Restriction of Dietary Phosphate Ameliorates Skeletal Abnormalities in a Mouse Model for Craniometaphyseal Dysplasia

Abstract

Craniometaphyseal dysplasia (CMD), a rare genetic bone disorder, is characterized by lifelong progressive thickening of craniofacial bones and metaphyseal flaring of long bones. The autosomal dominant form of CMD is caused by mutations in the progressive ankylosis gene ANKH (mouse ortholog Ank), encoding a pyrophosphate (PPi) transporter. We previously reported reduced formation and function of osteoblasts and osteoclasts in a knockin (KI) mouse model for CMD (Ank KI/KI) and in CMD patients. We also showed rapid protein degradation of mutant ANK/ANKH. Mutant ANK protein displays reduced PPi transport, which may alter the inorganic phosphate (Pi) and PPi ratio, an important regulatory mechanism for bone mineralization. Here we investigate whether reducing dietary Pi intake can ameliorate the CMD-like skeletal phenotype by comparing male and female Ank +/+ and Ank KI/KI mice exposed to a low (0.3%) and normal (0.7%) Pi diet for 13 weeks from birth. Serum Pi and calcium (Ca) levels were not significantly changed by diet, whereas PTH and 25-hydroxy vitamin D (25-OHD) were decreased by low Pi diet but only in male Ank +/+ mice. Importantly, the 0.3% Pi diet significantly ameliorated mandibular hyperostosis in both sexes of Ank KI/KI mice. A tendency of decreased femoral trabeculation was observed in male and female Ank +/+ mice as well as in male Ank KI/KI mice fed with the 0.3% Pi diet. In contrast, in female Ank KI/KI mice the 0.3% Pi diet resulted in increased metaphyseal trabeculation. This was also the only group that showed increased bone formation rate. Low Pi diet led to increased osteoclast numbers and increased bone resorption in all mice. We conclude that lowering but not depleting dietary Pi delays the development of craniofacial hyperostosis in CMD mice without severely compromising serum levels of Pi, Ca, PTH, and 25-OHD. These findings may have implications for better clinical care of patients with CMD.

Ankylosis homologue mediates cellular efflux of ATP, not pyrophosphate

“…Our analyses indicated PPi concentrations in plasma of Ank ank/ank mice were about 25% lower than levels detected in their wild type littermates (wild type: 1.7 ± 0.4 µM vs Ank ank/ank 1.3 ± 0.4 µM, Fig. 3B), in line with recent data of Fujii et al (4). As all PPi detected in plasma depends on ENPP1 ( 16), the fraction (~25%)…”

“…Affected joints show massive ectopic calcification resulting in degradation of articular cartilage and ankylosis. For many years it was thought that ANKH prevents ectopic calcification by mediating cellular release of the calcification inhibitor inorganic pyrophosphate (PPi) into the joint space (2,4,5). Very recently we discovered that ANKH has an unexpected role in the extrusion of a range of cellular organic anions (6).…”

The Mineralization Regulator ANKH Mediates Cellular Efflux of ATP, Not Pyrophosphate

“…3 B ), in line with recent data of Fujii and colleagues. ( 13 ) As all PPi detected in plasma depends on ENPP1, ( 5 ) the fraction (~25%) that depends on Ank activity must be due to NTPs released into the circulation and cannot be explained by direct, Ank‐dependent, cellular efflux of PPi. This provides additional evidence that ANKH is involved in release of NTPs, not of PPi.…”

“…(11,12) For many years it was thought that ANKH directly shuttles PPi from the intracellular milieu into the extracellular environment. (1,13,14) Very recently we discovered that ANKH has an unexpected role in the extrusion of a range of cellular organic anions. (15) These include malate, succinate and especially citrate, but also nucleoside triphosphates (NTPs).…”

Hypophosphatemic rickets: An unexplained early feature of craniometaphyseal dysplasia

“…Nonetheless, supplementation with high phosphorus diet has not improved the skeletal phenotype in mice ( Liu et al, 2016 ). Conversely, a low-phosphorus diet, attenuated the craniofacial hyperostosis in ANK F377del knock-in mice ( Fujii et al, 2020 ).…”