Delineating the mechanisms underlying rare disorders of ectopic calcification to reveal novel therapeutic strategies
Ziegler, Shira Gillian
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Biallelic mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), a disease characterized by calcification in the skin, eyes, and blood vessels. While the function of ABCC6 and pathogenesis of PXE remains unclear, the mechanisms of related ectopic calcification disorders are better understood. Generalized arterial calcification of infancy (GACI) is caused by biallelic mutations in ENPP1, which encodes an enzyme that converts ATP to AMP and pyrophosphate (PPi), a major inhibitor of tissue calcification. AMP is further degraded to adenosine and inorganic phosphate by CD73, encoded by NT5E. We recently discovered that biallelic mutations in NT5E cause calcification of joints and arteries. We reasoned that a test for genetic interaction in mouse models of ectopic calcification might inform the pathogenesis of PXE. Enpp1-/- and Abcc6-/- mice showed fibrous capsule calcification of the vibrissae (an early marker of ectopic calcification) at 15 weeks of age, while Nt5e-/- mice calcified after one year. Abcc6-/- mice with one mutated Enpp1 or two defective Nt5e alleles showed accentuated calcification with strong statistical evidence for synergy. Additionally, ABCC6, ENPP1, and CD73 exhibited regulatory interactions; human fibroblasts with biallelic ABCC6 mutations had increased ENPP1 and decreased CD73 activity. Taken together, these data suggest that ABCC6 participates with ENPP1 and CD73 in ATP metabolism. Under osteogenic culture conditions, ABCC6 mutant cells calcified, suggesting a provoked cell-autonomous defect. Using a conditional Abcc6 knockout mouse model, we excluded the prevailing hypothesis that singularly invokes failure of hepatic secretion of an endocrine inhibitor of calcification. Instead, deficiency of Abcc6 in both local and distant cells was necessary to achieve the early onset and penetrant ectopic calcification observed upon constitutive gene targeting. Given their ability to recapitulate pathogenic events, PXE patient fibroblasts emerged as a viable model for investigating therapies. ABCC6 mutant cells additionally had increased expression and activity of tissue non-specific alkaline phosphatase (TNAP), an enzyme that degrades PPi, a major inhibitor of calcification. A novel, selective, and orally bioavailable TNAP inhibitor prevented calcification in ABCC6 mutant cells in vitro and attenuated both the development and progression of calcification in Abcc6-/- mice in vivo, without the deleterious effects on bone associated with other proposed treatment strategies. In summary, our studies provide evidence that ectopic calcification in PXE integrates both local and systemic perturbations of extracellular ATP metabolism, which can be attenuated in patient cells or Abcc6 knockout mice with a TNAP inhibitor.