vitamin-k-semiquinone-radical and Pseudoxanthoma-Elasticum

The ABCC6 gene encodes an organic anion transporter protein, ABCC6/MRP6. Mutations in the gene cause a rare, recessive genetic disease, pseudoxanthoma elasticum, while the loss of one ABCC6 allele is a genetic risk factor in coronary artery disease. We review here the information available on gene structure, evolution as well as the present knowledge on its transcriptional regulation. We give a detailed description of the characteristics of the protein, and analyze the relationship between the distributions of missense disease-causing mutations in the predicted three-dimensional structure of the transporter, which suggests functional importance of the domain-domain interactions. Though neither the physiological function of the protein nor its role in the pathobiology of the diseases are known, a current hypothesis that ABCC6 may be involved in the efflux of one form of Vitamin K from the liver is discussed. Finally, we analyze potential strategies how the gene can be targeted on the transcriptional level to increase protein expression in order to compensate for reduced activity. In addition, pharmacologic correction of trafficking-defect mutants or suppression of stop codon mutations as potential future therapeutic interventions are also reviewed.

Topics: Alleles; Animals; ATP-Binding Cassette Transporters; Disease Models, Animal; Genetic Therapy; Humans; Mice; Molecular Targeted Therapy; Multidrug Resistance-Associated Proteins; Mutation; Pseudoxanthoma Elasticum; Vitamin K

The mineralisation disorder pseudoxanthoma elasticum (PXE) is associated with mutations in the transporter protein ABCC6. Patients with PXE suffer from calcified lesions in the skin, eyes and vasculature, and PXE is related to a more severe vascular calcification syndrome called generalised arterial calcification of infancy (GACI). Mutations in ABCC6 are linked to reduced levels of circulating vitamin K. Here, we describe a mutation in the zebrafish (Danio rerio) orthologue abcc6a, which results in extensive hypermineralisation of the axial skeleton. Administration of vitamin K to embryos was sufficient to restore normal levels of mineralisation. Vitamin K also reduced ectopic mineralisation in a zebrafish model of GACI, and warfarin exacerbated the mineralisation phenotype in both mutant lines. These data suggest that vitamin K could be a beneficial treatment for human patients with PXE or GACI. Additionally, we found that abcc6a is strongly expressed at the site of mineralisation rather than the liver, as it is in mammals, which has significant implications for our understanding of the function of ABCC6.

Topics: Animals; Anthraquinones; ATP-Binding Cassette Transporters; Calcinosis; Chromosomes, Artificial, Bacterial; DNA Primers; In Situ Hybridization; Mutation; Pseudoxanthoma Elasticum; Transgenes; Vascular Calcification; Vitamin K; Warfarin; Zebrafish; Zebrafish Proteins

Gamma-glutamyl carboxylase (GGCX) mutations have been reported in patients with a pseudoxanthoma elasticum (PXE)-like phenotype, loose redundant skin, and multiple vitamin K-dependent coagulation factor deficiencies. We report on the clinical findings and molecular results in 13 affected members of two families who had a uniform phenotype consisting of (PXE)-like skin manifestations in the neck and trunk, loose sagging skin of the trunk and upper limbs, and retinitis pigmentosa confirmed by electroretinographies in 10 affected individuals. There were no coagulation abnormalities. Molecular investigations of the ATP-binding cassette subfamily C member 6 did not yield causative mutations. All 13 affected family members were found to be homozygous for the splice-site mutation c.373+3G>T in the GGCX gene. All tested parents were heterozygous for the mutation, and healthy siblings were either heterozygous or had the wild type. We suggest that the present patients represent a hitherto unreported phenotype associated with GGCX mutations. Digenic inheritance has been suggested to explain the variability in phenotype in GGCX mutation carriers. Consequently, the present phenotype may not be explained only by the GGCX mutations only but may be influenced by variants in other genes or epigenetic and environmental factors.

