ABCC6 is a member of the large ABC gene subfamily C. In this group of transmembrane proteins, in addition to active transporters such as ABCC1, −2, −3, −4, and −5, there are also ion channel-forming proteins and ion channel regulators like ABCC7 (CFTR), ABCC8 and −9 (SUR1 and −2). The work of Ilias et al. (2002) and Belinsky et al. (2002) have shown that ABCC6 is a genuine active efflux transporter that uses ATP to effectively pump a glutathione conjugate of N-ethylmaleimide (NEM-GS) and also leukotriene C4 (LTC4). The affinity of ABCC6 for LTC4 was much lower than for NEM-GS and overall the maximal rate of NEM-GS transport was markedly inferior to two other well-known glutathione conjugate-transporting proteins, ABCC1 and ABCC2. Interestingly, ABCC6 failed to effectively transport 17-β-estradiol-17-β-D-glucuronide that is otherwise a recognized a substrate for ABCC3 (Hirohashi et al., 1999). The same study also found that the most effective inhibitors for ABCC6 were benzbromarone and indomethacin. These in vitro observations demonstrated that ABCC6 has a defined perhaps restricted, substrate specificity. Though the actual endogenous substrate(s) for this transporter and hence the contributing factor to ectopic calcification in PXE, β-thalassemia, GACI, and DCC remains unknown.

A 3D configuration of ABCC6 was successfully modeled using the X-ray structure of the Staphylococcus aureus Sav1866 export pump (Dawson and Locher, 2007). This prokaryote pump has already been used as template to build other homology models for the several human ABCC transporters such as ABCC1 (DeGorter et al., 2008), ABCC4 (Ravna and Sager, 2008), ABCC5 (Ravna et al., 2007), ABCC7/CFTR (Serohijos et al., 2008) as well as ABCB1 (Zolnerciks et al., 2007), ABCG2 (Li et al., 2007). Fülöp et al. have used their model of ABCC6 and the distribution of PXE-causing mutations to demonstrate the strict relevance of the transmission interface (ICL-ABC contacts) as well as the ABC-ABC domain contacts for the function of the transporter (Fulop et al., 2009). For more information on the structure/function relation of ABCC6, see the review of Arányi et al. in this issue.

ABCC6 protein has thus far been unambiguously associated with the basolateral membrane of hepatocytes in mice, rat, and human samples (Madon et al., 2000; Beck et al., 2003; Le Saux et al., 2011) as well as in the proximal kidney tubules and a kidney cell line (Sinko et al., 2003; Beck et al., 2005). Although a recent publication is unconvincingly attempting to challenge the already well-defined cellular localization of ABCC6 (Martin et al., 2012), it is interesting to note that PXE-causing missense mutations in ABCC6 do lead to defective cellular localization of the protein along with other functional alterations of the translated proteins (Le Saux et al., 2011). Although a relatively small number of mutants were analyzed, two possible outcomes of pathological mutations were described: (1) failure to use ATP causing transport deficiency and (2) the altered folding and/or protein stability leading to intracellular retention and reduced trafficking or a combination thereof. Therefore, the various structural and functional alterations of mutated ABCC6 presumably all result in the loss of physiological function, which provides a reasonable explanation for the observed lack of phenotype–genotype correlation in PXE (Le Saux et al., 2001; Chassaing et al., 2005; Pfendner et al., 2007).

