Altered Bone Development and an Increase in FGF-23 Expression in <em>Enpp1-</em>/- Mice

Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required for the conversion of extracellular ATP into inorganic pyrophosphate (PP(i)), a recognised inhibitor of hydroxyapatite (HA) crystal formation. A detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1(-/-)) was completed to determine the role of NPP1 in skeletal and soft tissue mineralization in juvenile and adult mice. Histopathological assessment of Enpp1(-/-) mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind-limb showed hyper-mineralization in the talocrural joint and hypo-mineralization in the femur and tibia. MicroCT analysis of the tibia and femur disclosed altered trabecular architecture and bone geometry at 6 and 22 weeks of age in Enpp1(-/-) mice. Trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness were all significantly reduced in tibiae and femurs from Enpp1(-/-) mice (P<0.05). Bone stiffness as determined by 3-point bending was significantly reduced in Enpp1(-/-) tibiae and femurs from 22-week-old mice (P<0.05). Circulating phosphate and calcium levels were reduced (P<0.05) in the Enpp1(-/-) null mice. Plasma levels of osteocalcin were significantly decreased at 6 weeks of age (P<0.05) in Enpp1(-/-) mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (Ratlaps™) and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1(-/-) mice at 22 weeks of age (P<0.05). Fgf-23 messenger RNA expression in cavarial osteoblasts was increased 12-fold in Enpp1(-/-) mice compared to controls. These results indicate that Enpp1(-/-) mice are characterized by severe disruption to the architecture and mineralization of long-bones, dysregulation of calcium/phosphate homeostasis and changes in Fgf-23 expression. We conclude that NPP1 is essential for normal bone development and control of physiological bone mineralization

Body weight, body length and long bone length taken at 6 week and 22 weeks of age from male and female and <i>Enpp1^−/−</i> and wild-type mice.

<p>Data are presented as mean +/− SEM (n = 10). Significance is denoted by</p><p>*P<0.05,</p><p>**P<0.01.</p><p>***P<0.001.</p

MicroCT analysis of cortical bone in male wild-type and <i>Enpp1^−/−</i> mice.

<p>6 week femur (WT <i>n = 7</i>, <i>Enpp1^−/− n = 6</i>) and tibia (WT <i>n = 6</i>, <i>Enpp1^−/− n = 7</i>) and 22 week femur (WT and <i>Enpp1^−/− n = 9</i>) and tibia (WT <i>n = 8</i>, <i>Enpp1^−/− n = 9</i>) were tested. SEM is shown in brackets, significance is denoted by</p><p>*P<0.05,</p><p>**P<0.01.</p><p>***P<0.001.</p

MicroCT analysis of trabecular bone in male wild-type and <i>Enpp1^−/−</i> mice.

<p>6 week femur (WT and <i>Enpp1^−/− n = 7</i>) and tibia (WT <i>n = 6</i>, <i>Enpp1^−/− n = 7</i>) and 22 week femur (WT and <i>Enpp1^−/− n = 9</i>) and tibia (WT and <i>Enpp1^−/− n = 9</i>) were tested. SEM is shown in brackets, significance is denoted by</p><p>*P<0.05,</p><p>**P<0.01.</p><p>***P<0.001.</p

Measurements taken of blood serum biochemistry of female 22 week old wild-type and <i>Enpp1^−/−</i> mice.

<p>SEM is shown in brackets, significance is denoted by</p><p>*P<0.05,</p><p>**P<0.01.</p><p>*** P<0.005.</p

3-point bending shows a reduction in mechanical strength of long bones.

<p>Values are shown for tibias and femurs taken from wild-type and <i>Enpp1^−/−</i> mice at 6 weeks and 22 weeks of age. (A) Maximum stiffness was calculated from the point of maximum gradient of a polynomial curve fitted to the load-extension curve. (B) Yield is the point at which the gradient is reduced to 90% of the maximum stiffness. (C) Maximum load was defined as the highest point on the Load-extension curve. Error bars show SEM, significance is denoted by * P<0.05, ** P<0.01 *** P<0.005.</p

3D reconstruction of the trabecular bone scanned using microCT.

<p>(A) Wild-type and (B) <i>Enpp1^−/−</i> mice femurs were collected at i) 6 weeks and ii) 22 weeks of age. These reconstructions illustrate the reduction of trabecular number in the <i>Enpp1^−/−</i> mice.</p

Levels of serum markers and mRNA.

<p>(A) Osteocalcin, a marker of bone formation, (B) CTx (RatLaps TM), a marker of bone resorption, and (C) FGF-23 were measured by ELISA in serum samples taken from wild-type and <i>Enpp1^−/−</i> mice at 6 weeks and 22 weeks of age. (D) <i>Fgf-23</i> mRNA expression was assessed by RT-qPCR in calvarial bone of wild-type and <i>Enpp1^−/−</i> mice at 12 weeks of age. Error bars show SEM, significance is denoted by *P<0.05, ** P<0.01 *** P<0.005.</p