Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice

Authors

Emily G. Farrow, Xijie Yu, Lelia J. Summers, Siobhan I. Davis, James C. Fleet, Matthew R. Allen, Alexander G. Robling, Keith R. Stayrook, Victoria Jideonwo, Martin J. Magers, Holly J. Garringer, Ruben Vidal, Rebecca J. Chan, Charles B. Goodwin, Siu L. Hui, Munro Peacock, and Kenneth E. White

Abstract

Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 176RXXR179/S180 proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene–environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR.

Link to Article

http://dx.doi.org/10.1073/pnas.1110905108

A murine model of neurofibromatosis type 1 tibial pseudarthrosis featuring proliferative fibrous tissue and osteoclast-like cells

Authors

Jad El-Hoss MSc1,2,*, Kate Sullivan PhD1,2, Tegan Cheng1, Nicole YC Yu BEng(Hons)1, Justin D Bobyn BSc, MBBS2, Lauren Peacock1, Kathy Mikulec1, Paul Baldock PhD3, Ian E Alexander MBBS, FRACP, PhD2, Aaron Schindeler PhD1,2, David G Little MBBS, FRACS(Orth), PhD

Abstract

Neurofibromatosis type 1 is a common genetic condition caused by mutations in the NF1 gene. Patients often suffer from tissue specific lesions associated with local double-inactivation of NF1. In this study, we generated a novel fracture model to investigate the mechanism underlying congenital pseudarthrosis of the tibia (CPT) associated with NF1. We used a Cre-expressing adenovirus (AdCre) to inactivate Nf1 in vitro in cultured osteoprogenitors and osteoblasts, and in vivo in the fracture callus of Nf1flox/flox and Nf1flox/- mice. The effects of the presence of Nf1null cells were extensively examined. Cultured Nf1null committed osteoprogenitors from neonatal calvaria failed to differentiate and express mature osteoblastic markers, even with rhBMP-2 treatment. Similarly, Nf1null inducible osteoprogenitors obtained from Nf1 mouse muscle were also unresponsive to rhBMP-2. In both closed and open fracture models in Nf1flox/flox and Nf1flox/- mice, local AdCre injection significantly impaired bone healing, with fracture union being <50% that of wild type controls. No significant difference was seen between Nf1flox/flox and Nf1flox/- mice. Histological analyses showed invasion of the Nf1null fractures by fibrous and highly proliferative tissue. Mean amounts of fibrous tissue were increased upwards of 10-fold in Nf1null fractures and BrdU staining in closed fractures showed increased numbers of proliferating cells. In Nf1null fractures, TRAP+ cells were frequently observed within the fibrous tissue, not lining a bone surface. In summary, we report that local Nf1 deletion in a fracture callus is sufficient to impair bony union and recapitulate histological features of clinical CPT. Cell culture findings support the concept that Nf1 double inactivation impairs early osteoblastic differentiation. This model provides valuable insight into the pathobiology of the disease, and will be helpful for trialing therapeutic compounds.

Link to Article

htt://dx.doi.org/10.1002/jbmr.528/

Osteoblast extracellular Ca2+-sensing receptor regulates bone development, mineralization and turnover

Authors

Melita M. Dvorak-Ewell, Tsui-Hua Chen, Nathan Liang, Caitlin Garvey, Betty Liu, Chialing Tu, Wenhan Chang, Daniel D. Bikle, Dolores M. Shoback

Abstract

The extracellular Ca2+-sensing receptor (CaR), a G protein-coupled receptor responsible for maintenance of calcium homeostasis, is implicated in regulation of skeletal metabolism. To discern the role of the osteoblast CaR in regulation of bone development and remodeling, we generated mice in which the CaR is excised in a broad population of osteoblasts expressing the 3.6 kb a1(I) collagen promoter. Conditional knockouts had abnormal skeletal histology at birth and developed progressively reduced mineralization secondary to retarded osteoblast differentiation, evident by significantly reduced numbers of osteoblasts and decreased expression of collagen I, osteocalcin and sclerostin mRNAs. Elevated expression of ankylosis protein, ectonucleotide pyrophosphatase/phosphodiesterase 1, and osteopontin mRNAs in the conditional knockout indicate altered regulation of genes important in mineralization. Knockout of the osteoblast CaR also resulted in increased expression of the receptor activator of nuclear factor kappa B ligand (RANK-L), the major stimulator of osteoclast differentiation and function, consistent with elevated osteoclast numbers in vivo. Osteoblasts from the conditional knockouts exhibited delayed differentiation, reduced mineralizing capacity, altered expression of regulators of mineralization and increased ability to promote osteoclastogenesis in co-culture experiments. We conclude that CaR signaling in a broad population of osteoblasts is essential for bone development and remodeling and plays an important role in the regulation of differentiation and expression of regulators of bone resorption and mineralization.

