyrosomus regius: calcified cartilage and not bone as the primary site of MGP accumulation in fish.

Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius, separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5'rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non-osteocytic teleost fish, but only in calcified cartilage. In addition, our results also indicate that the presence of MGP mRNA in heart tissue is due, at least in fish, to the expression of the MGP gene in only two specific cell types, smooth muscle and endothelial cells, whereas no expression was found in the striated muscle fibers of the ventricle. In light of these results and recent information on expression of MGP gene in these same cell types in mammalian aorta, it is likely that the levels of MGP mRNA previously detected in Xenopus, birds, and mammalian heart tissue may be restricted to regions rich in smooth muscle and endothelial cells. Our results also emphasize the need to re-evaluate which cell types are involved in MGP gene expression in other soft tissues and bring further evidence that fish are a valuable model system to study MGP gene expression and regulation.

Basic fibroblast growth factor as a selective inducer of matrix Gla protein gene expression in proliferative chondrocytes.

Matrix Gla protein (MGP) is a member of the vitamin K-dependent gamma carboxylase protein family expressed in cartilage. Insulin-like growth factor I (IGF1) stimulates chondrocyte differentiation, whereas basic fibroblast growth factor (FGF2) acts in an opposite manner. We explored the differential expression and regulation by IGF1 and FGF2 of the MGP gene during chondrocyte differentiation. We used a primary culture system of rabbit epiphyseal chondrocytes to show that MGP mRNA is mainly expressed during serum-induced proliferation. Much lower MGP mRNA content is observed in post-mitotic chondrocytes, which newly express alpha 1X procollagen mRNA, a marker of late-differentiated cells. From studies of a series of growth factors, it was shown that IGF1 decreased chondrocyte MGP transcripts, whereas FGF2 had the opposite effect. FGF2 stimulated chondrocyte MGP production in a dose- and time-dependent manner at the mRNA and protein levels. FGF2 acted in a dose- and time-dependent manner, reaching a maximum at 10 ng/ml at 20 h. The protein synthesis inhibitor cycloheximide did not modify FGF2 action, in agreement with a direct effect. Actinomycin D abolished FGF2-induced stimulation, strongly suggesting that FGF2 modulated MGP gene transcription. We transiently transfected chondrocytes with a construct containing the mouse MGP promoter from -5000 to -168 base pairs, relative to the transcription start site of the gene linked to the luciferase gene (MGP-Luc). In transfected cells, FGF2 stimulated luciferase activity up to sevenfold while IGF1 had no effect. Hence, FGF2 induces transcription of the MGP gene via the 5'-flanking region of the gene. Using a series of deleted MGP-Luc constructs, we identified a sequence of 748 base pairs which was sufficient for transcriptional activation by FGF2. These results led us to postulate that the inhibitory chondrogenic action of FGF2 involves a mechanism whereby MGP gene transcription and protein are induced.

Down-regulation of matrix Gla protein messenger RNA in human colorectal adenocarcinomas

Matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein commonly found in a variety of tissues. In this study, we describe the potential use of MGP gene expression as the tumor marker of colorectal cancer. A decrease in expression of the MGP gene was also discovered in colorectal cancer using differential screening of cDNA libraries. The MGP expression in 80 human colorectal adenocarcinomas was quantified by a Northern blot analysis to better define the expression pattern of MGP in colorectal cancer. The expression of MGP mRNA was reduced in 63 of 80 (79%) colorectal adenocarcinomas ( P<0.001) as compared to the mRNA in adjacent normal tissue, implying that a decrease in MGP expression is associated with colorectal cancer development. The proportion of tumors with downregulated expression of MGP was lower in Duke's A/B than Duke's C/D (34 of 47 versus 26 of 33, respectively) tumors and was lower in moderate differentiation than poor differentiation (44 of 64 versus 16 of 16, respectively). However, χ 2 analysis does not reveal any correlation between a loss of MGP expression and tumor progression or differentiation state. In conclusion, the downregulation of MGP mRNA generally occurs in colorectal adenocarcinomas. Although the role of MGP in cancer development is unknown, the reduced expression of MGP may be used to distinguish the normal colorectal cells from malignant cells.

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb.

