Function Of Matrix Gla Protein Research Articles

Randomized Controlled Clinical Trial of the Effect of Treatment with Vitamin K2 on Vascular Calcification in Hemodialysis Patients (Trevasc-HDK)

Vitamin K deficiency among patients on hemodialysis (HD) affects the function of matrix GLA protein (MGP), a potent vitamin K-dependent inhibitor of vascular calcification (VC). We conducted a single-center randomized controlled trial (RCT) on maintenance HD patients to examine if vitamin K2 supplementation can reduce progression of coronary artery calcification (CAC) over an 18-month study period. Patients were randomized to vitamin K2 group receiving menaquinone-7360 μg 3 times/wk or control group. The primary outcome was CAC scores at the end of the study period. The secondary outcomes were aortic valve calcification (AVC), carotid-femoral pulse wave velocity (cfPWV), aortic augmentation index (AIx), dephosphorylated undercarboxylated MGP (dp-ucMGP) levels, major adverse cardiac events (MACE), and vascular access events. Of the 178 patients randomized, follow-up was completed for 138 patients. The CAC scores between the 2 groups were not statistically different at the end of 18 months (relative mean difference [RMD] 0.85, 95% CI 0.55-1.31). The secondary outcomes did not differ significantly in AVC (RMD 0.82, 95% CI 0.34-1.98), cfPWV (absolute mean difference [AMD] 0.55, 95% CI-0.50 to 1.60), and AIx (AMD 0.13, 95% CI-3.55 to 3.80). Supplementation with vitamin K2 did reduce dp-ucMGP levels (AMD-86, 95% CI-854 to-117). The composite outcome of MACE and mortality was not statistically different between the 2 groups (Hazard ratio= 0.98, 95% CI 0.50-1.94). Our study did not demonstrate a beneficial effect of vitamin K2 in reducing progression of VC in this population at the studied dose and duration.

Prevention of Arterial Elastocalcinosis: Differential Roles of the Conserved Glutamic Acid and Serine Residues of Matrix Gla Protein.

Inactivating mutations in matrix Gla protein (MGP) lead to Keutel syndrome, a rare disease hallmarked by ectopic calcification of cartilage and vascular tissues. Although MGP acts as a strong inhibitor of arterial elastic lamina calcification (elastocalcinosis), its mode of action is unknown. Two sets of conserved residues undergoing posttranslational modifications-4 glutamic acid residues, which are γ-carboxylated by gamma-glutamyl carboxylase; and 3 serine residues, which are phosphorylated by yet unknown kinase(s)-are thought to be essential for MGP's function. We pursued a genetic approach to study the roles of MGP's conserved residues. First, a transgenic line (SM22a-GlamutMgp) expressing a mutant form of MGP, in which the conserved glutamic acid residues were mutated to alanine, was generated. The transgene was introduced to Mgp-/- mice to generate a compound mutant, which produced the mutated MGP only in the vascular tissues. We generated a second mouse model (MgpS3mut/S3mut) to mutate MGP's conserved serine residues to alanine. The initiation and progression of vascular calcification in these models were analyzed by alizarin red staining, histology, and micro-computed tomography imaging. On a regular diet, the arterial walls in the Mgp-/-; SM22α-GlamutMgp mice were not calcified. However, on a high phosphorus diet, these mice showed wide-spread arterial calcification. In contrast, MgpS3mut/S3mut mice on a regular diet recapitulated arterial calcification traits of Mgp-/- mice, although with lesser severity. For the first time, we show here that MGP's conserved serine residues are indispensable for its antimineralization function in the arterial tissues. Although the conserved glutamic acid residues are not essential for this function on a regular diet, they are needed to prevent phosphate-induced arterial elastocalcinosis.

