pyrophosphate and Bone-Diseases--Developmental

ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. This study investigated the effects of these ANKH mutants on cellular localisation and associated biochemistry. We generated four ANKH overexpression-plasmids containing either calcium pyrophosphate deposition disease or craniometaphyseal dysplasia linked mutations: P5L, E490del and S375del, G389R. They were transfected into CH-8 articular chondrocytes and HEK293 cells. The ANKH mutants dynamic differential localisations were imaged and we investigated the interactions with the autophagy marker LC3. Extracellular inorganic pyrophosphate, mineralization, ENPP1 activity expression of ENPP1, TNAP and PIT-1 were measured. P5L delayed cell membrane localisation but once recruited into the membrane it increased extracellular inorganic pyrophosphate, mineralization, and ENPP1 activity. E490del remained mostly cytoplasmic, forming punctate co-localisations with LC3, increased mineralization, ENPP1 and ENPP1 activity with an initial but unsustained increase in TNAP and PIT-1. S375del trended to decrease extracellular inorganic pyrophosphate, increase mineralization. G389R delayed cell membrane localisation, trended to decrease extracellular inorganic pyrophosphate, increased mineralization and co-localised with LC3. Our results demonstrate a link between pathological localisation of ANKH mutants with different degrees in mineralization. Furthermore, mutant ANKH functions are related to synthesis of defective proteins, inorganic pyrophosphate transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1.

Topics: Alkaline Phosphatase; Autophagy; Bone Diseases, Developmental; Carrier Proteins; Chondrocalcinosis; Chondrocytes; Craniofacial Abnormalities; Diphosphates; HEK293 Cells; Humans; Hyperostosis; Hypertelorism; Microscopy, Confocal; Mutation; Phosphate Transport Proteins; Phosphoric Diester Hydrolases; Protein Domains; Pyrophosphatases; Transcription Factor Pit-1

Individuals with neurofibromatosis type-1 (NF1) can manifest focal skeletal dysplasias that remain extremely difficult to treat. NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase-activating protein neurofibromin. We report here that ablation of Nf1 in bone-forming cells leads to supraphysiologic accumulation of pyrophosphate (PPi), a strong inhibitor of hydroxyapatite formation, and that a chronic extracellular signal-regulated kinase (ERK)-dependent increase in expression of genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank, causes this phenotype. Nf1 ablation also prevents bone morphogenic protein-2-induced osteoprogenitor differentiation and, consequently, expression of alkaline phosphatase and PPi breakdown, further contributing to PPi accumulation. The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization. They also suggest that altered PPi homeostasis contributes to the skeletal dysplasias associated with NF1 and that some of the NF1 skeletal conditions could be prevented pharmacologically.

Topics: Adolescent; Alkaline Phosphatase; Animals; Bone Development; Bone Diseases, Developmental; Bone Morphogenetic Protein 2; Calcification, Physiologic; Cells, Cultured; Child; Child, Preschool; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type II; Diphosphates; Disease Models, Animal; Durapatite; Humans; Immunoglobulin G; Infant; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinases; Neurofibromatosis 1; Neurofibromin 1; Osteoblasts; Osteogenesis; Phosphate Transport Proteins; Phosphoric Diester Hydrolases; Pyrophosphatases; Recombinant Fusion Proteins; Sp7 Transcription Factor; Transcription Factors

Topics: Adolescent; Adult; Bone Diseases, Developmental; Child; Diagnosis, Differential; Diphosphates; Female; Humans; Male; Middle Aged; Skull; Skull Neoplasms; Technetium; Technetium Tc 99m Pyrophosphate; Tomography, Emission-Computed