Despina Sitara

Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process

Fibroblast growth factor 23 null mice (Fgf‐23−/−) have a short lifespan and show numerous biochemical and morphological features consistent with premature aging‐like phenotypes, including kyphosis, severe muscle wasting, hypogonadism, osteopenia, emphysema, uncoordinated movement, T cell dysregulation, and atrophy of the intestinal villi, skin, thymus, and spleen. Furthermore, increased vitamin D activities in homozygous mutants are associated with severe atherosclerosis and widespread soft tissue calcifications; ablation of vitamin D activity from Fgf‐23−/− mice, by genetically deleting the 1α(OH)ase gene, eliminates atherosclerosis and ectopic calcifications and significantly rescues premature aging‐like features of Fgf‐23−/− mice, resulting in prolonged survival of Fgf‐23−/−/1α(OH)ase−/− double mutants. Our results indicate a novel role of Fgf‐23 in developing premature aging‐like features through regulating vitamin D homeostasis. Finally, our data support a new model of interactions among Fgf‐23, vitamin D, and klotho, a gene described as being associated with premature aging process.

The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice

Nearly every extracellular ligand that has been found to play a role in regulating bone biology acts, at least in part, through MAPK pathways. Nevertheless, much remains to be learned about the contribution of MAPKs to osteoblast biology in vivo. Here we report that the p38 MAPK pathway is required for normal skeletogenesis in mice, as mice with deletion of any of the MAPK pathway member-encoding genes MAPK kinase 3 (Mkk3), Mkk6, p38a, or p38b displayed profoundly reduced bone mass secondary to defective osteoblast differentiation. Among the MAPK kinase kinase (MAP3K) family, we identified TGF-beta-activated kinase 1 (TAK1; also known as MAP3K7) as the critical activator upstream of p38 in osteoblasts. Osteoblast-specific deletion of Tak1 resulted in clavicular hypoplasia and delayed fontanelle fusion, a phenotype similar to the cleidocranial dysplasia observed in humans haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2). Mechanistic analysis revealed that the TAK1-MKK3/6-p38 MAPK axis phosphorylated Runx2, promoting its association with the coactivator CREB-binding protein (CBP), which was required to regulate osteoblast genetic programs. These findings reveal an in vivo function for p38beta and establish that MAPK signaling is essential for bone formation in vivo. These results also suggest that selective p38beta agonists may represent attractive therapeutic agents to prevent bone loss associated with osteoporosis and aging.

Genetic evidence of serum phosphate-independent functions of FGF-23 on bone

Maintenance of physiologic phosphate balance is of crucial biological importance, as it is fundamental to cellular function, energy metabolism, and skeletal mineralization. Fibroblast growth factor-23 (FGF-23) is a master regulator of phosphate homeostasis, but the molecular mechanism of such regulation is not yet completely understood. Targeted disruption of the Fgf-23 gene in mice (Fgf-23−/−) elicits hyperphosphatemia, and an increase in renal sodium/phosphate co-transporter 2a (NaPi2a) protein abundance. To elucidate the pathophysiological role of augmented renal proximal tubular expression of NaPi2a in Fgf-23−/− mice and to examine serum phosphate–independent functions of Fgf23 in bone, we generated a new mouse line deficient in both Fgf-23 and NaPi2a genes, and determined the effect of genomic ablation of NaPi2a from Fgf-23−/− mice on phosphate homeostasis and skeletal mineralization. Fgf-23−/−/NaPi2a−/− double mutant mice are viable and exhibit normal physical activities when compared to Fgf-23−/− animals. Biochemical analyses show that ablation of NaPi2a from Fgf-23−/− mice reversed hyperphosphatemia to hypophosphatemia by 6 weeks of age. Surprisingly, despite the complete reversal of serum phosphate levels in Fgf-23−/−/NaPi2a−/−, their skeletal phenotype still resembles the one of Fgf23−/− animals. The results of this study provide the first genetic evidence of an in vivo pathologic role of NaPi2a in regulating abnormal phosphate homeostasis in Fgf-23−/− mice by deletion of both NaPi2a and Fgf-23 genes in the same animal. The persistence of the skeletal anomalies in double mutants suggests that Fgf-23 affects bone mineralization independently of systemic phosphate homeostasis. Finally, our data support (1) that regulation of phosphate homeostasis is a systemic effect of Fgf-23, while (2) skeletal mineralization and chondrocyte differentiation appear to be effects of Fgf-23 that are independent of phosphate homeostasis.