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

Expression of matrix Gla protein (MGP) in an in vitro model of vascular calcification

To investigate the role of matrix Gla protein (MGP), which can bind mineral ions through γ-carboxylated glutamic acid residues, in vascular calcification, we examined the expression of MGP in an in vitro calcification model by using bovine vascular smooth muscle cells (BVSMC). The expression of MGP mRNA was decreased during BVSMC calcification and its levels were inversely correlated with the quantities of BVSMC calcification. MGP mRNA expression was restored to the level of uncalcified control by inhibiting BVSMC calcification with bisphosphonates. These data suggest that the expression of MGP gene is modulated in the development of vascular calcification.

Gene expression of noncollagenous bone matrix proteins in the limb joints and intervertebral disks of the twy mouse.

The twy (tiptoe walking Yoshimura) mouse is an autosomal recessive mutant manifesting multiple osteochondral lesions characterized by pathologic calcium deposition. To elucidate the pathophysiology of the limb joint lesions and the intervertebral disk lesions of the twy mouse, we assessed the mRNA expression of noncollagenous bone matrix proteins such as osteocalcin, osteonectin, osteopontin, and matrix Gla protein (MGP) by in situ hybridization, but only expression of MGP was observed in association with the pathologic calcium deposits in twy mice. Mild degeneration and abnormal growth of the cartilage in contact with the joint capsule was observed at 5 weeks in the articular cartilage of the ankle joint of the twy mouse, and MGP gene expression was observed at the same time. Simultaneous growth of synovial membrane cells and relatively undifferentiated articular cartilage cells in the knee joint, and of cartilage-like cells near the insertion of the cruciate ligament was observed in the twy mouse, and MGP gene expression was found to be present at the same time. Hypertrophy of abnormally proliferated chondrocyte-like cells, which are different from fibrocartilaginous cells of the annulus fibrosus, was observed in the intervertebral disks of the twy mouse at 3 weeks of age, and MGP gene expression was noted at the same time. These findings suggest that abnormal expression of MGP plays a major role in the pathologic calcification of the twy mouse.

Matrix Gla protein gene expression is induced by transforming growth factor-beta in embryonic lung culture.

Matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein with a wide tissue distribution. The current study was designed to investigate the possible regulation of MGP by exogenous transforming growth factor-beta (TGF-beta) during lung development. Using reverse transcription coupled competitive polymerase chain reaction methodology, we determined that exogenous TGF-beta 1 increases MGP mRNA levels in embryonic mouse lung culture in a concentration-dependent manner. MGP mRNA levels were elevated by 5.0-fold at 50 ng/ml TGF-beta 1 in E11 embryonic mouse lungs explanted for 4 days in serumless culture. MGP mRNA stimulation by TGF-beta 1 was a time-dependent event: MGP message increment was initially detected after 1 day in culture, and MGP mRNA levels continued to increase up to 4 days in the presence of TGF-beta 1. In addition, embryonic lungs in serumless medium without exogenously added TGF-beta 1 showed an increase, although to a lesser extent, in pulmonary MGP mRNA level during culture, indicating that MGP expression is also developmentally regulated. The present results indicate that MGP gene expression can be stimulated by exogenous TGF-beta 1 during early embryonic mouse lung branching morphogenesis in culture.

Identification of a novel negative retinoic acid responsive element in the promoter of the human matrix Gla protein gene.

The vitamin K-dependent matrix Gla protein (MGP) is synthesized in a wide variety of tissues such as lung, heart, kidney, cartilage, and bone. Expression of the MGP gene is regulated by various growth factors, steroid hormones, and the vitamin A metabolite retinoic acid (RA). In this report, we present evidence that RA down-regulates MGP gene expression in different rat and human cell lines via endogenous retinoid receptors [RA receptor (RAR) and retinoid X receptor (RXR)]. Repression of the human MGP (hMGP) gene is specifically mediated by ligand-activated RAR and RXR. Deletion analysis led to the identification of a novel negative response element (NRE) within the hMGP promoter. DNA binding studies performed with bacterially expressed RAR/RXR reveal the formation of a specific heterodimer/NRE complex. Furthermore, electrophoretic mobility-shift assays performed with proteins from RA-treated cells show that endogenous RAR/RXR binds to the NRE. We demonstrate that the NRE contains a CCAAT box and that both RAR/RXR and CCAAT-binding proteins such as c/EBP beta recognize this common regulatory sequence in the hMGP promoter. Our results indicate that RA-mediated repression of the hMGP gene is due to binding of liganded RAR/RXR to a novel negative RA response element.