Topics: Adolescent; Adult; Carbon-Carbon Ligases; Child; Cutis Laxa; Family Health; Female; Heterozygote; Homozygote; Humans; Male; Middle Aged; Pedigree; Phenotype; Pseudoxanthoma Elasticum; Retinitis Pigmentosa; RNA Splice Sites; Skin; Vitamin K; Young Adult

Pseudoxanthoma elasticum (PXE) is a multisystem ectopic mineralization disorder caused by mutations in the ABCC6 gene. Warfarin, a commonly used anticoagulant, is associated with increased mineralization of the arterial blood vessels and cardiac valves. We hypothesized that warfarin may accelerate ectopic tissue mineralization in PXE, with clinical consequences. To test this hypothesis, we developed a model in which Abcc6(-/-) mice, which recapitulate features of PXE, were fed a diet supplemented with warfarin and vitamin K1. Warfarin action was confirmed by significantly increased serum levels of oxidized vitamin K. For mice placed on a warfarin-containing diet, quantitative chemical and morphometric analyses revealed massive accumulation of mineral deposits in a number of tissues. Mice fed a warfarin-containing diet were also shown to have abundant uncarboxylated form of matrix Gla protein, which allowed progressive tissue mineralization to ensue. To explore the clinical relevance of these findings, 1747 patients with PXE from the approximately 4000 patients in the PXE International database were surveyed about the use of warfarin. Of the 539 respondents, 2.6% reported past or present use of warfarin. Based on the prevalence of PXE (approximately 1:50,000), thousands of patients with PXE worldwide may be at risk for worsening of PXE as a result of warfarin therapy.

Topics: Animals; ATP-Binding Cassette Transporters; Calcification, Physiologic; Calcium; Calcium-Binding Proteins; Dermis; Diet; Dietary Supplements; Disease Models, Animal; Extracellular Matrix Proteins; Humans; Magnesium; Matrix Gla Protein; Mice; Minerals; Multidrug Resistance-Associated Proteins; Organ Specificity; Phosphates; Phosphorus; Pseudoxanthoma Elasticum; Vitamin K; Warfarin; X-Ray Microtomography

A number of beta-thalassemia (β-thal) patients in the course of the disease exhibit ectopic calcification affecting skin, eyes and the cardiovascular system. Clinical and histopathological features have been described similar to those in pseudoxanthoma elasticum (PXE), although different genes are affected in the two diseases. Cultured dermal fibroblasts from β-thal patients with and without PXE-like clinical manifestations have been compared for parameters of redox balance and for the expression of proteins, which have been already associated with the pathologic mineralisation of soft connective tissues. Even though oxidative stress is a well-known condition of β-thal patients, our results indicate that the occurrence of mineralized elastin is associated with a more pronounced redox disequilibrium, as demonstrated by the intracellular increase of anion superoxide and of oxidized proteins and lipids. Moreover, fibroblasts from β-thal PXE-like patients are characterized by decreased availability of carboxylated matrix Gla protein (MGP), as well as by altered expression of proteins involved in the vitamin K-dependent carboxylation process. Results demonstrate that elastic fibre calcification is promoted when redox balance threshold levels are exceeded and the vitamin K-dependent carboxylation process is affected decreasing the activity of MGP, a well-known inhibitor of ectopic calcification. Furthermore, independently from the primary gene defect, these pathways are similarly involved in fibroblasts from PXE and from β-thal PXE-like patients as well as in other diseases leading to ectopic calcification, thus suggesting that can be used as markers of pathologic mineralisation.

Topics: Adult; Advanced Oxidation Protein Products; beta-Thalassemia; Blotting, Western; Calcinosis; Calcium-Binding Proteins; Carboxylic Acids; Dermis; DNA Methylation; Elastic Tissue; Elastin; Extracellular Matrix Proteins; Female; Fibroblasts; Flow Cytometry; Glutathione Peroxidase; Glutathione Transferase; Humans; Lipid Peroxides; Male; Malondialdehyde; Matrix Gla Protein; Oxidative Stress; Pseudoxanthoma Elasticum; Superoxide Dismutase; Vitamin K

Mineralization of elastic fibers in pseudoxanthoma elasticum (PXE) has been associated with low levels of carboxylated matrix gla protein (MGP), most likely as a consequence of reduced vitamin K (vit K) availability. Unexpectedly, vit K supplementation does not exert beneficial effects on soft connective tissue mineralization in the PXE animal model. To understand the effects of vit K supplementation and in the attempt to interfere with pathways leading to the accumulation of calcium and phosphate within PXE-mineralized soft connective tissues, we have conducted in vitro studies on dermal fibroblasts isolated from control subjects and from PXE patients. Cells were cultured in standard conditions and in calcifying medium (CM) in the presence of vit K1 and K2, or levamisole, an alkaline phosphatase (ALP) inhibitor. Control and PXE fibroblasts were characterized by a similar dose-dependent uptake of both vit K1 and vit K2, thus promoting a significant increase of total protein carboxylation in all cell lines. Nevertheless, MGP carboxylation remained much less in PXE fibroblasts. Interestingly, PXE fibroblasts exhibited a significantly higher ALP activity. Consistently, the mineralization process induced in vitro by a long-term culture in CM appeared unaffected by vit K, whereas it was abolished by levamisole.

