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Targeted Disruption of NF1 in Osteocytes Increases FGF23 and Osteoid With Osteomalacia-like Bone Phenotype
Abstract
Neurofibromatosis type 1 (NF1, OMIM 162200), caused by NF1 gene mutations, exhibits multi-system abnormalities including skeletal deformities in humans. Osteocytes play critical roles in controlling bone modeling and remodeling. However, the role of neurofibromin, the protein product of the NF1 gene, in osteocytes is largely unknown. This study investigated the role of neurofibromin in osteocytes by disrupting Nf1 under the Dmp1-promoter. The conditional knockout (Nf1 cKO) mice displayed serum profile of a metabolic bone disorder with an osteomalacia-like bone phenotype. Serum FGF23 levels were 4-times increased in cKO mice compared to age-matched controls. In addition, calcium-phosphorus metabolism were significantly altered (calcium; reduced, phosphorus; reduced, PTH; increase, 1,25(OH)2 D; decrease). Bone histomorphometry showed dramatically increased osteoid parameters including osteoid volume, surface, and thickness. Dynamic bone histomorphometry revealed reduced bone formation rate and mineral apposition rate in the cKO mice. TRAP staining showed a reduced osteoclast number. Micro-CT demonstrated thinner and porous cortical bones in the cKO mice, in which osteocyte dendrites were disorganized as assessed by electron microscopy. Interestingly, the cKO mice exhibited spontaneous fractures in long bones, as seen in NF1 patients. Mechanical testing of femora revealed significantly reduced maximum force and stiffness. Immunohistochemistry showed significantly increased FGF23 protein in the cKO bones. Moreover, primary osteocytes from cKO femora showed about 8-fold increase in FGF23 mRNA levels compared to control cells. The upregulation of FGF23 was specifically and significantly inhibited by PI3K inhibitor Ly294002, indicating upregulation of FGF23 through PI3K in Nf1 deficient osteocytes. Taken together, these results indicate that Nf1 deficiency in osteocytes dramatically increases FGF23 production and causes a mineralization defect (i.e. hyperosteoidosis) via the alteration of calcium-phosphorus metabolism. This study demonstrates critical roles of neurofibromin in osteocytes for osteoid mineralization. This article is protected by copyright. All rights reserved.
... Indeed, in conditional knockout for neurofibromin mice model, it was observed that primary osteocytes showed remarkable increase in the expression of FGF-23 in the serum and in the femur [32]. This was linked to abnormal calcium-phosphorus metabolism and to reduced bone formation and mineral apposition rate [34]. In the same study, micro-ct examination, also, demonstrated thinner and porous cortical bones with disorganized osteocyte dendrites that exhibited reduced strength in mechanical forces leading to spontaneous fractures [34]. ...
... This was linked to abnormal calcium-phosphorus metabolism and to reduced bone formation and mineral apposition rate [34]. In the same study, micro-ct examination, also, demonstrated thinner and porous cortical bones with disorganized osteocyte dendrites that exhibited reduced strength in mechanical forces leading to spontaneous fractures [34]. This was supported by the clinical findings of increased circulating levels of FGF-23 in two patients with NF1 that appeared severe HO [23,25]. ...
... This was supported by the clinical findings of increased circulating levels of FGF-23 in two patients with NF1 that appeared severe HO [23,25]. A possible explanation was that the increased serum concentration of FGF-23 inhibited renal reabsorption of phosphorus and decreased the production of 1,25--dihydroxyvitamin D leading to increased phosphate wasting and lower levels of phosphorus in the serum [34]. ...
Introduction:
Neurofibromatosis Type 1 (Nf1), also termed von Recklinghausen disease, is a rare autosomal dominant genetic disorder accompanied by several osseous and skeletal manifestations. In NF, hypophosphatemia linked to secondary hyperparathyroidism due to Vitamin D deficiency and low calcium intake has been reported as a risk factor for low bone mass density (BMD), but reports of NF1 associated oncogenic hypophosphatemic osteomalacia (HO) are extremely rare.
Case report:
We report a patient with NF1 associated with intracranial low-grade gliomas and congenital renal agenesis suffering from HO. Bone defects and deformities such as generalized bone pains located in feet, ankles and lower limbs, thoracic scoliosis, mild bowing of long bones of lower limbs, stress fractures, and old fractures as well as with altered bone metabolic serum markers were present. After 8 weeks of follow-up, it was observed that the combination of oral administration of phosphate and Vitamin D improved her medical symptoms without significant changes in phosphate levels or BMD.
