Conditional Deletion of Murine Fgf23: Interruption of the Normal Skeletal Responses to Phosphate Challenge and Rescue of Genetic Hypophosphatemia

Authors

Erica L. Clinkenbeard, Taryn A. Cass, Pu Ni, Julia M. Hum, Teresita Bellido, Matthew R. Allen and Kenneth E. White

Abstract

The transgenic and knock out (KO) animals involving Fgf23 have been highly informative in defining novel aspects of mineral metabolism, but are limited by shortened life span, inability of spatial/temporal FGF23 control, and infertility of the global KO. To more finely test the role of systemic and genetic influences in FGF23 production, a mouse was developed that carried a floxed (‘f’)-Fgf23 allele (exon 2 floxed) which demonstrated in vivo recombination when bred to global-Cre transgenic mice (eIIa-cre). Mice homozygous for the recombined allele (‘Δ’) had undetectable serum intact FGF23, elevated serum phosphate (p < 0.05), and increased kidney Cyp27b1 mRNA (p < 0.05) similar to global Fgf23-KO mice. To isolate cellular FGF23 responses during phosphate challenge Fgf23Δ/f mice were mated with early osteoblast type Iα1 collagen 2.3kb promoter-cre mice (Col2.3-cre) and the late osteoblast/early osteocyte Dentin matrix protein-1-cre (Dmp1-cre). Fgf23Δ/f/Col2.3-cre+ and Fgf23Δ/f/Dmp1-cre+ exhibited reduced baseline serum intact FGF23 versus controls. After challenge with high phosphate diet Cre- mice had 2.1-2.5 fold increased serum FGF23 (p < 0.01), but Col2.3-cre+ mice had no significant increase, and Dmp1-cre+ mice had only a 37% increase (p < 0.01) despite prevailing hyperphosphatemia in both models. The Fgf23Δ/f/Col2.3-cre was bred onto the Hyp (murine XLH model) genetic background to test the contribution of osteoblasts and osteocytes to elevated FGF23 and Hyp disease phenotypes. Whereas Hyp mice maintained inappropriately elevated FGF23 considering their marked hypophosphatemia, Hyp/Fgf23Δ/f/Col2.3-cre+ mice had serum FGF23 <4% of Hyp (p < 0.01), and this targeted restriction normalized serum phosphorus and ricketic bone disease. In summary, deleting FGF23 within early osteoblasts and osteocytes demonstrated that both cell types contribute to baseline circulating FGF23 concentrations, and that targeting osteoblasts/osteocytes for FGF23 production can modify systemic responses to changes in serum phosphate concentrations and rescue the Hyp genetic syndrome.

Link to Article

http://dx.doi.org/10.1002/jbmr.2792

Sclerostin antibody (Scl-Ab) improves osteomalacia phenotype in dentin matrix protein 1(Dmp1) knockout mice with little impact on serum levels of phosphorus and FGF23

Authors

Yinshi Rena, Xianglong Hana, Yan Jinga, Baozhi Yuanc, Huazhu Ked, Min Liud, Jian Q. Feng

Abstract

Unlike treatments for most rickets, the treatment using 1,25-(OH)2 vitamin D3 has little efficacy on patients with hypophosphatemic rickets, a set of rare genetic diseases. Thus, understanding the local cause for osteomalacia in hypophosphatemic rickets and developing an effective treatment to restore mineralization in this rare disease has been a longstanding goal in medicine. Here, we used Dmp1 knockout (KO) mice (whose mutations led to the same type of autosomal recessive hypophosphatemic rickets in humans) as the model in which the monoclonal antibody of sclerostin (Scl-Ab) was tested in two age groups for 8 weeks: the prevention group (starting at age 4 weeks) and the treatment group (starting at age 12 weeks). Applications of Scl-Ab greatly improved the osteomalacia phenotype (> 15%) and the biomechanical properties (3-point bending, ~ 60%) in the treated long-bone group. Our studies not only showed improvement of the osteomalacia in the alveolar bone, which has the highest bone metabolism rate, as well as the long bone phenotypes in treated mice. All these improvements attributed to the use of Scl-Ab are independent of the change in serum levels of phosphorus and FGF23, since Scl-Ab had little efficacy on those parameters. Finally, we propose a model to explain how Scl-Ab can improve the Dmp1 KO osteomalacia phenotype, in which the sclerostin level is already low.