Topics: Adjuvants, Immunologic; Adult; Alkaline Phosphatase; Antifibrinolytic Agents; Calcification, Physiologic; Calcinosis; Calcium; Calcium-Binding Proteins; Cell Survival; Cells, Cultured; Dermis; Enzyme Activation; Extracellular Matrix Proteins; Female; Fibroblasts; Humans; Levamisole; Matrix Gla Protein; Middle Aged; Phosphates; Pseudoxanthoma Elasticum; Vitamin K

Pseudoxanthoma elasticum (PXE) is a heritable multisystem disorder manifesting with ectopic calcification of peripheral connective tissues, caused by mutations in the ABCC6 gene. Alterations in vitamin K metabolism have been suggested to contribute to the pathomechanisms of the mineralization process. In this study we administered vitamin K or its glutathione conjugate (K3-GSH) into Abcc6 (-/-) mice which recapitulate features of PXE. Oral administration of vitamin K2 in dosages, which vastly exceed the amounts in control diet or the recommended amounts for humans, did not alter the ectopic mineralization in Abcc6 (-/-) mice. Similarly, intravenous administration of K3-GSH did not alter the degree of mineralization. Testing of vitamin K2, K3 and K3-GSH in an in vitro calcification system provided no evidence of mineralization inhibition. Collectively, our data suggest that vitamin K deficiency in the peripheral tissues is not a simple explanation for development of mineral deposits in PXE.

Topics: Animals; ATP-Binding Cassette Transporters; Calcinosis; Cell Line; Disease Models, Animal; Elastic Tissue; Female; Humans; Male; Mice; Mice, Knockout; Multidrug Resistance-Associated Proteins; Mutation; Pseudoxanthoma Elasticum; Vitamin K; Vitamin K Deficiency

Pseudoxanthoma elasticum (PXE) is a heritable disease characterized by calcified elastic fibers in cutaneous, ocular, and vascular tissues. PXE is caused by mutations in ABCC6, which encodes a protein of the ATP-driven organic anion transporter family. The inability of this transporter to secrete its substrate into the circulation is the likely cause of PXE. Vitamin K plays a role in the regulation of mineralization processes as a co-factor in the carboxylation of calcification inhibitors such as Matrix Gla Protein (MGP). Vitamin K precursor or a conjugated form has been proposed as potential substrate(s) for ABCC6. We investigated whether an enriched diet of vitamin K1 or vitamin K2 (MK4) could stop or slow the disease progression in Abcc6 (-/-) mice. Abcc6 (-/-) mice were placed on a diet of either vitamin K1 or MK4 at 5 or 100 mg/kg at prenatal, 3 weeks or 3 months of age. Disease progression was quantified by measuring the calcium content of one side of the mouse muzzle skin and histological staining for calcium of the opposing side. Raising the vitamin K1 or MK4 content of the diet increased the concentration of circulating MK4 in the serum. However, this increase did not significantly affect the MGP carboxylation status or reduce its abnormal abundance, the total calcium content or the pathologic calcification in the whiskers of the 3 treatment groups compared to controls. Our findings showed that raising the dietary intake of vitamin K1 or MK4 was not beneficial in the treatment of PXE and suggested that the availability of vitamin K may not be a limiting factor in this pathology.

Topics: Animals; ATP-Binding Cassette Transporters; Calcinosis; Dietary Supplements; Disease Models, Animal; Mice; Mice, Knockout; Multidrug Resistance-Associated Proteins; Pseudoxanthoma Elasticum; Treatment Failure; Vitamin K

Pseudoxanthoma elasticum (PXE), a prototypic heritable disorder with ectopic mineralization, manifests with characteristic skin findings, ocular involvement and cardiovascular problems, with considerable morbidity and mortality. The classic forms of PXE are due to loss-of-function mutations in the ABCC6 gene, which encodes ABCC6, a transmembrane efflux transporter expressed primarily in the liver. Several lines of evidence suggest that PXE is a primary metabolic disorder, which in the absence of ABCC6 transporter activity, displays reduced plasma anti-mineralization capacity due to reduced fetuin-A and matrix gla-protein (MGP) levels. MGP requires to be activated by gamma-glutamyl carboxylation, a vitamin K-dependent reaction, to serve in an anti-mineralization role in the peripheral connective tissue cells. Although the molecules transported from the hepatocytes to circulation by ABCC6 in vivo remain unidentified, it has been hypothesized that a critical vitamin K derivative, such as reduced vitamin K conjugated with glutathione, is secreted to circulation physiologically, but not in the absence of ABCC6 transporter activity. As a result, activation of MGP by gamma-glutamyl carboxylase is diminished, allowing slow yet progressive mineralization of connective tissues characteristic of PXE. Understanding of the pathomechanistic details of PXE provides a basis for the development of targeted molecular therapies for this currently intractable disease.