Conclusion:
Although renal agenesis is not correlated with hypophosphatemia, the coexistence of NF1, renal congenital deformities, and low-grade gliomas may contribute to disease severity. Conventional treatment with high doses of oral calcitriol associated with phosphate is efficient to improve the clinical and laboratory symptoms of the disease.
... Similarly, immunohistochemical (IHC) staining for FGF23 and a point mutation in KRAS protooncogene has been demonstrated in a patient of metastatic adenocarcinoma of colon [8]. In a recent study involving a Nf1 conditional knockout mouse model (Nf1 cKO), it was demonstrated that bone was the source of excessive FGF23 production as assessed by immunohistochemistry (IHC) and RTPCR [9]. The FGF23 production was specifically inhibited by an inhibitor of PI3 kinase (one of the downstream signaling pathway of activated RAS) [9]. ...
... In a recent study involving a Nf1 conditional knockout mouse model (Nf1 cKO), it was demonstrated that bone was the source of excessive FGF23 production as assessed by immunohistochemistry (IHC) and RTPCR [9]. The FGF23 production was specifically inhibited by an inhibitor of PI3 kinase (one of the downstream signaling pathway of activated RAS) [9]. Furthermore, an association between the activation of AKT pathway and FGF23 production was shown in a patient with PTEN-negative Cowden syndrome [10]. ...
... Taken together, IHC data suggests neurofibromas are unlikely source of excess circulatory FGF23 levels and the bone is probably the primary source of circulatory FGF23 in the proband. Supporting this hypothesis, a recent study has shown that bone is the source of FGF23 in a Nf1 cKO mouse model [9]. ...
Context:
The mechanism behind hypophosphatemia in the setting of neurofibromatosis type 1 (NF1) is not known. We describe a possible role of fibroblast growth factor 23 (FGF23) in the pathophysiology of hypophosphatemia in a patient with NF1.
Case description:
A 34-year woman with NF1 presented with severe hypophosphatemia, osteomalacia, and elevated plasma FGF23. The patient had considerable improvement on replacement of oral phosphate. Two Ga68 DOTANOC PET-CT scans over a period of 2 years failed to detect any localized uptake. Immuno-staining for FGF23 was absent in the neural-derived tumour cells of the neurofibromas in the proband.
Conclusion:
The patient with NF1 had elevated had elevated circulating FGF23. Tumour cells in the neurofibroma tissues did not stain for FGF23 on IHC. It is unlikely for neurofibromas to contribute to high circulating FGF23 levels in the proband.
... FGF23, which is a phosphotropic hormone produced by bones [56], is mainly expressed in bony tissues, especially in osteoblasts/osteocytes, and exerts its action, after proteolytic activation, by binding to the FGF receptor-Klotho complex. Increased secretion of FGF23 from Nf-deficient osteocytes results in mineral defects and an osteomalacia-like bone phenotype [53] and has been associated with abnormal calcium-phosphorus metabolism and reduced bone formation and mineral apposition rate [57]. A possible explanation could be that the increased serum concentration of FGF-23 inhibited renal reabsorption of phosphorus and decreased the production of 1,25-dihydroxy-vitamin D leading to increased phosphate wasting and lower levels of phosphorus in the serum [57]. ...
... Increased secretion of FGF23 from Nf-deficient osteocytes results in mineral defects and an osteomalacia-like bone phenotype [53] and has been associated with abnormal calcium-phosphorus metabolism and reduced bone formation and mineral apposition rate [57]. A possible explanation could be that the increased serum concentration of FGF-23 inhibited renal reabsorption of phosphorus and decreased the production of 1,25-dihydroxy-vitamin D leading to increased phosphate wasting and lower levels of phosphorus in the serum [57]. ...