Link to Article

http://dx.doi.org/10.1016/j.matbio.2015.12.009

Immediate effects of retinoic acid on gene expression in primary murine osteoblasts

Authors

Timur A. Yorgan, Timo Heckt, Carsten Rendenbach, Christina Helmis, Sebastian Seitz, Thomas Streichert, Michael Amling, Thorsten Schinke

Abstract

Consistent with clinical observations demonstrating that hypervitaminosis A is associated with increased skeletal fracture risk, we have previously found that dietary retinol deprivation partially corrects the bone mineralization defects in a mouse model of X-linked hypophosphatemic rickets. That retinol-dependent signaling pathways impact the skeleton is further supported by various findings demonstrating a negative influence of retinoic acid (RA) on bone-forming osteoblasts. We hypothesized that RA would directly regulate the expression of specific target genes in osteoblasts, and we aimed to identify these by genome-wide expression analyses. Here we show that high dietary retinol intake in mice causes low bone mass associated with increased osteoclastogenesis and decreased osteoblastogenesis, but intact bone matrix mineralization. We additionally found that short-term treatment of primary osteoblasts with RA causes a rapid induction of specific genes involved in either retinol-dependent signaling (i.e. Rara, Crabp2) or skeletal remodeling (i.e. Twist2,Tnfsf11). In contrast, neither expression of established osteoblast differentiation markers nor the proliferation rate was immediately affected by RA administration. Collectively, our data suggest that the negative effects of vitamin A on skeletal integrity are explainable by an immediate influence of RA signaling on specific genes in osteoblasts that in turn influence bone remodeling.

Link To Article

http://dx.doi.org/10.1007/s00774-015-0666-2

Improvement of the skeletal and dental hypophosphatasia phenotype in Alpl−/− mice by administration of soluble (non-targeted) chimeric alkaline phosphatase

Authors

Kellen Gasque, Brian L. Foster, Pia Kuss, Manisha C. Yadav, Jin Liu, Tina Kiffer-Moreira, Andrea van Elsas, Nan Hatch, Martha J. Somerman, Jose Luis Millan

Abstract

Hypophosphatasia (HPP) results from ALPL gene mutations, which lead to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP), and accumulation of inorganic pyrophosphate, a potent inhibitor of mineralization that is also a natural substrate of TNAP, in the extracellular space. HPP causes mineralization disorders including soft bones (rickets or osteomalacia) and defects in teeth and periodontal tissues. Enzyme replacement therapy using mineral-targeting recombinant TNAP has proven effective in preventing skeletal and dental defects in TNAP knockout (Alpl−/−) mice, a model for life-threatening HPP. Here, we show that the administration of a soluble, intestinal-like chimeric alkaline phosphatase (ChimAP) improves the manifestations of HPP in Alpl−/− mice. Mice received daily subcutaneous injections of ChimAP at doses of 1, 8 or 16 mg/kg, from birth for up to 53 days. Lifespan and body weight of Alpl−/− mice were normalized, and vitamin B6-associated seizures were absent with 16 mg/kg/day of ChimAP. Radiographs, μCT and histological analyses documented improved mineralization in cortical and trabecular bone and secondary ossification centers in long bones of ChimAP16-treated mice. There was no evidence of craniosynostosis in the ChimAP16-treated mice and we did not detect ectopic calcification by radiography and histology in the aortas, stomachs, kidneys or lungs in any of the treatment groups. Molar tooth development and function improved with the highest ChimAP dose, including enamel, dentin, and tooth morphology. Cementum remained deficient and alveolar bone mineralization was reduced compared to controls, though ChimAP-treated Alpl−/− mice featured periodontal attachment and retained teeth. This study provides the first evidence for the pharmacological efficacy of ChimAP for use in the treatment of skeletal and dental manifestations of HPP.