Since the identification of ABCC6 as the causative gene for PXE (Bergen et al., 2000; Le Saux et al., 2000), the question of its substrate(s) has thus far eluded all interested parties, and indeed the identification of an endogenous substrate(s) for an ABC transporter is not an easy task. Our knowledge to date is based on limited experimental data showing ABCC6's ability to use ATP to extrude conjugated metabolites in vitro (Belinsky et al., 2002; Ilias et al., 2002). Because the protein rests in the basolateral membrane of polarized cells, the prevailing hypothesis stipulates that the inability of this transporter to secrete its unknown substrate(s) for systemic circulation is the primary cause of the ectopic calcification phenotype of PXE, some GACI, and β-thalassemia patients and for DCC. This has prompted some to describe PXE as a metabolic disorder (Uitto et al., 2001), which could also apply to DCC and the ABCC6-dependent GACI and β-thalassemia cases. The metabolic hypothesis implies that the ABCC6 substrate(s) ultimately acts as an inhibitor of calcification in peripheral tissues. Does it work as a signaling molecule(s) that diffuses from the circulation into connective tissues where it contributes the normal phenotype of resident cells such as fibroblasts, or SMCs (Quaglino et al., 2000; Boraldi et al., 2003)? Or does it interact directly with the extracellular matrix, collagen fibrils, elastic fibers thereby precluding oxidative stress (Garcia-Fernandez et al., 2008), abnormal assembly/deposition of collagen fibrils (Gheduzzi et al., 2003) and elastic fiber (Le Saux et al., 2006) calcification and fragmentation? As multiple structural and molecular alterations have been noted in PXE notably the so-called “collagen flowers,” elastorrhexis along with abnormal glycoaminoglycans depositions (Lebwohl et al., 1993; Passi et al., 1996; Quaglino et al., 2000; Baccarani-Contri et al., 2001; Gheduzzi et al., 2003; Gotting et al., 2005) it is possible that a combination of molecular and cellular processes contribute to all the molecular and structural changes that have been described up to now (Figure 1). These interrogations clearly reflect a lack of sufficient data to even speculate as to the nature of the ABCC6 physiological substrate(s). And, in such situations, there are two possible approaches. Either one tests candidate molecules or performs a systematic search.

Reports by Vanakker and co-workers prompted several laboratories to test one such candidate molecule. The first of two publications described a PXE-like syndrome caused by a deficiency in gamma-glutamyl carboxylase (GGCX) (Vanakker et al., 2007) and a second article reported a deficit in the carboxylation (activation) of Gla proteins, including the calcification inhibitor Matrix Gla Protein (MGP), secondary to reduced vitamin K levels in the circulation of PXE patients (Vanakker et al., 2010). Because the common denominator between both reports is the vitamin K, the speculation that this co-factor could be an ABCC6 substrate was proposed (Borst et al., 2008) and tested in both indirect and direct experiments. The indirect approaches focused on phenotype correction in animal models of PXE, namely the calcification of vibrissae, with diets enriched in phylloquinone (vitamin K₁) and menaquinones (vitamin K₂). Three independent studies were carried out and all obtained negative results (Brampton et al., 2011; Gorgels et al., 2011; Jiang et al., 2011). These results were later confirmed by the direct observation that ABCC6 failed to effectively transport a glutathione conjugate of vitamin K₃, unlike ABCC1 (Fulop et al., 2011). Vitamin K₃ is an intermediate generated during the conversion of the dietary vitamin K₁ to the most abundant form vitamin K₂.

More recently, another candidate molecule was tested on the premise that adenosine has a key role in the etiology of arterial calcification due to deficiency of CD73 (ACDC), a rare condition that has phenotypic similarities with PXE (Markello et al., 2011). The ACDC mineralization primarily affects the large lower limb arteries (femoral, popliteal, and tibial arteries) and cartilage tissues but spares the main arterial vessels of the upper body. ACDC results from mutations in the NT5E gene that encodes the 5′ exonucleotidase CD73. This protein is a glycosyl phosphatidylinositol-anchored plasma membrane protein that generates extracellular adenosine, downstream of ENPP1 as part of the extracellular degradation pathway from ATP to adenosine and inorganic phosphate. The calcification in ACDC patients derives from an increase tissue non-specific alkaline phosphatase (TNAP) expression as a consequence of the lack of adenosine signaling. The partial overlap between the PXE and ACDC phenotypes prompted Markello et al. to suggest that adenosine might be an ABCC6 substrate (Markello et al., 2011). However, the limitations of this theory were quickly apparent as CD73 contributed to calcification only in specific arterial territories without dermal or ocular involvement (Leftheriotis et al., 2011b). Subsequent in vitro testing showed that indeed ABCC6 did not efficiently transport adenosine (Szabo et al., 2011).

As for systematic approaches, at present three groups of laboratories are reportedly undertaking experiments using animal or human tissue extracts and fluids that potentially contain the ABCC6 substrate(s). Though no publications or public reports has been made thus far, two of these groups have based their technical approaches on the principle described by Krumpochova et al. (2012) which use of a combination of inverted membrane vesicles from Spodoptera frugiperda (Sf9) insect cells overproducing ABCC6 and liquid chromatography/mass spectroscopy-based metabolomics to determine the compounds transported into the vesicles (Personal communications, O. Le Saux, A. Varadi, and P. Borst). The third group of laboratories actively seeking the ABCC6 substrate(s) uses the methodology reported by Van et al. (2008): NMR spectroscopy with animal and/or human urine and serum (Personal communications M. Dean).