Characterization of dynamic changes in Matrix Gla Protein (MGP) gene expression as function of genetic risk alleles, osteoarthritis relevant stimuli, and the vitamin K inhibitor warfarin

We here aimed to characterize changes of Matrix Gla Protein (MGP) expression in relation to its recently identified OA risk allele rs1800801-T in OA cartilage, subchondral bone and human ex vivo osteochondral explants subjected to OA related stimuli. Given that MGP function depends on vitamin K bioavailability, we studied the effect of frequently prescribed vitamin K antagonist warfarin. Differential (allelic) mRNA expression of MGP was analyzed using RNA-sequencing data of human OA cartilage and subchondral bone. Human osteochondral explants were used to study exposures to interleukin one beta (IL-1β; inflammation), triiodothyronine (T3; Hypertrophy), warfarin, or 65% mechanical stress (65%MS) as function of rs1800801 genotypes. We confirmed that the MGP risk allele rs1800801-T was associated with lower expression and that MGP was significantly upregulated in lesioned as compared to preserved OA tissues, mainly in risk allele carriers, in both cartilage and subchondral bone. Moreover, MGP expression was downregulated in response to OA like triggers in cartilage and subchondral bone and this effect might be reduced in carriers of the rs1800801-T risk allele. Finally, warfarin treatment in cartilage increased COL10A1 and reduced SOX9 and MMP3 expression and in subchondral bone reduced COL1A1 and POSTN expression. Our data highlights that the genetic risk allele lowers MGP expression and upon OA relevant triggers may hamper adequate dynamic changes in MGP expression, mainly in cartilage. The determined direct negative effect of warfarin on human explant cultures functionally underscores the previously found association between vitamin K deficiency and OA.

Vascular Calcification in Chronic Kidney Disease: The Role of Vitamin K- Dependent Matrix Gla Protein.

Arterial calcification is highly prevalent in chronic kidney disease (CKD) patients and is associated with cardiovascular (CV) morbidity and mortality. Patients at early CKD stages are more likely to suffer a fatal CV event than to develop end-stage renal disease and require hemodialysis treatment. The heavy CV burden of these patients cannot be solely explained by traditional calcification risk factors. Moreover, the pathophysiologic mechanisms underlying this association are complex and yet not fully understood. Although vascular calcification was regarded as a passive degenerative process for over a century, this theory changed by recent evidence that pointed toward an active process, where calcification promoters and inhibitors were involved. Matrix Gla Protein (MGP) has been established as a strong inhibitor of calcification both in vitro and in vivo. Not only it prevents mineralization of the arterial wall, but it is the only factor that can actually reverse it. To become fully active, MGP must undergo carboxylation of specific protein bound glutamate residues, a process fully dependent on the availability of vitamin K. Low vitamin K status leads to inactive, uncarboxylated forms of MGP and has been repeatedly associated with accelerated vascular calcification. Aim of this review is to present the pathophysiologic mechanisms underlying the activation and function of MGP and review the existing, accumulating data regarding the association between vitamin K, MGP and vascular calcification/CV disease in CKD patients.

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.

VKORC1 gene (vitamin K epoxide reductase) polymorphisms are associated with cardiovascular disease in chronic kidney disease patients on hemodialysis

Vitamin K is necessary for the carboxylation of clotting factors and matrix Gla protein (MGP). Vitamin K epoxide reductase (VKOR) is the enzyme responsible for recirculation of Vitamin K increasing its tissue availability. Polymorphisms of VKOR may alter the function of MGP, thereby influencing vascular calcification. We conducted this study to investigate the relationship of VKORC1 gene single nucleotide polymorphisms (SNP's) to vascular calcification and clinically overt cardiovascular disease in chronic kidney disease (CKD) patients on hemodialysis (HD). The study included 54 CKD patients on HD. We excluded those with diabetes or on anticoagulant therapy. Vascular calcifications were measured using computerized tomography scans and roentgenograms. Prevalent clinically overt cardiovascular disease was reported based on the evidence of documented preexisting major cardiovascular events. Genotype detection for the gene VKORC1 C1173T and G-1639A polymorphisms was carried out by polymerase chain reaction. We found a significant association between C1173T polymorphisms and vascular calcification (odds ratio [OR] = 43, P = 0.001). The mutant T allele was also linked with higher odds of vascular calcification (OR = 8.880, 95% confidence interval [CI] = 3.1-25.4, P = 0.001) and clinically overt cardiovascular disease (OR = 4.7, 95% CI = 1.5-14.7, P = 0.005). VKORC1 G-1639A polymorphisms were not associated with vascular calcification and had lower prevalence of clinically overt cardiovascular disease (OR = 0.07, 95% CI = 0.01-0.4, P = 0.001). In patients with CKD on HD, we found that VKORC1 gene polymorphisms did have an association with prevalent cardiovascular calcification and clinically overt cardiovascular disease, C1173T polymorphisms with higher risk for disease, and G-1639A with lower risk.

