Bone Marrow-Derived ABCC6 Is an Essential Regulator of Ectopic Calcification In Pseudoxanthoma Elasticum.

Physiological calcification of soft tissues is a common occurrence in aging and various acquired and inherited disorders. ABCC6 sequence variations cause the calcification phenotype of pseudoxanthoma elasticum (PXE) as well as some cases of generalized arterial calcification of infancy, which is otherwise caused by defective ENPP1. ABCC6 is primarily expressed in the liver, which has given the impression that the liver is central to the pathophysiology of PXE/generalized arterial calcification of infancy. The emergence of inflammation as a contributor to the calcification in PXE suggested that peripheral tissues play a larger role than expected. In this study, we investigated whether bone marrow-derived ABCC6 contributes to the calcification in PXE. In Abcc6‒/‒ mice, we observed prevalent mineralization in several lymph nodes and surrounding connective tissues and an extensive network of lymphatic vessels within vibrissae, a calcified tissue in Abcc6‒/‒ mice. Furthermore, we found evidence of lymphangiogenesis in patients with PXE and mouse skin, suggesting an inflammatory process. Finally, restoring wild-type bone marrow in Abcc6‒/‒ mice produced a significant reduction of calcification, suggesting that the liver alone is not sufficient to fully inhibit mineralization. With evidence that ABCC6 is expressed in lymphocytes, we suggest that the adaptative immune system and inflammation largely contribute to the calcification in PXE/generalized arterial calcification of infancy.

ABCC6 deficiency promotes dyslipidemia and atherosclerosis.

ABCC6 deficiency promotes ectopic calcification; however, circumstantial evidence suggested that ABCC6 may also influence atherosclerosis. The present study addressed the role of ABCC6 in atherosclerosis using Ldlr-/- mice and pseudoxanthoma elasticum (PXE) patients. Mice lacking the Abcc6 and Ldlr genes were fed an atherogenic diet for 16 weeks before intimal calcification, aortic plaque formation and lipoprotein profile were evaluated. Cholesterol efflux and the expression of several inflammation, atherosclerosis and cholesterol homeostasis-related genes were also determined in murine liver and bone marrow-derived macrophages. Furthermore, we examined plasma lipoproteins, vascular calcification, carotid intima-media thickness and atherosclerosis in a cohort of PXE patients with ABCC6 mutations and compared results to dysmetabolic subjects with increased cardiovascular risk. We found that ABCC6 deficiency causes changes in lipoproteins, with decreased HDL cholesterol in both mice and humans, and induces atherosclerosis. However, we found that the absence of ABCC6 does not influence overall vascular mineralization induced with atherosclerosis. Decreased cholesterol efflux from macrophage cells and other molecular changes such as increased pro-inflammation seen in both humans and mice are likely contributors for the phenotype. However, it is likely that other cellular and/or molecular mechanisms are involved. Our study showed a novel physiological role for ABCC6, influencing plasma lipoproteins and atherosclerosis in a haploinsufficient manner, with significant penetrance.

Pyrophosphate Supplementation Prevents Chronic and Acute Calcification in ABCC6-Deficient Mice.

Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6-/- mice fully inhibited the acute dystrophic cardiac calcification phenotype, whereas alendronate had no significant effect. We also found that daily injection of PPi to Abcc6-/- mice over several months prevented the development of pseudoxanthoma elasticum-like spontaneous calcification, but failed to reverse already established lesions. Furthermore, we found that the expression of low amounts of the human ABCC6 in liver of transgenic Abcc6-/- mice, resulting in only a 27% increase in plasma PPi levels, led to a major reduction in acute and chronic calcification phenotypes. This proof-of-concept study shows that the development of both acute and chronic calcification associated with ABCC6 deficiency can be prevented by compensating PPi deficits, even partially. Our work indicates that PPi substitution represents a promising strategy to treat ABCC6-dependent calcification disorders.

Functional Rescue of ABCC6 Deficiency by 4-Phenylbutyrate Therapy Reduces Dystrophic Calcification in Abcc6-/- Mice.

