The distribution of Abcc6 in normal mouse tissues suggests multiple functions for this ABC transporter.

We have studied the tissue distribution of Abcc6, a member of the ABC transmembrane transporter subfamily C, in normal C57BL/6 mice. RNase protection assays revealed that although almost all tissues studied contained detectable levels of the mRNA encoding Abcc6, the highest levels of Abcc6 mRNA were found in the liver. In situ hybridization (ISH) demonstrated abundant Abcc6 mRNA in epithelial cells from a variety of tissues, including hepatic parenchymal cells, bile duct epithelia, kidney proximal tubules, mucosa and gland cells of the stomach, intestine, and colon, squamous epithelium of the tongue, corneal epithelium of the eye, keratinocytes of the skin, and tracheal and bronchial epithelium. Furthermore, we detected Abcc6 mRNA in arterial endothelial cells, smooth muscle cells of the aorta and myocardium, in circulating leukocytes, lymphocytes in the thymus and lymph nodes, and in neurons of the brain, spinal cord, and the specialized neurons of the retina. Immunohistochemical analysis using a polyclonal Abcc6 rabbit antibody confirmed the tissue distribution of Abcc6 suggested by our ISH studies and revealed the cellular localization of Abcc6 in the basolateral plasma membrane in the epithelial cells of proximal convoluted tubules in the kidney. Although the function of Abcc6 is unknown, mutations in the human ABCC6 gene result in a heritable disorder of connective tissue called pseudoxanthoma elasticum (PXE). Our results demonstrating the presence of Abcc6 in epithelial and endothelial cells in a variety of tissues, including those tissues affected in PXE patients, suggest a possible role for Abcc6 in the normal assembly of extracellular matrix components. However, the presence of Abcc6 in neurons and leukocytes, two cell populations not associated with connective tissue, also suggests a more complex multifunctional role for Abcc6.     

The tissue distribution of murine Abcc6 (Mrp6) during embryogenesis indicates that the presence of Abcc6 in elastic tissues is not required for elastic fiber assembly

Summary Mutations in the gene coding for the ABC transporter, ABCC6, in humans cause Pseudoxanthoma elasticum, which is characterized by the deposition of aberrant elastic fibers. To investigate whether the presence of ABCC6 in tissues synthesizing elastin is required for elastin deposition and elastic fiber assembly, we have compared the steady-state levels and tissue distribution of Abcc6 and tropoelastin mRNAs during mouse embryogenesis. Whereas tropoelastin mRNA levels rose during embryogenesis and were the highest in neonatal mice, Abcc6 mRNA levels remained constantly low throughout embryogenesis. In some tissues, both Abcc6 and tropoelastin mRNA were detected. However, Abcc6 mRNA and protein were not detected in neonatal aorta and arteries, which produce large amounts of elastin indicating that the presence of Abcc6 in elastic tissues is not required for elastic fiber assembly.     

Clustering of disease-causing mutations on the domain-domain interfaces of ABCC6

Mutations in ABCC6 are responsible for pseudoxanthoma elasticum (PXE), a rare genetic disease affecting the elastic tissues of the body. ABCC6 encodes a 1503 amino acid long ABC transporter, ABCC6/MRP6. The functional link between the impaired activity of the protein and the disease is not known. We have built a homology model of this transporter, and analyzed the distribution of the known 119 missense PXE-associated mutations within the predicted structure. Significant clustering of the missense mutations has been found at complex domain-domain interfaces: at the transmission interface that involves four intracellular loops and the two ABC domains as well as at the ABC-ABC interacting surfaces. The mutations affecting these regions are 2.75 and 3.53-fold more frequent than the average mutational rate along the transporter protein sequence. These data provide a genetic proof of the importance of these domain-domain interactions in the ABCC6 transporter.     

Vitamin K does not prevent soft tissue mineralization in a mouse model of pseudoxanthoma elasticum

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.     

Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6)

Mutations in the ABCC6 (MRP6) gene cause pseudoxanthoma elasticum (PXE), a rare heritable disorder resulting in the calcification of elastic fibers. In the present study a cDNA encoding a full-length normal variant of ABCC6 was amplified from a human kidney cDNA library, and the protein was expressed in Sf9 insect cells. In isolated membranes ATP binding as well as ATP-dependent active transport by ABCC6 was demonstrated. We found that glutathione conjugates, including leukotriene C(4) and N-ethylmaleimide S-glutathione (NEM-GS), were actively transported by human ABCC6. Organic anions (probenecid, benzbromarone, indomethacin), known to interfere with glutathione conjugate transport of human ABCC1 and ABCC2, inhibited the ABCC6-mediated NEM-GS transport in a specific manner, indicating that ABCC6 has a unique substrate specificity. We have also expressed three missense mutant forms of ABCC6, which have recently been shown to cause PXE. MgATP binding was normal in these proteins; ATP-dependent NEM-GS or leukotriene C(4) transport, however, was abolished. Our data indicate that human ABCC6 is a primary active transporter for organic anions. In the three ABCC6 mutant forms examined, the loss of transport activity suggests that these mutations result in a PXE phenotype through a direct influence on the transport activity of this ABC transporter.     