Link To Article

http://dx.doi.org/10.1016/j.bone.2014.11.017

Ablation of Osteopontin Improves the Skeletal Phenotype of Phospho1-/- Mice

Authors

Manisha C. Yadav PhD., Carmen Huesa PhD., Sonoko Narisawa PhD., Marc F. Hoylaerts PhD., Alain Moreau PhD., Colin Farquharson PhD. and José Luis Millán PhD

Abstract

PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) have non-redundant functions during skeletal mineralization. While TNAP deficiency (Alpl-/- mice) leads to hypophosphatasia, caused by accumulation of the mineralization inhibitor inorganic pyrophosphate (PPi), comparably elevated levels of PPi in Phospho1-/- mice do not explain their stunted growth, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis. We have previously shown that elevated PPi in Alpl-/- mice is accompanied by elevated osteopontin (OPN), another potent mineralization inhibitor, and that the amount of OPN correlates with the severity of hypophosphatasia in mice. Here we demonstrate that plasma OPN is elevated and OPN expression is upregulated in the skeleton, particularly in the vertebrae, of Phospho1-/- mice. Liquid chromatography/tandem mass spectrometry showed an increased proportion of phosphorylated OPN (p-OPN) peptides in Phospho1-/- mice, suggesting that accumulation of p-OPN causes the skeletal abnormalities in Phospho1-/- mice. We also show that ablation of the OPN gene, Spp1, leads to improvements in the skeletal phenotype in Phospho1-/- as they age. In particular, their scoliosis is ameliorated at 1 month of age and is completely rescued at 3 months of age. There is also improvement in the long bone defects characteristic of Phospho1-/- mice at 3 months of age. Mineralization assays comparing [Phospho1-/-; Spp1-/-], Phospho1-/- and Spp1-/- chondrocytes display corrected mineralization by the double knockout cells. Expression of chondrocyte differentiation markers was also normalized in the [Phospho1-/-; Spp1-/-] mice. Thus, while Alpl and Phospho1 deficiencies lead to similar skeletal phenotypes and comparable changes in the expression levels of PPi and OPN, there is a clear dissociation in the hierarchical roles of these potent inhibitors of mineralization, with elevated PPi and elevated p-OPN levels causing the respective skeletal phenotypes in Alpl-/- and Phospho1-/- mice.

Link To Article

http://dx.doi.org/10.1002/jbmr.2281

Neonatal iron deficiency causes abnormal phosphate metabolism by elevating FGF23 in normal and ADHR mice

Authors

Erica L. Clinkenbeard PhD, Emily G. Farrow PhD, Lelia J. Summers, Taryn A. Cass, Jessica L. Roberts, Christine A. Bayt, Tim Lahm MD, Marjorie Albrecht, Matthew R. Allen PhD, Munro Peacock MD, Kenneth E. White PhD

Abstract

FGF23 gain of function mutations can lead to autosomal dominant hypophosphatemic rickets (ADHR) disease onset at birth, or delayed onset following puberty or pregnancy. We previously demonstrated that the combination of iron deficiency and a knock-in R176Q FGF23 mutation in mature mice induced FGF23 expression and hypophosphatemia that paralleled the late onset ADHR phenotype. As anemia in pregnancy and in premature infants is common, the goal of this study was to test whether iron deficiency alters phosphate handling in neonatal life. Wild type (WT) and ADHR female breeder mice were provided control or iron-deficient diets during pregnancy and nursing. Iron-deficient breeders were also made iron replete. Iron deficient WT and ADHR pups were hypophosphatemic, with ADHR pups having significantly lower serum phosphate (P < 0.01) and widened growth plates. Both genotypes increased bone FGF23 mRNA (>50 fold; P < 0.01). WT and ADHR pups receiving low iron had elevated intact serum FGF23 with ADHR mice affected to a greater degree (P < 0.01). Iron deficient mice also showed increased Cyp24a1 and reduced Cyp27b1, and low serum 1,25(OH)2 vitamin D. Iron repletion normalized most abnormalities. Because iron deficiency can induce tissue hypoxia, oxygen deprivation was tested as a regulator of FGF23, and was shown to stimulate FGF23 mRNA in vitro and serum C-terminal FGF23 in normal rats in vivo. These studies demonstrate that FGF23 is modulated by iron status in young WT and ADHR mice and that hypoxia independently controls FGF23 expression in situations of normal iron. Therefore, disturbed iron and oxygen metabolism in neonatal life may have important effects on skeletal function and structure through FGF23 activity on phosphate regulation.

Link to Article

http://dx.doi.org/10.1002/jbmr.2049