Neurofibromatosis type 1 (NF1), which is the most common phacomatoses, is an autoso-mal dominant disorder characterized by clinical presentations in various tissues and organs, such as the skin, eyes and nervous and skeletal systems. The musculoskeletal implications of NF1 include a variety of deformities, including scoliosis, kyphoscoliosis, spondylolistheses, congenital bony bowing, pseudarthrosis and bone dysplasia. Scoliosis is the most common skeletal problem, affecting 10-30% of NF1 patients. Although the pathophysiology of spinal deformities has not been elucidated yet, defects in bone metabolism have been implicated in the progression of scoliotic curves. Measurements of Bone Mineral Density (BMD) in the lumbar spine by using dual energy absorp-tiometry (DXA) and quantitative computer tomography (QCT) have demonstrated a marked reduction in Z-score and osteoporosis. Additionally, serum bone metabolic markers, such as vitamin D, calcium, phosphorus, osteocalcin and alkaline phosphatase, have been found to be abnormal. In-traoperative and histological vertebral analysis confirmed that alterations of the trabecular micro-architecture are associated with inadequate bone turnover, indicating generalized bone metabolic defects. At the molecular level, loss of function of neurofibromin dysregulates Ras and Transforming Growth factor-β1 (TGF-β1) signaling and leads to altered osteoclastic proliferation, osteoblastic activity and collagen production. Correlation between clinical characteristics and molecular pathways may provide targets for novel therapeutic approaches in NF1.
(J. Clin. Med. 2022, 11, 444)
... -/mice [9], and osteocyte-targeted Nf1 Dmp1 -/mice [23]. A common critique of these studies is to question the relevance of double-knockout models. ...
Neurofibromatosis type 1 (NF1) is a complex genetic disorder that affects a range of tissues including muscle and bone. Recent preclinical and clinical studies have shown that Nf1 deficiency in muscle causes metabolic changes resulting in intramyocellular lipid accumulation and muscle weakness. These can be subsequently rescued by dietary interventions aimed at modulating lipid availability and metabolism. It was speculated that the modified diet may rescue defects in cortical bone as NF1 deficiency has been reported to affect genes involved with lipid metabolism. Bone specimens were analyzed from wild type control mice as well as Nf1Prx1-/- (limb-targeted Nf1 knockout mice) fed standard chow versus a range of modified chows hypothesized to influence lipid metabolism. Mice were fed from 4 weeks to 12 weeks of age. MicroCT analysis was performed on the cortical bone to examine standard parameters (bone volume, tissue mineral density, cortical thickness) and specific porosity measures (closed pores corresponding to osteocyte lacunae, and larger open pores). Nf1Prx1-/- bones were found to have inferior bone properties to wild type bones, with a 4-fold increase in the porosity attributed to open pores. These measures were rescued by dietary interventions including a L-carnitine + medium-chain fatty acid supplemented chow previously shown to improve muscle histology function. Histological staining visualized these changes in bone porosity. These data support the concept that lipid metabolism may have a mechanistic impact on bone porosity and quality in NF1.
... Moreover, the canaliculi are disorganised and coarse, and the number of canaliculi per cell is significantly reduced. Overall, Nf1 deficiency in osteocytes appears to disrupt the mineralisation process, leading to an osteopenic change and osteomalacia-like bone phenotype [58]. ...
Recent years have witnessed an evolution of imaging technologies towards sophisticated approaches for visualising cells within their natural environment(s) and for investigating their interactions with other cells, with adjacent anatomical structures, and with implanted biomaterials. Resin cast etching (RCE) is an uncomplicated technique involving sequential acid etching and alkali digestion of resin embedded bone to observe the osteocyte lacuno-canalicular network using scanning electron microscopy. This review summarises the applicability of RCE to bone and the bone-implant interface. Quantitative parameters such as osteocyte size, osteocyte density, and number of canaliculi per osteocyte, and qualitative metrics including osteocyte shape, disturbances in the arrangement of osteocytes and canaliculi, and physical communication between osteocytes and implant surfaces can be investigated. Ageing, osteoporosis, long-term immobilisation, spinal cord injury, osteoarthritis, irradiation, and chronic kidney disease have been shown to impact osteocyte lacuno-canalicular network morphology. In addition to titanium, calcium phosphates, and bioactive glass, observation of direct connectivity between osteocytes and cobalt chromium provides new insights into the osseointegration potential of materials conventionally viewed as non-osseointegrating. Other applications include in vivo and in vitro testing of polymer-based tissue engineering scaffolds and tissue-engineered ossicles, validation of ectopic osteochondral defect models, ex vivo organ culture of whole bones, and observing the effects of gene dysfunction/deletion on the osteocyte lacuno-canalicular network. Without additional contrast staining, any resin embedded specimen (including clinical biopsies) can be used for RCE. The multitude of applications described here attest to the versatility of RCE for routine use within correlative analytical workflows, particularly in biomaterials science.