Warfarin Use and Prevalence of Coronary Artery Calcification Assessed by Multislice Computed Tomography

Warfarin inhibits the synthesis and function of matrix Gla protein, a vitamin K-dependent protein, which is a potent inhibitor of tissue calcification. We had earlier reported the association of warfarin use with valvular calcification in patients with nonvalvular atrial fibrillation. The aim of our present study was to investigate the association of warfarin use with the presence and severity of coronary artery calcification. A total of 233 patients underwent computed tomography scan (CT) at our institution for the assessment of coronary artery calcium score (CACS). Of 233 patients, the mean age was 63 years, 28 patients (12%) were treated with warfarin, and 205 patients (88%) were not on warfarin. Based on their total CACS, the patients were subsequently stratified into 59 with no coronary calcium (CACS = 0), 63 with low CACS (1-100), 49 with moderate CACS (101-400), 33 with severe CACS (410-1000), and 29 with very severe CACS (>1000). The χ test and Student t-test were used for the comparison of categorical and continuous variables, respectively, between warfarin users and nonusers. Using the variables age, gender, race, smoking, hypertension, diabetes, dyslipidemia, glomerular filtration rate, calcium-phosphorus product, alkaline phosphatase, use of aspirin, beta blockers, angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, and statins, stepwise logistic regression analysis did not show any association of coronary calcification with use of warfarin. In our study, warfarin use was not associated with a higher prevalence or severity of CACS assessed by coronary computed tomography.

Keutel syndrome: Report of two novel MGP mutations and discussion of clinical overlap with arylsulfatase E deficiency and relapsing polychondritis

Keutel syndrome is a rare, autosomal recessive disorder characterized by diffuse cartilage calcification, peripheral pulmonary artery stenosis, midface retrusion, and short distal phalanges. To date, 28 patients from 18 families have been reported, and five mutations in the matrix Gla protein gene (MGP) have been identified. The matrix Gla protein (MGP) is a vitamin K-dependent extracellular protein that functions as a calcification inhibitor through incompletely understood mechanisms. We present the clinical manifestations of three affected siblings from a consanguineous Turkish family, in whom we detected the sixth MGP mutation (c.79G>T, which predicts p.E27X) and a fourth unrelated patient in whom we detected the seventh MGP mutation, a partial deletion of exon 4. Both mutations predict complete loss of MGP function. One of the patients presented initially with a working diagnosis of relapsing polychondritis. Clinical features suggestive of Keutel syndrome were also observed in one additional unrelated patient who was later found to have a deletion of arylsulfatase E, consistent with a diagnosis of X-linked recessive chondrodysplasia punctata. Through a discussion of these cases, we highlight the clinical overlap of Keutel syndrome, X-linked chondrodysplasia punctata, and the inflammatory disease relapsing polychondritis.