Link to Article

http://dx.doi.org/10.1002/jbmr.520

Abrogation of Cbl–PI3K Interaction Increases Bone Formation and Osteoblast Proliferation

Authors

Tracy Brennan, Naga Suresh Adapala, Mary F. Barbe, Vanessa Yingling, and Archana Sanjay

Abstract

Cbl is an adaptor protein and E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and phosphorylated by Src family kinases. Phosphorylated CblY737 creates a binding site for the p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) that also plays an important role in the regulation of bone homeostasis. To investigate the role of Cbl–PI3K interaction in bone homeostasis, we examined knock-in mice in which the PI3K binding site on Cbl was ablated due to the substitution of tyrosine 737 to phenylalanine (CblYF/YF, YF mice). We previously reported that bone volume in these mice is increased due to decreased osteoclast function (Adapala et al., J Biol Chem 285:36745–36758, 19). Here, we report that YF mice also have increased bone formation and osteoblast numbers. In ex vivo cultures bone marrow-derived YF osteoblasts showed increased Col1A expression and their proliferation was also significantly augmented. Moreover, proliferation of MC3T3-E1 cells was increased after treatment with conditioned medium generated by culturing YF bone marrow stromal cells. Expression of stromal derived factor-1 (SDF-1) was increased in YF bone marrow stromal cells compared to wild type. Increased immunostaining of SDF-1 and CXCR4 was observed in YF bone marrow stromal cells compared to wild type. Treatment of YF condition medium with neutralizing anti-SDF-1 and anti-CXCR4 antibodies attenuated MC3T3-E1 cell proliferation. Cumulatively, these results show that abrogation of Cbl–PI3K interaction perturbs bone homeostasis, affecting both osteoclast function and osteoblast proliferation.

Link to Article

http://dx.doi.org/10.1007/s00223-011-9531-z

Genetic Ablation of CD68 Results in Mice with Increased Bone and Dysfunctional Osteoclasts

Authors

Jason W. Ashley, Zhenqi Shi, Haibo Zhao, Xingsheng Li, Robert A. Kesterson, Xu Feng

Abstract

CD68 is a member of the lysosome associated membrane protein (LAMP) family that is restricted in its expression to cells of the monocyte/macrophage lineage. This lineage restriction includes osteoclasts, and, while previous studies of CD68 in macrophages and dendritic cells have proposed roles in lipid metabolism, phagocytosis, and antigen presentation, the expression and function of CD68 in osteoclasts have not been explored. In this study, we investigated the expression and localization of CD68 in macrophages and osteoclasts in response to the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). We found that M-CSF stimulates CD68 expression and RANKL alters the apparent molecular weight of CD68 as measured by Western immunoblotting. In addition, we explored the significance of CD68 expression in osteoclasts by generating mice that lack expression of CD68. These mice have increased trabecular bone, and in vitro assessment of CD68−/− osteoclasts revealed that, in the absence of CD68, osteoclasts demonstrate an accumulation of intracellular vesicle-like structures, and do not efficiently resorb bone. These findings demonstrate a role for CD68 in the function of osteoclasts, and future studies will determine the mechanistic nature of the defects seen in CD68−/− osteoclasts.

Link to Article

http://dx.doi.org/10.1371/journal.pone.0025838

Trabecular bone histomorphometry in humans with type 1 diabetes Mellitus

Authors

Laura A.G. Armas, Mohammed P. Akhter, Andjela Drincic, Robert R. Recker

Abstract

Patients with Type 1 Diabetes Mellitus (DM) have markedly increased risk of fracture, but little is known about abnormalities in bone micro-architecture or remodeling properties that might give insight into the pathogenesis of skeletal fragility in these patients. We report here a case–control study comparing bone histomorphometric and micro-CT results from iliac biopsies in 18 otherwise healthy subjects with Type 1 Diabetes Mellitus with those from healthy age- and sex- matched non-diabetic control subjects. Five of the diabetics had histories of low-trauma fracture. Transilial bone biopsies were obtained after tetracycline labeling. The biopsy specimens were fixed, embedded, and scanned using a desktop μCT at 16 micron resolution. They were then sectioned and quantitative histomorphometry was performed as previously described by Recker et al. 1988.[1] Two sections, > 250 μm apart, were read from the central part of each biopsy. Overall there were no significant differences between diabetics and controls in histomorphometric or micro-CT measurements. However, fracturing diabetics had structural and dynamic trends different from nonfracturing diabetics by both methods of analysis. In conclusion, Type 1 Diabetes Mellitus does not result in abnormalities in bone histomorphometric or micro-CT variables in the absence of manifest complications from the diabetes. However, diabetics suffering fractures may have defects in their skeletal microarchitecture that may underlie the presence of excess skeletal fragility.

Link to Article

http://dx.doi.org/10.1016/j.bone.2011.09.055