Topics: alpha-2-HS-Glycoprotein; Animals; ATP-Binding Cassette Transporters; Blood Proteins; Calcium-Binding Proteins; Carbon-Carbon Ligases; Connective Tissue; Extracellular Matrix Proteins; Humans; Liver; Matrix Gla Protein; Mice; Mice, Knockout; Multidrug Resistance-Associated Proteins; Phenotype; Pseudoxanthoma Elasticum; Vitamin K

Topics: Animals; ATP-Binding Cassette Transporters; Carbon-Carbon Ligases; Diet; Disease Models, Animal; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Multidrug Resistance-Associated Proteins; Mutation; Pseudoxanthoma Elasticum; Vitamin K

Soft-tissue mineralization is a tightly regulated process relying on the activity of systemic and tissue-specific inhibitors and promoters of calcium precipitation. Many of these, such as matrix gla protein (MGP) and osteocalcin (OC), need to undergo carboxylation to become active. This post-translational modification is catalyzed by the gammaglutamyl carboxylase GGCX and requires vitamin K (VK) as an essential co-factor. Recently, we described a novel phenotype characterized by aberrant mineralization of the elastic fibers resulting from mutations in GGCX. Because of the resemblance with pseudoxanthoma elasticum (PXE), a prototype disorder of elastic fiber mineralization, it was coined the PXE-like syndrome. As mutations in GGCX negatively affect protein carboxylation, it is likely that inactive inhibitors of calcification contribute to ectopic mineralization in PXE-like syndrome. Because of the remarkable similarities with PXE, we performed a comparative study of various forms of VK-dependent proteins in serum, plasma (using ELISA), and dermal tissues (using immunohistochemistry) of PXE-like and PXE patients using innovative, conformation-specific antibodies. Furthermore, we measured VK serum concentrations (using HPLC) in PXE-like and PXE samples to evaluate the VK status. In PXE-like patients, we noted an accumulation of uncarboxylated Gla proteins, MGP, and OC in plasma, serum, and in the dermis. Serum levels of VK were normal in these patients. In PXE patients, we found similar, although not identical results for the Gla proteins in the circulation and dermal tissue. However, the VK serum concentration in PXE patients was significantly decreased compared with controls. Our findings allow us to conclude that ectopic mineralization in the PXE-like syndrome and in PXE results from a deficient protein carboxylation of VK-dependent inhibitors of calcification. Although in PXE-like patients this is due to mutations in the GGCX gene, a deficiency of the carboxylation co-factor VK is at the basis of the decreased activity of calcification inhibitors in PXE.

Topics: Adult; Aged; Animals; Calcinosis; Calcium-Binding Proteins; Extracellular Matrix Proteins; Humans; Matrix Gla Protein; Mice; Mice, Knockout; Middle Aged; Mixed Function Oxygenases; Mutation; Proteins; Pseudoxanthoma Elasticum; Syndrome; Vitamin K; Vitamin K Deficiency; Vitamin K Epoxide Reductases

Pseudoxanthoma elasticum (PXE) is a multisystem disorder characterized by ectopic mineralization of connective tissues with primary manifestations in the skin, eyes, and cardiovascular system. The classic forms of PXE are due to mutations in the ABCC6 gene that encodes the ABCC6 protein, a putative transmembrane transporter expressed primarily in the liver and the kidneys. PXE-like clinical findings have been encountered in association with vitamin K-dependent coagulation factor deficiency, an autosomal recessive disorder that is due to mutations in either the GGCX or VKORC1 genes. In this study, we investigated a family with two siblings with characteristic features of PXE and vitamin K-dependent coagulation factor deficiency. Mutation analysis identified two GGCX mutations in the affected individuals (p. R83W and p.Q374X); however, no mutations in either ABCC6 or VKORC1 could be found. GGCX encodes a gamma-glutamyl carboxylase necessary for activation of both coagulation factors in the liver and matrix gla protein, which, in fully carboxylated form, is able to prevent ectopic mineralization. Analysis of skin by specific antibodies demonstrated that matrix gla protein was found predominantly in undercarboxylated form and was associated with the mineralized areas in the patients' lesional skin. These observations pathomechanistically suggest that, in our patients, reduced carboxylase activity results in a reduction of matrix gla protein carboxylation, thus allowing peripheral mineralization to occur. Our findings also confirm GGCX as the second gene locus causing PXE.

