Abstract
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.
Highlights
Found in aging tissue, dystrophic calcification is defined as the abnormal deposition of calcium salts in altered or diseased tissues
As we have previously demonstrated that glutathione-conjugated N-ethylmaleimide (NEM-GS) and Leukotriene C4 (LTC4) were both efficiently transported by ABCC6 in vitro (13), we continued the characterization of the mutants by measuring their transport activity with these two substrates
Pharmacological rescue of ABCC6 mutants As it was shown that 4-PBA can partially restore cellular trafficking of the ABCC7 cystic fibrosis mutant protein (DF508), we studied whether pre-treating mice with 4-PBA
Summary
Found in aging tissue, dystrophic calcification is defined as the abnormal deposition of calcium salts in altered or diseased tissues It occurs in pathologies such as diabetes, hypercholesterolemia, chronic renal failure and certain genetic conditions. Pseudoxanthoma elasticum (PXE, OMIM 26480) in human and dystrophic cardiac calcification (DCC) in mice are similar pathologies both defined by dystrophic mineralization of cardiovascular, ocular and dermal tissues Both conditions derive from loss-of-function mutations in the human ABCC6 and mouse Abcc genes [1,2,3,4,5]. Because ABCC6 is predominantly found in liver and kidney and with little or no expression in tissues affected by PXE [8,16,17] this pathology appears to be systemic in nature This implies the presence of an abnormal circulating molecule(s) that results from the failure of ABCC6 to export its substrate(s), promoting calcification in peripheral tissues. We’ve detected the presence of such circulating molecules in the serum of adult PXE patients through their effects on elastic fibers deposited in cultures [18] and others have reached similar conclusions using a mouse model [19,20]
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