Soft-tissue calcification is associated with aging, common conditions such as diabetes or hypercholesterolemia, and with certain genetic disorders. ABCC6 is an efflux transporter primarily expressed in liver facilitating the release of adenosine triphosphate from hepatocytes. Within the liver vasculature, adenosine triphosphate is converted into pyrophosphate, a major inhibitor of ectopic calcification. ABCC6 mutations thus lead to reduced plasma pyrophosphate levels, resulting in the calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Most mutations in ABCC6 are missense, and many preserve transport activity but are retained intracellularly. We have previously shown that the chemical chaperone 4-phenylbutyrate (4-PBA) promotes the maturation of ABCC6 mutants to the plasma membrane. In a humanized mouse model of pseudoxanthoma elasticum, we investigated whether 4-PBA treatments could rescue the calcification inhibition potential of selected ABCC6 mutants. We used the dystrophic cardiac calcification phenotype of Abcc6-/- mice as an indicator of ABCC6 function to quantify the effect of 4-PBA on human ABCC6 mutants transiently expressed in the liver. We showed that 4-PBA administrations restored the physiological function of ABCC6 mutants, resulting in enhanced calcification inhibition. This study identifies 4-PBA treatment as a promising strategy for allele-specific therapy of ABCC6-associated calcification disorders.

The level of hepatic ABCC6 expression determines the severity of calcification after cardiac injury.

Because vascular or cardiac mineralization is inversely correlated with morbidity and long-term survival, we investigated the role of ABCC6 in the calcification response to cardiac injury in mice. By using two models of infarction, nonischemic cryoinjury and the pathologically relevant coronary artery ligation, we confirmed a large propensity to acute cardiac mineralization in Abcc6−/− mice. Furthermore, when the expression of ABCC6 was reduced to approximately 38% of wild-type levels in Abcc6+/− mice, no calcium deposits in injured cardiac tissue were observed. In addition, we used a gene therapy approach to deliver a functional human ABCC6 via hydrodynamic tail vein injection to approximately 13% of mouse hepatocytes, significantly reducing the calcification response to cardiac cryoinjury. We observed that the level and distribution of known regulators of mineralization, such as osteopontin and matrix Gla protein, but not osteocalcin, were concomitant to the level of hepatic expression of human and mouse ABCC6. We notably found that undercarboxylated matrix Gla protein precisely colocalized within areas of mineralization, whereas osteopontin was more diffusely distributed in the area of injury, suggesting a prominent association for matrix Gla protein and osteopontin in ABCC6-related dystrophic cardiac calcification. This study showed that the expression of ABCC6 in liver is an important determinant of calcification in cardiac tissues in response to injuries and is associated with changes in the expression patterns of regulators of mineralization.

Analysis of pseudoxanthoma elasticum-causing missense mutants of ABCC6 in vivo; pharmacological correction of the mislocalized proteins.

Mutations in the ABCC6 gene cause soft-tissue calcification in pseudoxanthoma elasticum (PXE) and, in some patients, generalized arterial calcification of infancy (GACI). PXE is characterized by late onset and progressive mineralization of elastic fibers in dermal, ocular, and cardiovascular tissues. GACI patients present a more severe, often prenatal arterial calcification. We have tested 10 frequent disease-causing ABCC6 missense mutants for the transport activity by using Sf9 (Spodoptera frugiperda) cells, characterized the subcellular localization in MDCKII (Madin-Darby canine kidney (cell line)) cells and in mouse liver, and tested the phenotypic rescue in zebrafish. We aimed at identifying mutants with preserved transport activity but with improper plasma membrane localization for rescue by the chemical chaperone 4-phenylbutyrate (4-PBA). Seven of the mutants were transport-competent but mislocalized in mouse liver. The observed divergence in cellular localization of mutants in MDCKII cells versus mouse liver underlined the limitations of this 2D in vitro cell system. The functionality of ABCC6 mutants was tested in zebrafish, and minimal rescue of the morpholino-induced phenotype was found. However, 4-PBA, a drug approved for clinical use, restored the plasma membrane localization of four ABCC6 mutants (R1114P, S1121W, Q1347H, and R1314W), suggesting that allele-specific therapy may be useful for selected patients with PXE and GACI.