As of today, the sum of current publications only paints a relatively fragmented picture of the pathophysiological role of ABCC6 that seem to favor a signaling/hormone-type of activity for ABCC6 substrate(s) toward a variety of tissues, including but not limited to, the connective tissue. For example, the observations that Fetuin-A, MGP (Hendig et al., 2006, 2008b), vitamin K (Vanakker et al., 2010), osteogenic makers, and oxidative stress (Pasquali-Ronchetti et al., 2006; Garcia-Fernandez et al., 2008; Hendig et al., 2008a) are altered in the circulation of PXE patients points indirectly to unbalanced homeostasis of multiple organs and tissues. More direct evidence have shown the presence of pathological metabolite(s) in the serum of PXE patients and Abcc6^−/− mice that promoted elastic fibers structural alterations and ectopic calcification (Le Saux et al., 2006; Jiang et al., 2007). Further, we have also described a significant expansion of the lymphatic vessel network in Abcc6^−/− mice (Le Corre et al., 2012) that emphasize the systemic and global nature of the physiological changes that accompany and/or lead to the mineralization phenotype linked to ABCC6 deficiency. This calcification has always been presented as the hallmark of PXE and the major pathological development DCC and GACI, but it might in fact be only the visible tip of the iceberg.

PXE (MIM 264800) is a recessive disease affecting the connective tissue and is defined by the calcification and fragmentation of elastic fibers (Chassaing et al., 2005). The PXE clinical signs primarily involve the skin, the Bruch's membrane of the eyes and cardiovascular system resulting in skin sagging and redundancy, visual impairment caused by retinal hemorrhages and peripheral arterial disease (PAD) associated with gastrointestinal bleeding and intermittent claudication. The localization of the transmembrane ABCC6 protein into the basolateral plasma membrane of hepatocytes and proximal kidney tubules cells is very relevant to its physiological function. It is its inability to secrete its unknown substrate toward the circulation that is the most likely cause of the ectopic calcification in PXE, which describes this disease as metabolic rather than a connective tissue disease.

The second calcification phenotype linked to ABCC6 is in fact a phenocopy of PXE. β-thalassemia (MIM 141900) is a monogenic disorder caused by mutations in the β-globin gene that leads to the underproduction of β-globin chains. The stoichiometric excess of α-chains unbound to β-globin is unstable and precipitate in red blood cell precursors forming inclusion bodies. These are responsible for the intramedullary destruction of the erythroid precursors and the ineffective erythropoiesis that characterize the β-thalassemias. Ineffective erythropoeisis in thalassemia major and certain intermedia patients results in considerable marrow expansion causing bone deformities and iron overload that is further exacerbated by frequent blood transfusions (Thein, 1998). The β-thalassemias are widespread throughout the Mediterranean, Africa, the Middle East, the Indian subcontinent, and Southeast Asia. In the past decade, it has become apparent that a large number of Mediterranean patients affected by β-thalassemia or sickle cell anemia also develop manifestations similar to PXE (Aessopos et al., 2002). β-thalassemia and PXE are distinct genetic disorders yet, the ectopic mineralization phenotype of seen in β-thalassemia patients is clinically and structurally identical to inherited PXE (Baccarani-Contri et al., 2001; Cianciulli et al., 2002; Farmakis et al., 2003, 2004). As we have established that the PXE-like mineralization in β-thalassemia patients arises independently of ABCC6 mutations (Hamlin et al., 2003), we hypothesized that the expression of the ABCC6 gene or the biological properties of its product could be disrupted in liver and/or kidneys as a secondary consequence of the hemoglobinopathy. We have tested this possibility by following the synthesis of ABCC6 in the liver and kidneys of a β-thalassemia mouse model (Hbb^th3/+). We found a progressive liver-specific downregulation in the Abcc6 gene expression and the corresponding protein levels. This downregulation became significant at 6 months of age and stabilized at older ages at ~25% of the wild type protein levels. Studying the transcriptional regulation of the Abcc6 gene revealed that the main cause of the downregulation resided with the absence of a single transcription factor, the erythroid-specific NF-E2 from the Abcc6 promoter. Coincidentally, NF-E2 is a major transcription factor for the expression of several hemoglobin-related genes (Andrews, 1998). Hbb^th3/+mice did not develop spontaneous calcification as seen in Abcc6^−/− mice probably because the ABCC6 protein decrease occurred late in life and/or was insufficient to promote mineralization in the Hbb^th3/+ mouse with the DCC-resistant C57BL/6J genetic background (Martin et al., 2011). Nevertheless, as the transcriptional regulation of the mouse and human ABCC6 genes is similar (Aranyi et al., 2005; Douet et al., 2006, 2007; de Boussac et al., 2010; Ratajewski et al., 2012), it is likely that the human β-thalassemia phenotype could induce comparable molecular changes leading to a suboptimal endowment in ABCC6 and increased susceptibility to ectopic mineralization in a PXE-like manner.

