Biochemical and Biophysical Research Communications
ABCC6 does not transport vitamin K3-glutathione conjugate from the liver: Relevance to pathomechanisms of pseudoxanthoma elasticum
Highlights
► The substrate of ABCC6 preventing soft tissue calcification is not known. ► It was proposed that vitamin K3-glutathione conjugate is the physiological substrate. ► We tested VK3GS transport by several assay systems. ► We found that this conjugate is not a substrate of ABCC6. ► Vitamin K3-glutathione conjugate may not prevent pseudoxanthoma-related symptoms.
Introduction
Mutations in the ABCC6 gene are responsible for the development of pseudoxanthoma elasticum (PXE, OMIM 26480) [1], [2], [3]. PXE is a multisystem disease characterized by ectopic mineralization of dermal, cardiovascular and ocular tissues (for recent reviews, see Refs. [4], [5], [6]). Carriers of one loss-of-function mutant ABCC6 allele have an increased risk of developing coronary artery disease [7], [8]. A fraction of beta-thalassemic patients develops PXE-like phenotype [9], [10] and this is most probably due to the down-regulation of ABCC6 [11]. A PXE-like phenotype can also develop in ABCC6 mutation carriers when they are carriers of mutations in the GGCX gene as well [12].
ABCC6 is an organic anion transporter [13], [14] predominantly present in the basolateral membrane of hepatocytes. Evidence that liver ABCC6 is essential and sufficient to prevent PXE came from transplantation studies in which skin grafts were exchanged between Abcc6−/− and Abcc6+/+ mice [15] as well as from a parabiotic mouse model [16]. These experiments show that PXE is a metabolic disease due to the absence of a substance that needs to be secreted from the liver by ABCC6 to prevent the development of mineral deposits in the peripheral soft tissues of PXE patients [5]. Unfortunately, the identity of this substance remains unknown.
An autosomal recessive syndrome with PXE-like cutaneous features is also associated with multiple coagulation factor deficiency caused by mutations in the GGCX gene [17], [18]. Since vitamin K is an essential cofactor of gamma-glutamyl carboxylation, insufficient supply of peripheral vitamin K may prevent the gamma-carboxylation of proteins, which are known to be required for counteracting calcification of connective tissue. Based on these observations, Borst and coworkers proposed that the ABCC6 substrate is vitamin K or one of its derivatives [19]. An obvious candidate is VK3GS, since this vitamin K derivate is formed in many cell types including hepatocytes, and since ABCC6 is known to transport glutathione conjugates [13].
We have now tested VK3GS in several independent ABCC6-dependent transporter systems and find that it is not substrate of ABCC6.
Section snippets
Liver perfusion with vitamin K3 (VK3)
The Abcc6−/− mouse was developed by targeted inactivation of the Abcc6 gene and maintained as described [20]. Twelve month-old mice were used for in situ non-recirculating (ex vivo) liver perfusion experiments as described [21]. Mice were starved overnight, anaesthetized with an i.p. injection of ketamine/xylazine (2 mg/0.3 mg per g body weight) and fixed to an operation table. After laparotomy, the portal vein and the inferior vena cava were cannulated with 22-gauge and 24-gauge Teflon cannula,
Liver perfusion
First, we characterized glutathione conjugation of vitamin K3 (VK3) in mouse liver. Livers of wild type (WT) and Abcc6−/− mice were perfused with 0.5 μM VK3 through the portal vein, and the outflow was collected via the inferior vena cava (corresponding to secretion toward the blood through the basolateral membranes of hepatocytes) and analyzed by HPLC-MS. We detected the glutathione-monoconjugate form (VK3GS) of VK3 in the outflow fluid only when VK3 was added to the perfusing fluid. Fig. 1
Acknowledgments
This study was supported by NIH/NIAMS grants R01 AR28450, R01 AR55225, and K08 AR57099, by the Hungarian research grants (from the National Research Foundation, OTKA) OTKA CK 80135, OTKA NK 81204, OTKA PD 79183, by PXE International Inc., and by a Top grant (40-00812-98-07-028) of ZonMw to KvdW and PB. T. A. is a recipient of Bolyai Fellowship of the Hungarian Academy of Sciences.
References (31)
- et al.
Pseudoxanthoma elasticum: molecular genetics and putative pathomechanisms
J. Invest. Dermatol.
(2010) - et al.
A mouse model of beta-thalassemia shows a liver-specific down-regulation of Abcc6 expression
Am. J. Pathol.
(2011) - et al.
Mutations in the GGCX and ABCC6 genes in a family with pseudoxanthoma elasticum-like phenotypes
J. Invest. Dermatol.
(2009) - et al.
Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6)
J. Biol. Chem.
(2002) - et al.
Pseudoxanthoma elasticum is a metabolic disease
J. Invest. Dermatol.
(2009) - et al.
Parabiotic heterogenetic pairing of Abcc6−/−/Rag1-/- mice and their wild-type counterparts halts ectopic mineralization in a murine model of pseudoxanthoma elasticum
Am. J. Pathol.