Zfp521 Is a Target Gene and Key Effector of Parathyroid Hormone-Related Peptide Signaling in Growth Plate Chondrocytes

In the growth plate, the interplay between parathyroid hormone-related peptide (PTHrP) and Indian hedgehog (Ihh) signaling tightly regulates chondrocyte proliferation and differentiation during longitudinal bone growth. We found that PTHrP increases the expression of Zfp521, a zinc finger transcriptional coregulator, in prehypertrophic chondrocytes. Mice with chondrocyte-targeted deletion of Zfp521 resembled PTHrP(-/-) and chondrocyte-specific PTHR1(-/-) mice, with decreased chondrocyte proliferation, early hypertrophic transition, and reduced growth plate thickness. Deleting Zfp521 increased expression of Runx2 and Runx2 target genes, and decreased Cyclin D1 and Bcl-2 expression while increasing Caspase-3 activation and apoptosis. Zfp521 associated with Runx2 in chondrocytes, antagonizing its activity via an HDAC4-dependent mechanism. PTHrP failed to upregulate Cyclin D1 and to antagonize Runx2, Ihh, and collagen X expression when Zfp521 was absent. Thus, Zfp521 is an important PTHrP target gene that regulates growth plate chondrocyte proliferation and differentiation.

Transcriptional regulation of bone and joint remodeling by NFAT

Summary:  Osteoporosis and arthritis are highly prevalent diseases and a significant cause of morbidity and mortality worldwide. These diseases result from aberrant tissue remodeling leading to weak, fracture‐prone bones or painful, dysfunctional joints. The nuclear factor of activated T cells (NFAT) transcription factor family controls diverse biologic processes in vertebrates. Here, we review the scientific evidence that links NFAT‐regulated gene transcription to bone and joint pathology. A particular emphasis is placed on the role of NFATs in bone resorption and formation by osteoclasts and osteoblasts, respectively. In addition, emerging data that connect NFATs with cartilage biology, angiogenesis, nociception, and neurogenic inflammation are explored. The goal of this article is to highlight the importance of tissue remodeling in musculoskeletal disease and situate NFAT‐driven cellular responses within this context to inspire future research endeavors.

Biological activity of FGF-23 fragments.

The phosphaturic activity of intact, full-length, fibroblast growth factor-23 (FGF-23) is well documented. FGF-23 circulates as the intact protein and as fragments generated as the result of proteolysis of the full-length protein. To assess whether short fragments of FGF-23 are phosphaturic, we compared the effect of acute, equimolar infusions of full-length FGF-23 and various FGF-23 fragments carboxyl-terminal to amino acid 176. In rats, intravenous infusions of full-length FGF-23 and FGF-23 176–251 significantly and equivalently increased fractional phosphate excretion (FE Pi) from 14 ± 3 to 32 ± 5% and 15 ± 2 to 33 ± 2% (p < 0.001), respectively. Chronic administration of FGF-23 176–251 reduced serum Pi and serum concentrations of 1α,25-dihydroxyvitamin D. Shorter forms of FGF-23 (FGF-23 180–251 and FGF-23 184–251) retained phosphaturic activity. Further shortening of the FGF-23 carboxyl-terminal domain, however, abolished phosphaturic activity, as infusion of FGF-23 206–251 did not increase urinary phosphate excretion. Infusion of a short fragment of the FGF-23 molecule, FGF-23 180–205, significantly increased FE Pi in rats and reduced serum Pi in hyperphosphatemic Fgf-23−/− knockout mice. The activity of FGF-23 180–251 was confirmed in opossum kidney cells in which the peptide reduced Na+-dependent Pi uptake and enhanced internalization of the Na+-Pi IIa co-transporter. We conclude that carboxyl terminal fragments of FGF-23 are phosphaturic and that a short, 26-amino acid fragment of FGF-23 retains significant phosphaturic activity.

FGF-23 Is a Negative Regulator of Prenatal and Postnatal Erythropoiesis

Background: FGF-23, a bone-derived hormone, regulates phosphate and vitamin D in the kidney. Results: Genetic and pharmacological manipulations of FGF-23 alter erythropoiesis and HSC frequency both in young adult age and embryonically. Conclusion: Fgf-23 regulates erythropoiesis through Epo and independent of vitamin D. Significance: These findings provide a new target for treating blood disorders associated with bone and renal defects. Abnormal blood cell production is associated with chronic kidney disease (CKD) and cardiovascular disease (CVD). Bone-derived FGF-23 (fibroblast growth factor-23) regulates phosphate homeostasis and bone mineralization. Genetic deletion of Fgf-23 in mice (Fgf-23−/−) results in hypervitaminosis D, abnormal mineral metabolism, and reduced lymphatic organ size. Elevated FGF-23 levels are linked to CKD and greater risk of CVD, left ventricular hypertrophy, and mortality in dialysis patients. However, whether FGF-23 is involved in the regulation of erythropoiesis is unknown. Here we report that loss of FGF-23 results in increased hematopoietic stem cell frequency associated with increased erythropoiesis in peripheral blood and bone marrow in young adult mice. In particular, these hematopoietic changes are also detected in fetal livers, suggesting that they are not the result of altered bone marrow niche alone. Most importantly, administration of FGF-23 in wild-type mice results in a rapid decrease in erythropoiesis. Finally, we show that the effect of FGF-23 on erythropoiesis is independent of the high vitamin D levels in these mice. Our studies suggest a novel role for FGF-23 in erythrocyte production and differentiation and suggest that elevated FGF-23 levels contribute to the pathogenesis of anemia in patients with CKD and CVD.