Research Progress of Warfarin-associated Vascular Calcification and Its Possible Therapy

Vascular calcification is a common comorbidity in elderly patients with diabetes mellitus or renal insufficiency. A large number of studies have shown that vascular calcification can be induced and accelerated in patients undergoing long-term treatment with warfarin, leading to some severe complications, such as hypertension, atherosclerosis, valvular calcification, and coronary calcification, especially in the population with atrial fibrillation, hemodialysis, and chronic kidney disease. Warfarin inhibits the activation of vitamin K-dependent coagulation factors and affects the function of vitamin K-dependent proteins via interference of the vitamin K cycle by antagonizing vitamin K. One of its consequences is adverse effects on the expression and function of matrix Gla protein, one of the important vitamin K-dependent proteins. Matrix Gla protein acts as an inhibitor of vascular calcification by blocking bone morphogenetic protein signaling or promoting the phagocytosis of apoptotic bodies; moreover, it restrains the formation of calcification directly resulting in the promotion of vascular calcification. This article also discusses the various treatments for vascular calcification caused by warfarin.

Peptides of Matrix Gla Protein Inhibit Nucleation and Growth of Hydroxyapatite and Calcium Oxalate Monohydrate Crystals

Matrix Gla protein (MGP) is a phosphorylated and γ-carboxylated protein that has been shown to prevent the deposition of hydroxyapatite crystals in the walls of blood vessels. MGP is also expressed in kidney and may inhibit the formation of kidney stones, which mainly consist of another crystalline phase, calcium oxalate monohydrate. To determine the mechanism by which MGP prevents soft-tissue calcification, we have synthesized peptides corresponding to the phosphorylated and γ-carboxylated sequences of human MGP in both post-translationally modified and non-modified forms. The effects of these peptides on hydroxyapatite formation and calcium oxalate crystallization were quantified using dynamic light scattering and scanning electron microscopy, respectively. Peptides YGlapS (MGP1-14: YγEpSHEpSMEpSYELNP), YEpS (YEpSHEpSMEpSYELNP), YGlaS (YγESHESMESYELNP) and SK-Gla (MGP43-56: SKPVHγELNRγEACDD) inhibited formation of hydroxyapatite in order of potency YGlapS > YEpS > YGlaS > SK-Gla. The effects of YGlapS, YEpS and YGlaS on hydroxyapatite formation were on both crystal nucleation and growth; the effect of SK-Gla was on nucleation. YGlapS and YEpS significantly inhibited the growth of calcium oxalate monohydrate crystals, while simultaneously promoting the formation of calcium oxalate dihydrate. The effects of these phosphopeptides on calcium oxalate monohydrate formation were on growth of crystals rather than nucleation. We have shown that the use of dynamic light scattering allows inhibitors of hydroxyapatite nucleation and growth to be distinguished. We have also demonstrated for the first time that MGP peptides inhibit the formation of calcium oxalate monohydrate. Based on the latter finding, we propose that MGP function not only to prevent blood-vessel calcification but also to inhibit stone formation in kidney.

Elastin Haploinsufficiency Impedes the Progression of Arterial Calcification in MGP-Deficient Mice

Matrix gla protein (MGP) is a potent inhibitor of extracellular matrix (ECM) mineralization. MGP-deficiency in humans leads to Keutel syndrome, a rare genetic disease hallmarked by abnormal soft tissue calcification. MGP-deficient (Mgp(-/-)) mice show progressive deposition of hydroxyapatite minerals in the arterial walls and die within 2 months of age. The mechanism of antimineralization function of MGP is not fully understood. We examined the progression of vascular calcification and expression of several chondrogenic/osteogenic markers in the thoracic aortas of Mgp(-/-) mice at various ages. Although cells with chondrocyte-like morphology have been reported in the calcified aorta, our gene expression data indicate that chondrogenic/osteogenic markers are not upregulated in the arteries prior to the initiation of calcification. Interestingly, arterial calcification in Mgp(-/-) mice appears first in the elastic laminae. Considering the known mineral scaffolding function of elastin (ELN), a major elastic lamina protein, we hypothesize that elastin content in the laminae is a critical determinant for arterial calcification in Mgp(-/-) mice. To investigate this, we performed micro-computed tomography (µCT) and histological analyses of the aortas of Mgp(-/-);Eln(+/-) mice and show that elastin haploinsufficiency significantly reduces arterial calcification in this strain. Our data suggest that MGP deficiency leads to alterations of vascular ECM that may in turn initiate arterial calcification.