Vitamin K does not prevent soft tissue mineralization in a mouse model of pseudoxanthoma elasticum

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.Key words: pseudoxanthoma elasticum, vitamin K, mineralization, Abcc6, mouse     

Molecular genetics of pseudoxanthoma elasticum: a metabolic disorder at the environment-genome interface?

Pseudoxanthoma elasticum (PXE) is a relatively rare heritable disorder affecting the skin, eyes and cardiovascular system, with considerable morbidity and mortality. The disease affects the elastic fibers of affected organs, which become progressively calcified. Thus, PXE has been considered as a prototypic heritable connective tissue disorder affecting the elastic fiber system. Recently, PXE has been linked to mutations in the MRP6/ABCC6 gene, a member of the ABC transporter family, expressed primarily in the liver and the kidneys. This information, together with clinical observations suggesting environmental, hormonal and/or dietary modulation of the disease, raises the intriguing possibility that PXE is a primary metabolic disorder at the environment-genome interface.     

Targeted ablation of the abcc6 gene results in ectopic mineralization of connective tissues

Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene. ABCC6 encodes multidrug resistance-associated protein 6 (MRP6), which is expressed primarily in the liver and kidneys. Mechanisms producing ectopic mineralization as a result of these mutations remain unclear. To elucidate this complex disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Mrp6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice. Particularly striking was the mineralization of vibrissae, as confirmed by von Kossa and alizarin red stains. Electron microscopy revealed mineralization affecting both elastic structures and collagen fibers. Mineralization of vibrissae was noted as early as 5 weeks of age and was progressive with age in Abcc6(-/-) mice but was not observed in Abcc6(+/-) or Abcc6(+/+) mice up to 2 years of age. A total body computerized tomography scan of Abcc6(-/-) mice revealed mineralization in skin and subcutaneous tissue as well as in the kidneys. These data demonstrate aberrant mineralization of soft tissues in PXE-affected organs, and, consequently, these mice recapitulate features of this complex disease.     

Molecular Docking Simulations Provide Insights in the Substrate Binding Sites and Possible Substrates of the ABCC6 Transporter

The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein (ABCC6), primarily expressed in liver and kidney. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive connective tissue disease characterized by ectopic mineralization of the elastic fibers. The pathophysiology underlying PXE is incompletely understood, which can at least partly be explained by the undetermined nature of the ABCC6 substrates as well as the unknown substrate recognition and binding sites. Several compounds, including anionic glutathione conjugates (N-ethylmaleimide; NEM-GS) and leukotriene C4 (LTC4) were shown to be modestly transported in vitro; conversely, vitamin K3 (VK3) was demonstrated not to be transported by ABCC6. To predict the possible substrate binding pockets of the ABCC6 transporter, we generated a 3D homology model of ABCC6 in both open and closed conformation, qualified for molecular docking and virtual screening approaches. By docking 10 reported in vitro substrates in our ABCC6 3D homology models, we were able to predict the substrate binding residues of ABCC6. Further, virtual screening of 4651 metabolites from the Human Serum Metabolome Database against our open conformation model disclosed possible substrates for ABCC6, which are mostly lipid and biliary secretion compounds, some of which are found to be involved in mineralization. Docking of these possible substrates in the closed conformation model also showed high affinity. Virtual screening expands this possibility to explore more compounds that can interact with ABCC6, and may aid in understanding the mechanisms leading to PXE.     

Large-Scaled Metabolic Profiling of Human Dermal Fibroblasts Derived from Pseudoxanthoma Elasticum Patients and Healthy Controls

Mutations in the ABC transporter ABCC6 were recently identified as cause of Pseudoxanthoma elasticum (PXE), a rare genetic disorder characterized by progressive mineralization of elastic fibers. We used an untargeted metabolic approach to identify biochemical differences between human dermal fibroblasts from healthy controls and PXE patients in an attempt to find a link between ABCC6 deficiency, cellular metabolic alterations and disease pathogenesis. 358 compounds were identified by mass spectrometry covering lipids, amino acids, peptides, carbohydrates, nucleotides, vitamins and cofactors, xenobiotics and energy metabolites. We found substantial differences in glycerophospholipid composition, leucine dipeptides, and polypeptides as well as alterations in pantothenate and guanine metabolism to be significantly associated with PXE pathogenesis. These findings can be linked to extracellular matrix remodeling and increased oxidative stress, which reflect characteristic hallmarks of PXE. Our study could facilitate a better understanding of biochemical pathways involved in soft tissue mineralization.