Deletion of the Pyrophosphate Generating Enzyme ENPP1 Rescues Craniofacial Abnormalities in the TNAP−/− Mouse Model of Hypophosphatasia and Reveals FGF23 as a Marker of Phenotype Severity

Abstract

Hypophosphatasia is a rare heritable metabolic disorder caused by deficient Tissue Non-specific Alkaline Phosphatase (TNAP) enzyme activity. A principal function of TNAP is to hydrolyze the tissue mineralization inhibitor pyrophosphate. ENPP1 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 1) is a primary enzymatic generator of pyrophosphate and prior results showed that elimination of ENPP1 rescued bone hypomineralization of skull, vertebral and long bones to different extents in TNAP null mice. Current TNAP enzyme replacement therapy alleviates skeletal, motor and cognitive defects but does not eliminate craniosynostosis in pediatric hypophosphatasia patients. To further understand mechanisms underlying craniosynostosis development in hypophosphatasia, here we sought to determine if craniofacial abnormalities including craniosynostosis and skull shape defects would be alleviated in TNAP null mice by genetic ablation of ENPP1. Results show that homozygous deletion of ENPP1 significantly diminishes the incidence of craniosynostosis and that skull shape abnormalities are rescued by hemi- or homozygous deletion of ENPP1 in TNAP null mice. Skull and long bone hypomineralization were also alleviated in TNAP−/−/ENPP1−/− compared to TNAP−/−/ENPP1+/+ mice, though loss of ENPP1 in combination with TNAP had different effects than loss of only TNAP on long bone trabeculae. Investigation of a relatively large cohort of mice revealed that the skeletal phenotypes of TNAP null mice were markedly variable. Because FGF23 circulating levels are known to be increased in ENPP1 null mice and because FGF23 influences bone, we measured serum intact FGF23 levels in the TNAP null mice and found that a subset of TNAP−/−/ENPP1+/+ mice exhibited markedly high serum FGF23. Serum FGF23 levels also correlated to mouse body measurements, the incidence of craniosynostosis, skull shape abnormalities and skull bone density and volume fraction. Together, our results demonstrate that balanced expression of TNAP and ENPP1 enzymes are essential for microstructure and mineralization of both skull and long bones, and for preventing craniosynostosis. The results also show that FGF23 rises in the TNAP−/− model of murine lethal hypophosphatasia. Future studies are required to determine if the rise in FGF23 is a cause, consequence, or marker of disease phenotype severity.

Cranial Neural Crest Specific Deletion of Alpl (TNAP) via P0-Cre Causes Abnormal Chondrocyte Maturation and Deficient Cranial Base Growth

“…We were surprised to find that culture with P i increased ISS hypertrophic zone widths because, as stated above, P i was previously shown to be essential for promoting hypertrophic chondrocyte apoptosis in mice. Treatment with PP i increased the hypertrophic chondrocyte width and decreased apoptosis, demonstrating that high PP i levels downstream of TNAP deficiency [10,37] potentially caused the increased ISS hypertrophic zone widths seen in the Alpl fl/fl ; P0-Cre + mice.…”

“…It is also possible that the abnormal skull shape in Alpl-deficient mice occurs due to direct effects of TNAP on cranial bone growth. Nasal bones are shorter in both Alpl fl/fl ; P0-Cre + and Alpl −/− mice, and we previously reported diminished proliferation and a defect in the cell cycle progression in Alpl deficient primary calvarial cells in culture [36,37]. TNAP is expressed in cranial bone rudiments several days prior to mineralization, but its function there is not yet established [38][39][40].…”

Pro-Calcifying Role of Enzymatically Modified LDL (eLDL) in Aortic Valve Sclerosis via Induction of IL-6 and IL-33

“…Stimulation of VICs/myofibroblasts cultured in PM with eLDL resulted in upregulation of the expression of the osteogenic marker alkaline phosphatase (ALPL) (1.4-fold increase) and SP7/Osterix (5-fold increase). ALP is known to have a calcification-promoting effect by decreasing levels of the mineralization inhibitor inorganic pyrophosphate (PP i ) [45][46][47][48]. The ability of ALP to regulate pyrophosphate levels in vivo has been demonstrated by the elevated pyrophosphate levels in ALP-deficient humans [49].…”

Commensal Microbiota Effects on Craniofacial Skeletal Growth and Morphology

“…All other linear measurements were normalized to skull length (nasale to paro) to account for skull size differences, as reported previously. [ 42 , 43 , 44 ] Landmarks for craniometric analyses are depicted via schematics: skull length, skull width, and skull height (Fig. S1 ); skull length, nasal bone length, cranial vault bone lengths, and cranial base bone lengths (Fig.…”