Vitamin K status in chronic kidney disease: a report of a study and a mini-review

Hepatic vitamin K-dependent proteins (e.g., Factors II, VII, IX and X) form part of the clotting cascade. Factor II (FII)/Prothrombin incorporates 10 Glu residues on the N-terminal region that are γ-carboxylated to Gla residues by the action of γ-glutamyl carboxylase to confer biological activity. Vitamin K is also required for the normal function of Matrix Gla Protein (MGP)--one of several non-clotting-related extra-hepatic vitamin K-dependent proteins. MGP is known to have protective action against vascular calcification--indeed it is a powerful tissue-bound inhibitory mechanism and can be found in blood vessel walls. The mature protein is also dependent on activation by γ-glutamyl carboxylase enzyme to convert Glu residues in its amino acid sequence to Gla. This reaction can only take place when the enzyme is activated in the presence of vitamin K. It is of great potential interest to investigate whether subtle deficiencies of vitamin K may, through its effect on the action of MGP, be a contributing factor to vascular calcification in CKD patients, in whom CV disease is greatly accelerated and in whom vascular calcification is not only common, but progresses aggressively, and is something for which as yet there is no clinically applicable remedy.

Expression and function of matrix Gla protein in human peritoneal mesothelial cells

Chronic peritoneal dialysis (PD) is associated with peritoneal calcification. Studies in vascular tissue suggest that ectopic calcification is not merely a passive but a regulated process resembling bone mineralization. We investigated whether peritoneal calcification is controlled by matrix Gla protein (MGP) secreted by peritoneal mesothelial cells. Human primary mesothelial cells (HPMC) were exposed to constituents of PD fluids and to cytokines relevant to peritoneal integrity. Messenger RNA was quantitated by real-time reverse transcription polymerase chain reaction (RT-PCR), protein abundance by Western blot and in vivo protein expression immunohistochemically. To demonstrate functional relevance, MGP was silenced in HPMC by siRNA transfection and calcium phosphate matrix deposition measured by o-cresolphthalein complexone method and von Kossa staining. MGP was consistently detected in the mesothelial cell layer of peritoneal tissue specimens from uraemic and non-uraemic patients, in HPMC and in culture medium. MGP mRNA and protein abundance was increased by glucose and IGF1 and decreased by TGFß1. Suppression of MGP increased matrix calcium and phosphorus deposition by 90 ± 6% and 100 ± 4% at 1 mM ambient Ca(2+) and phosphorus concentration. Deposition was not increased any further by higher medium Ca(2+)/phosphorus concentrations nor reduced by inhibition of the phosphate cotransporter Pit1. MGP is expressed by HPMC and regulated by glucose, IGF1 and TGFß1. It is a potent inhibitor of calcification in vitro and may thus play a role in the regulation of peritoneal calcium homeostasis.

Gla-Rich Protein Is a Novel Vitamin K-Dependent Protein Present in Serum That Accumulates at Sites of Pathological Calcifications

Mineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization.

Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification.

Matrix Gla protein (MGP) is a small vitamin K-dependent protein containing five gamma-carboxyglutamic acid (Gla) residues that are believed to be important in binding Ca(2+), calcium crystals and bone morphogenetic protein. In addition, MGP contains phosphorylated serine residues that may further regulate its activity. In vivo, MGP has been shown to be a potent inhibitor of vascular calcification; however, the precise molecular mechanism underlying the function of MGP is not yet fully understood. We investigated the effects of MGP in human vascular smooth muscle cell (VSMC) monolayers that undergo calcification after exposure to an increase in Ca(2+) concentration. Increased calcium salt deposition was found in cells treated with the vitamin K antagonist warfarin as compared to controls, whereas cells treated with vitamin K(1) showed decreased calcification as compared to controls. With conformation-specific antibodies, it was confirmed that warfarin treatment of VSMCs resulted in uncarboxylated (Gla-deficient) MGP. To specifically test the effects of MGP on VSMC calcification, we used full-length synthetic MGP and MGP-derived peptides representing various domains in MGP. Full length MGP, the gamma-carboxylated motif (Gla) (amino acids 35-54) and the phosphorylated serine motif (amino acids 3-15) inhibited calcification. Furthermore, we showed that the peptides were not taken up by VSMCs but bound to the cell surface and to vesicle-like structures. These data demonstrate that both gamma-glutamyl carboxylation and serine phosphorylation of MGP contribute to its function as a calcification inhibitor and that MGP may inhibit calcification via binding to VSMC-derived vesicles.