ABCC6 is a basolateral plasma membrane protein.

RATIONALE: ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum and general arterial calcification of infancy. To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein. OBJECTIVE: In a recent article in Circulation Research, ABCC6 was reported to localize to the mitochondria-associated membrane and not the plasma membrane. As the suggested mitochondrial localization is inconsistent with published data and the presumed role of ABCC6, we performed experiments to determine the cellular localization of ABCC6 in its physiological environment. METHODS AND RESULTS: We performed immunofluorescent labeling of frozen mouse and human liver sections, as well as primary hepatocytes. We used several different antibodies recognizing human and mouse ABCC6. Our results unequivocally show that ABCC6 is in the basolateral membrane of hepatocytes and is not associated with the mitochondria, mitochondria-associated membrane, or the endoplasmic reticulum. CONCLUSIONS: Our findings support the model that ABCC6 is in the basolateral membrane, mediating the sinusoidal efflux of a metabolite from the hepatocytes to systemic circulation.

Transcriptional regulation of the ABCC6 gene and the background of impaired function of missense disease-causing mutations.

The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein expressed primarily in the liver and to a lesser extent in the kidneys and the intestines. We review here the mechanisms of this restricted tissue-specific expression and the role of hepatocyte nuclear factor 4α which is responsible for the expression pattern. Detailed analyses uncovered further regulators of the expression of the gene pointing to an intronic primate-specific regulator region, an activator of the expression of the gene by binding CCAAT/enhancer-binding protein beta, which interacts with other proteins acting in the proximal promoter. This regulatory network is affected by various environmental stimuli including oxidative stress and the extracellular signal-regulated protein kinases 1 and 2 pathway. We also review here the structural and functional consequences of disease-causing missense mutations of ABCC6. A significant clustering of the missense disease-causing mutations was found at the domain-domain interfaces. This clustering means that the domain contacts are much less permissive to amino acid replacements than the rest of the protein. We summarize the experimental methods resulting in the identification of mutants with preserved transport activity but failure in intracellular targeting. These mutants are candidates for functional rescue by chemical chaperons. The results of such research can provide the basis of future allele-specific therapy of ABCC6-mediated disorders like pseudoxanthoma elasticum or the generalized arterial calcification in infancy.

A novel telomerase activator suppresses lung damage in a murine model of idiopathic pulmonary fibrosis.

The emergence of diseases associated with telomere dysfunction, including AIDS, aplastic anemia and pulmonary fibrosis, has bolstered interest in telomerase activators. We report identification of a new small molecule activator, GRN510, with activity ex vivo and in vivo. Using a novel mouse model, we tested the potential of GRN510 to limit fibrosis induced by bleomycin in mTERT heterozygous mice. Treatment with GRN510 at 10 mg/kg/day activated telomerase 2-4 fold both in hematopoietic progenitors ex vivo and in bone marrow and lung tissue in vivo, respectively. Telomerase activation was countered by co-treatment with Imetelstat (GRN163L), a potent telomerase inhibitor. In this model of bleomycin-induced fibrosis, treatment with GRN510 suppressed the development of fibrosis and accumulation of senescent cells in the lung via a mechanism dependent upon telomerase activation. Treatment of small airway epithelial cells (SAEC) or lung fibroblasts ex vivo with GRN510 revealed telomerase activating and replicative lifespan promoting effects only in the SAEC, suggesting that the mechanism accounting for the protective effects of GRN510 against induced lung fibrosis involves specific types of lung cells. Together, these results support the use of small molecule activators of telomerase in therapies to treat idiopathic pulmonary fibrosis.

The molecular and physiological roles of ABCC6: more than meets the eye.