Amelioration of the premature ageing-like features of Fgf-23 knockout mice by genetically restoring the systemic actions of FGF-23

Genetic ablation of fibroblast growth factor 23 from mice (Fgf‐23−/−) results in a short lifespan with numerous abnormal biochemical and morphological features. Such features include kyphosis, hypogonadism and associated infertility, osteopenia, pulmonary emphysema, severe vascular and soft tissue calcifications, and generalized atrophy of various tissues. To determine whether these widespread anomalies in Fgf‐23−/− mice can be ameliorated by genetically restoring the systemic actions of FGF‐23, we generated Fgf‐23−/− mice expressing the human FGF‐23 transgene in osteoblasts under the control of the 2.3 kb α1(I) collagen promoter (Fgf‐23−/− /hFGF‐23‐Tg double mutants). This novel mouse model is completely void of all endogenous Fgf‐23 activity, but produces human FGF‐23 in bone cells that is subsequently released into the circulation. Our results suggest that lack of Fgf‐23 activities results in extensive premature ageing‐like features and early mortality of Fgf‐23−/− mice, while restoring the systemic effects of FGF‐23 significantly ameliorates these phenotypes, with the resultant effect being improved growth, restored fertility, and significantly prolonged survival of double mutants. With regard to their serum biochemistry, double mutants reversed the severe hyperphosphataemia, hypercalcaemia, and hypervitaminosis D found in Fgf‐23−/− littermates; rather, double mutants show hypophosphataemia and normal serum 1,25‐dihydroxyvitamin D3 levels similar to pure FGF‐23 Tg mice. These changes were associated with reduced renal expression of NaPi2a and 1α‐hydroxylase, compared to Fgf‐23−/− mice. FGF‐23 acts to prevent widespread abnormal features by acting systemically to regulate phosphate homeostasis and vitamin D metabolism. This novel mouse model provides us with an in vivo tool to study the systemic effects of FGF‐23 in regulating mineral ion metabolism and preventing multiple abnormal phenotypes without the interference of native Fgf‐23. Copyright

Aberrant cementum phenotype associated with the hypophosphatemic hyp mouse.

BACKGROUND Cementogenesis is sensitive to altered local phosphate levels; thus, we hypothesized a cementum phenotype, likely of decreased formation, would be present in the teeth of X-linked hypophosphatemic (Hyp) mice. Mutations in the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex) cause X-linked hypophosphatemia, characterized by rickets, osteomalacia, and hypomineralized dentin formation, a phenotype recapitulated in the Hyp mouse homolog. Here, we report a developmental study of tooth root formation in Hyp mouse molars, focusing on dentin and cementum. METHODS Light and transmission electron microscopy were used to study molar tissues from wild-type (WT) and Hyp mice. Demineralized and hematoxylin and eosin-stained tissues at developmental stages 23 to 96 days postcoital (dpc) were examined by light microscopy. Immunohistochemistry methods were used to detect bone sialoprotein (BSP) distribution in Hyp and WT mouse molar tissues, and transmission electron microscopy was used to study similar molar tissues in the non-demineralized state. RESULTS Dentin in Hyp mice exhibited mineralization defects by 33 dpc, as expected, but this defect was partially corrected by 96 dpc. In support of our hypothesis, a cementum phenotype was detected using a combination of immunohistochemistry and transmission electron microscopy, which included thinner BSP-positive staining within the cementum, discontinuous mineralization, and a globular appearance compared to WT controls. CONCLUSION Mutations in the phosphate-regulating Phex gene of the Hyp mouse resulted in defective cementum development.

PTH Ablation Ameliorates the Anomalies of Fgf23-Deficient Mice by Suppressing the Elevated Vitamin D and Calcium Levels

Fibroblast growth factor 23 (FGF23) is a key regulator of mineral ion homeostasis. Genetic ablation of Fgf23 in mice leads to severe biochemical disorders including elevated serum 1,25-dihydroxyvitamin D [1,25(OH)2D], hypercalcemia, hyperphosphatemia, and marked decreased PTH levels. Because PTH stimulates 1,25(OH)2D production and increases serum calcium levels, we hypothesized that ablation of PTH from the Fgf23 knockout (Fgf23-/-) mice could suppress these affects, thus ameliorating the soft tissue and skeletal anomalies in these animals. In this study, we generated a genetic mouse model with dual ablation of the Fgf23/PTH genes. The data show that deletion of PTH does suppress the markedly higher serum 1,25(OH)2D and calcium levels observed in Fgf23-/- mice and results in much larger, heavier, and more active double-knockout mice with improved soft tissue and skeletal phenotypes. On the contrary, when we infused PTH (1-34) peptide into Fgf23-/- mice using osmotic minipumps, serum 1,25(OH)2D and calcium levels were increased even further, leading to marked reduction in trabecular bone. These results indicate that PTH is able to modulate the anomalies of Fgf23-/- mice by controlling serum 1,25(OH)2D and calcium levels.