Topics: Amino Acid Sequence; Base Sequence; Blood Coagulation Factors; Carbon-Carbon Ligases; Coagulation Protein Disorders; DNA Mutational Analysis; Female; Heterozygote; Humans; Immunohistochemistry; Male; Microscopy, Electron, Transmission; Middle Aged; Mixed Function Oxygenases; Molecular Sequence Data; Multidrug Resistance-Associated Proteins; Pedigree; Polymerase Chain Reaction; Pseudoxanthoma Elasticum; Sequence Homology, Amino Acid; Vitamin K; Vitamin K Epoxide Reductases

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disease characterized by a progressive mineralization of connective tissue, resulting in skin, arterial and eye disease. Classical PXE is caused by mutations in the ABCC6 gene, which encodes a member of the ABCC (MRP) family of organic anion transporters. Recent studies on Abcc6(-/-) mice show that the absence of ABCC6 in the liver is crucial for PXE and confirm the "metabolic disease hypothesis" for PXE, which states that tissue calcification is due to the absence of a plasma factor secreted from the basolateral hepatocyte membrane. We propose that this plasma factor is vitamin K (precursor). We propose that vitamin K (precursor) is secreted by ABCC6 from the liver as a glutathione--(or glucuronide)--conjugate and that this supplements the vitamin K need of peripheral tissues that receive insufficient vitamin from the diet, because dietary vitamin K is effectively extracted from blood by the liver. Peripheral tissue vitamin K is needed for the gamma-carboxylation of glutamate residues in proteins known to be required for counteracting calcification of connective tissue throughout the body. Our hypothesis explains the known facts of PXE and also explains why PXE-like symptoms can occur in patients with mutations in the gamma-glutamyl carboxylase gene (encoding the enzyme responsible for protein carboxylase) and in rats treated with vitamin K antagonists. The hypothesis implies that the symptoms of PXE can be prevented or mitigated by providing patients (intravenously) with a form of plasma vitamin K (precursor) that can be used by peripheral tissues.

Topics: Glutamic Acid; Humans; Liver; Molecular Structure; Multidrug Resistance-Associated Proteins; Mutation; Pseudoxanthoma Elasticum; Vitamin K

Data on six patients with a Pseudoxanthoma Elasticum (PXE)-like phenotype, characterized by excessive skin folding (resembling cutis laxa) and a deficiency of the vitamin K-dependent clotting factors (II, VII, IX, and X) are presented. A comparison is made between the clinical, ultrastructural, and molecular findings in these patients and those seen in classic PXE and cutis laxa, respectively. Clinical overlap with PXE is obvious from the skin manifestations of yellowish papules or leathery plaques with dot-like depressions at presentation, angioid streaks and/or ocular peau d'orange, and fragmentation and calcification of elastic fibers in the dermis. Important phenotypic differences with PXE include much more severe skin laxity with spreading toward the trunk and limbs with thick, leathery skin folds rather than confinement to flexural areas, and no decrease in visual acuity. Moreover, detailed electron microscopic analyses revealed that alterations of elastic fibers as well as their mineralization were slightly different from those in classic PXE. Molecular analysis revealed neither causal mutations in the ABCC6 gene (ATP-binding cassette subfamily C member 6), which is responsible for PXE, nor in VKORC1 (vitamin K 2,3 epoxide reductase), known to be involved in vitamin K-dependent factor deficiency. However, the GGCX gene (gamma-glutamyl carboxylase), encoding an enzyme important for gamma-carboxylation of gla-proteins, harbored mutations in six out of seven patients analyzed. These findings all support the hypothesis that the disorder indeed represents a separate clinical and genetic entity, the molecular background of which remains to be unraveled.

Topics: Adult; Blood Coagulation Disorders; Blood Coagulation Factors; Cutis Laxa; Dermis; DNA Mutational Analysis; Female; Humans; Male; Middle Aged; Multidrug Resistance-Associated Proteins; Phenotype; Pseudoxanthoma Elasticum; Skin; Skin Diseases; Vitamin K