(2010) - et al.
Pseudoxanthoma elasticum-like phenotype with cutis laxa and multiple coagulation factor deficiency represents a separate genetic entity
J. Invest. Dermatol.
(2007) - et al.
Co-existent pseudoxanthoma elasticum and vitamin K-dependent coagulation factor deficiency: compound heterozygosity for mutations in the GGCX gene
Am. J. Pathol.
(2009) - et al.
Membrane topology and glycosylation of the human multidrug resistance-associated protein
J. Biol. Chem.
(1996) - et al.
Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase
J. Biol. Chem.
(1992)
Functional multidrug resistance protein (MRP1) lacking the N-terminal transmembrane domain
J. Biol. Chem.
Subcellular localization and N-glycosylation of human ABCC6, expressed in MDCKII cells
Biochem. Biophys. Res. Commun.
The role of the conserved glycines of ATP-binding cassette signature motifs of MRP1 in the communication between the substrate-binding site and the catalytic centers
J. Biol. Chem.
Increased levels of the multidrug resistance protein in lateral membranes of proliferating hepatocyte-derived cells
Gastroenterology
Mutations in ABCC6 cause pseudoxanthoma elasticum
Nat. Genet.
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PXE, a Mysterious Inborn Error Clarified
2019, Trends in Biochemical SciencesCitation Excerpt :However, the hypothesis was sufficiently plausible to be published [21], and it made sense to investigators in the field because there are known plasma proteins that bind calcium and that require vitamin K for post-translational modification. Indeed, several laboratories started to test the vitamin K hypothesis and conclusively proved that it was incorrect [22–25]. Nevertheless, vitamin K has remained an interesting player in the background because vitamin K is an essential cofactor for post-translational γ-carboxylation of several Ca-binding proteins [26].
A novel liquid chromatography-tandem mass spectrometry method for determination of menadione in human plasma after derivatization with 3-mercaptopropionic acid
2014, TalantaCitation Excerpt :Furthermore, VK3 is not endogenously fluorescent and requires a post-column zinc reduction for fluorescent detection. To the best of our knowledge, although the LC–MS/MS methods for determination of vitamin K3-glutathione conjugate in hepatocytes [28] and in liver [29] have been published over the years, no successful methods for the quantitative determination of VK3 using LC–MS/MS are reported so far. In recent decades, LC–MS/MS has been proven to be an extremely sensitive and specific technique for biomedical analysis.
Mineralization/anti-mineralization networks in the skin and vascular connective tissues
2013, American Journal of PathologyCitation Excerpt :However, feeding of Abcc6−/− mice with massive quantities of vitamin K did not prevent or reverse the mineralization process.31–33 Furthermore, subsequent liver perfusion experiments in Abcc6−/− mice and in Abcc6 vesicle transport systems failed to provide evidence that vitamin K would be transported by ABCC6.52 In addition to Abcc6−/− knockout mice, four naturally occurring inbred mouse strains have been recently shown to demonstrate connective tissue mineralization similar to Abcc6−/− mice.53,54
Pseudoxanthoma elasticum: Progress in research toward treatment: Summary of the 2012 PXE international research meeting
2013, Journal of Investigative DermatologyCitation Excerpt :This hypothesis has been tested in a number of laboratories by the administration of vitamin K to Abcc6−/- mice, which, however, did not counteract the aberrant mineralization of connective tissues (Brampton et al., 2011; Gorgels et al., 2011; Jiang et al., 2011). Furthermore, it was shown that ABCC6 does not transport vitamin K3–glutathione conjugate from the liver to circulation (Fülöp et al., 2011). Thus, the role of vitamin K in the pathogenesis of PXE, if any, remains unclear.
ABCC6 expression is regulated by CCAAT/enhancer-binding protein activating a primate-specific sequence located in the first intron of the gene
2012, Journal of Investigative DermatologyCitation Excerpt :This might indicate that the severity of PXE or various PXE-related phenotypes is directly linked to the metabolic status of the patients, potentially explaining the great variation of the clinical phenotype even within the same family (Varadi et al., 2011). This idea might serve as a basis of paradigm shift after the recently disproved hypothesis on the role of ABCC6 in vitamin K metabolism (Borst et al., 2008; Fulop et al., 2011) and help to decipher the pathomechanism of the disease. Finally, our results contribute to the development of treatments of PXE and PXE-related conditions as well.
Heritable Ectopic Mineralization Disorders: The Paradigm of Pseudoxanthoma Elasticum
2012, Journal of Investigative DermatologyCitation Excerpt :In this system, insect Sf9 cells are transfected with human ABCC6 expression vector, and the cells are then used to make inside-out vesicles that can be used in transport assays. This assay system has revealed that ABCC6 can transport anionic small molecular weight compounds, but specifically, ABCC6 does not transport vitamin K–glutathione conjugate (Fulop et al., 2011). Two lines of milestone studies have suggested potential treatment modalities for PXE, currently an intractable disorder.
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These authors equally contributed to the study.