ABCC6 does not transport vitamin K3-glutathione conjugate from the liver: Relevance to pathomechanisms of pseudoxanthoma elasticum

https://doi.org/10.1016/j.bbrc.2011.10.095Get rights and content

Abstract

Vitamin K is a cofactor required for gamma-glutamyl carboxylation of several proteins regulating blood clotting, bone formation and soft tissue mineralization. Vitamin K3 is an important intermediate during conversion of the dietary vitamin K1 to the most abundant vitamin K2 form. It has been suggested that ABCC6 may have a role in transporting vitamin K or its derivatives from the liver to the periphery. This activity is missing in pseudoxanthoma elasticum, a genetic disorder caused by mutations in ABCC6 characterized by abnormal soft tissue mineralization. Here we examined the efflux of the glutathione conjugate of vitamin K3 (VK3GS) from the liver in wild type and Abcc6−/− mice, and in transport assays in vitro. We found in liver perfusion experiments that VK3GS is secreted into the inferior vena cava, but we observed no significant difference between wild type and Abcc6−/− animals. We overexpressed the human ABCC6 transporter in Sf9 insect and MDCKII cells and assayed its vitamin K3-conjugate transport activity in vitro. We found no measurable transport of VK3GS by ABCC6, whereas ABCC1 transported this compound at high rate in these assays. These results show that VK3GS is not the essential metabolite transported by ABCC6 from the liver and preventing the symptoms 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)

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These authors equally contributed to the study.

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