Matrix GLA Protein Function in Human Trabecular Meshwork Cells: Inhibition of BMP2-Induced Calcification Process

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. 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). 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. 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.

Matrix GLA Protein Function in Human Trabecular Meshwork Cells: Inhibition of BMP2-Induced Calcification Process

Matrix GLA Protein Function in Human Trabecular Meshwork Cells: Inhibition of BMP2-Induced Calcification Process

Vitamin K and vascular calcification

Until recently, vitamin K has been exclusively related to blood coagulation. During the last decade, a second function for vitamin K-dependent proteins has become apparent : the regulation of tissue calcification. One of them is the function of Matrix Gla Protein (MGP) : potent inhibitors of vascular calcification. The function of MGP became clear from transgenic mice (MGP-deficient mice). Further research on MGP will resolve the complicated mechanism of atherosclerosis, especially of the arterial calcification. The recommended daily allowance for vitamin K to prevent vascular calcification should be evaluated.

Matrix Gla protein in Xenopus laevis: molecular cloning, tissue distribution, and evolutionary considerations.

Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins and in higher vertebrates, is found in the extracellular matrix of mineralized tissues and soft tissues. MGP synthesis is highly regulated at the transcription and posttranscription levels and is now known to be involved in the regulation of extracellular matrix calcification and maintenance of cartilage and soft tissue integrity during growth and development. However, its mode of action at the molecular level remains unknown. Because there is a large degree of conservation between amino acid sequences of shark and human MGP, the function of MGP probably has been conserved throughout evolution. Given the complexity of the mammalian system, the study of MGP in a lower vertebrate might be advantageous to relate the onset of MGP expression with specific events during development. Toward this goal, MGP was purified from Xenopus long bones and its N-terminal amino acid sequence was determined and used to clone the Xenopus MGP complementary DNA (cDNA) by a mixture of reverse-transcription (RT)- and 5'- rapid amplification of cDNA ends (RACE)-polymerase chain reaction (PCR). MGP messenger RNA (mRNA) was present in all tissues analyzed although predominantly expressed in Xenopus bone and heart and its presence was detected early in development at the onset of chondrocranium development and long before the appearance of the first calcified structures and metamorphosis. These results show that in this system, as in mammals, MGP may be required to delay or prevent mineralization of cartilage and soft tissues during the early stages of development and indicate that Xenopus is an adequate model organism to further study MGP function during growth and development.

Role of vitamin K and Gla proteins in the pathophysiology of osteoporosis and vascular calcification.

Among the proteins known or suspected to be involved in bone and vascular biology are several members of the vitamin K-dependent or Gla protein family. This review focuses on the role of two of these: osteocalcin and matrix Gla protein. Osteocalcin metabolism has been implicated in the pathogenesis of osteoporosis through an unknown mechanism that may be linked to suboptimal vitamin K status resulting in its undercarboxylation and presumed dysfunction. Recent studies that have investigated this hypothesis are discussed, as are recent promising clinical studies of vitamin K supplementation in osteoporosis. A recently delineated function of matrix Gla protein is as a powerful inhibitor of calcification of arteries and cartilage. In the period covered by this review there have been several landmark studies using cell systems, whole animals and genetic techniques that have consolidated and extended our knowledge of the role of matrix Gla protein in the prevention of ectopic calcification.