The third disease related to the ABCC6 deficiency is GACI, a rare autosomal-recessive disorder characterized by severe pathologic calcifications in the arterial media with intimal proliferation leading to vascular occlusion. GACI is associated with biallelic mutations in ENPP1 and affected patients suffer from hypertension, severe myocardial ischemia and congestive heart failure. Most affected patients die within the first 6 months of life. The obvious overlapping mineralization phenotype between GACI and PXE led to a recent study that correlated the phenotype to genotype in GACI and PXE patients (Nitschke et al., 2012). This work found clinical manifestations unique to PXE in GACI patients carrying ENPP1 mutations including angioid streaks and identical skin lesions. Additionally, mutations in ABCC6 accounted for a significant subset of GACI patients where no ENPP1 mutation was found. The authors concluded that PXE and GACI are in fact diseases with overlapping characteristics reflecting a spectrum of severity in ectopic calcification rather than two distinct entities (Nitschke et al., 2012). However, the clear resemblances between the GACI and PXE phenotypes rather suggests an underlying convergence of ENPP1 and ABCC6 molecular pathways toward a common inhibition of mineralization somewhere upstream of the phenotypic manifestations, i.e., calcification because ENPP1 deficiency leads to elastic fiber alterations typical of PXE in the vasculature, the skin, and ocular tissues (Figure 2A).

A somewhat similar convergence exists between the PXE-like syndrome in which GGCX mutations lead to insufficient MGP carboxylation (activation) and the classic inherited PXE (Gheduzzi et al., 2007; Vanakker et al., 2010). Unlike the PXE/GACI connection, the similarities between PXE and GGCX-associated syndrome represent a convergence of phenotypes rather than merging pathways (Figure 2B). The similarities are probably only due to the involvement of MGP deficiencies in both diseases. The patterns of elastic fiber mineralization are structurally different and the clinical evolution of the PXE-like syndrome diverges from PXE notably with a much greater laxity of the skin (Vanakker et al., 2007). A similar paradigm exists for other diseases such as cutis laxa, which can be either inherited through mutations in several genes related to elastic fibers or acquired through other processes affecting the structural integrity of these elastic fibers (Berk et al., 2012).

As discussed above, vitamin K or one of its derivatives is not a substrate transported by ABCC6, though the depleted levels of circulating vitamin K in PXE patients was thought to have a direct consequence in the carboxylation (activation) status of the calcification inhibitor MGP and the susceptibility to chronic calcification in PXE (Gheduzzi et al., 2007; Vanakker et al., 2007, 2010). We have shown that increasing the availability of vitamin K₁ or K₂ in peripheral tissues of Abcc6^−/− mice did not significantly affect the MGP carboxylation status in the calcified capsule of vibrissae (Brampton et al., 2011). And going further, Boraldi et al. (2012) have now established that dermal fibroblasts isolated from PXE patients were able to uptake and use vitamin K₁ or K₂ for overall protein carboxylation as efficiently as healthy fibroblasts but not for MGP, which remained specifically undercarboxylated. As described above, we found that variable ABCC6 expression levels in the liver modulated the amounts of undercarboxylated MGP in calcified cardiac tissues (Brampton et al., 2012). Taken together these results suggested that MGP or the regulation of its carboxylation process and possibly OPN correlate with ABCC6 signaling and/or the ectopic calcification status.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.