Abnormal mineralization occurs in the context of several common conditions, including advanced age, diabetes, hypercholesterolemia, chronic renal failure, and certain genetic conditions. Metabolic, mechanical, infectious, and inflammatory injuries promote ectopic mineralization through overlapping yet distinct molecular mechanisms of initiation and progression. The ABCC6 protein is an ATP-dependent transporter primarily found in the plasma membrane of hepatocytes. ABCC6 exports unknown substrates from the liver presumably for systemic circulation. ABCC6 deficiency is the primary cause for chronic and acute forms of ectopic mineralization described in diseases such as pseudoxanthoma elasticum (PXE), ß-thalassemia, and generalized arterial calcification of infancy (GACI) in humans and dystrophic cardiac calcification (DCC) in mice. These pathologies are characterized by mineralization of cardiovascular, ocular, and dermal tissues. PXE and to an extent GACI are caused by inactivating ABCC6 mutations, whereas the mineralization associated with ß-thalassemia patients derives from a liver-specific change in ABCC6 expression. DCC is an acquired phenotype resulting from cardiovascular insults (ischemic injury or hyperlipidemia) and secondary to ABCC6 insufficiency. Abcc6-deficient mice develop ectopic calcifications similar to both the human PXE and mouse DCC phenotypes. The precise molecular and cellular mechanism linking deficient hepatic ABCC6 function to distal ectopic mineral deposition is not understood and has captured the attention of many research groups. Our previously published work along with that of others show that ABCC6 influences other modulators of calcification and that it plays a much greater physiological role than originally thought.

Caveolin-1 deficiency protects from pulmonary fibrosis by modulating epithelial cell senescence in mice.

Idiopathic pulmonary fibrosis is associated with a decreased expression of caveolin-1 (cav-1), yet its role remains unclear. To investigate the role of cav-1, we induced pulmonary fibrosis in wild-type (WT) and cav-1-deficient (cav-1(-/-)) mice using intratracheal instillation of bleomycin. Contrary to expectations, significantly less collagen deposition was measured in tissue from cav-1(-/-) mice than in their WT counterparts, consistent with reduced mRNA expression of procollagen1a2 and procollagen3a1. Moreover, cav-1(-/-) mice demonstrated 77% less α-smooth muscle actin staining, suggesting reduced mesenchymal cell activation. Levels of pulmonary injury, assessed by tenascin-C mRNA expression and CD44v10 detection, were significantly increased at Day 21 after injury in WT mice, an effect significantly attenuated in cav-1(-/-) mice. The apparent protective effect against bleomycin-induced fibrosis in cav-1(-/-) mice was attributed to reduce cellular senescence and apoptosis in cav-1(-/-) epithelial cells during the early phase of lung injury. Reduced matrix metalloproteinase (MMP)-2 and MMP-9 expressions indicated a low profile of senescence-associated secretory phenotype (SASP) in the bleomycin-injured cav-1(-/-) mice. However, IL-6 and macrophage inflammatory protein 2 were increased in WT and cav-1(-/-) mice after bleomycin challenge, suggesting that bleomycin-induced inflammatory response substantiated the SASP pool. Thus, loss of cav-1 attenuates early injury response to bleomycin by limiting stress-induced cellular senescence/apoptosis in epithelial cells. In contrast, decreased cav-1 expression promotes fibroblast activation and collagen deposition, effects that may be relevant in later stages of reparative response. Hence, therapeutic strategies to modulate the expression of cav-1 should take into account cell-specific effects in the regenerative responses of the lung epithelium to injury.

Expression and in vivo rescue of human ABCC6 disease-causing mutants in mouse liver.

Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application.

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.

Biomembrane force probe investigation of RNA dissociation.

Investigations into the energy pathways of biomolecular interactions by use of dynamic force spectroscopy are limited by the range of loading rates accessible with a single technique. In the work discussed in this paper, this range has been extended for a previously studied system by using the biomembrane force probe (BFP). This work builds on our previous single-molecule atomic force microscopy (AFM) study of the dissociation of a bulge-motif-containing RNA complex. The disparity observed, at high loading rates, between the dissociation of a 12-base pair complex with and without a central three-base pair bulge was not observed at low rates. This suggests that the two species share a similar outer barrier to dissociation and that inclusion of the bulge motif creates an additional barrier at a distance closer to the bound state. Experiments performed in different buffer environments yielded similar results. The results, when combined with those of previous studies, suggest that the shared outer barrier to dissociation is that due to a rearrangement and fraying of the ends of the helix.