... For example, inhibiting the mevalonate pathway and RAS prenylation/activity with bone-targeted lovastatin, (15) inhibiting transforming growth factor β (TGF-β) signaling with a TGF-β receptor 1 (TβRI) kinase inhibitor (SD-208), (16) and inhibiting Wnt/β-catenin signaling with an adenovirus expressing Dickkopf-1 (17) or the analgesic Nefopam (18) all showed potential to promote bone healing in various mouse models of NF1 PA. Primarily assessing bones from 8 to 12 weeks of age, these models also showed that the loss of Nf1 in the osteoblast lineage leads to abnormalities in osteogenic differentiation, (19,20) collagen fiber formation, (21) increased cortical porosity (14,19,21) and reduced bone matrix mineralization, (13)(14)(15)19,21,22) the latter being associated with an osteoblastic upregulation of Ank and Enpp1, two genes directly involved in the extracellular transport and production of inorganic pyrophosphate (PPi), a strong inhibitor of matrix mineralization. (19) Tissue mineral density, mineral-to-matrix ratio, bending strength, and stiffness of mid-diaphysis cortical bone were reduced upon ablation of Nf1 in osteoprogenitor or osteochondroprogenitor cells. ...
Three‐to‐four percent of children with neurofibromatosis type 1 (NF1) present with unilateral tibia bowing, fracture, and recalcitrant healing. Alkaline phosphatase enzyme therapy prevented poor bone mineralization and poor mechanical properties in mouse models of NF1 skeletal dysplasia; but transition to clinical trials is hampered by the lack of a technique that i) identifies NF1 patients at risk of tibia bowing and fracture making them eligible for trial enrollment and ii) monitors treatment effects on matrix characteristics related to bone strength. Therefore, we assessed the ability of matrix‐sensitive techniques to provide characteristics that differentiate between cortical bone from mice characterized by postnatal loss of Nf1 in Osx‐creTet‐Off;Nf1flox/flox osteoprogenitors (cKO) and from wild‐type (WT) mice. Following euthanasia at two timepoints of bone disease progression, femur and tibia were harvested from both genotypes (n ≥ 8/age/sex/genotype). A reduction in the mid‐diaphysis ultimate force during three‐point bending at 20‐wks confirmed deleterious changes in bone induced by Nf1 deficiency, regardless of sex. Pooling females and males, low bound water (BW) and low cortical volumetric bone mineral density (Ct.vBMD) were the most accurate outcomes in distinguishing cKO from WT femurs with accuracy improving with age. Ct.vBMD and the average unloading slope (Avg‐US) from cyclic reference point indentation tests were the most sensitive in differentiating WT from cKO tibiae. Mineral‐to‐matrix ratio and carbonate substitution from Raman spectroscopy were not good classifiers. However, when combined with Ct.vBMD and BW (femur), they helped predict bending strength. Nf1 deficiency in osteoprogenitors negatively affected bone micro‐structure and matrix quality with deficits in properties becoming more pronounced with duration of Nf1 deficiency. Clinically measurable without ionizing radiation, BW and Avg‐US are sensitive to deleterious changes in bone matrix in a preclinical model of NF1 bone dysplasia and require further clinical investigation as potential indicators of an onset of bone weakness in children with NF1. This article is protected by copyright. All rights reserved.
... Studies have also reported that neuro brillin regulates the expression of FGF23( broblast growth factor 23) through the PI3K(phosphatidylinositol 3-kinase) pathway and plays an important role in bone cells. Bone cell NF1 loss leads to a signi cant increase in serum FGF23, and mineralization defects lead to bone hyperplasia, which leads to a signi cant decrease in bone mechanical strength (16). However, there is no report on the research of NF1 and OA. ...
Objective: To study the potential biomarkers and related pathways in osteoarthritis (OA) synovial lesions, and to provide theoretical basis and research directions for the pathogenesis and treatment of OA.
Methods: Download the microarray data sets GSE12021 and GSE82107 from Gene Expression Omnibus. GEO2R recognizes differentially expressed genes. Perform functional enrichment analysis of differentially expressed genes and construct protein-protein interaction network. Cytoscape performs module analysis and enrichment analysis of top-level modules. Further identify the Hub gene and perform functional enrichment analysis. TargetScan, miRDB and miRWalk three databases predict the target miRNAs of Hub gene and identify key miRNAs.
Results: Finally, 10 Hub genes and 17 key miRNAs related to the progression of OA synovitis were identified. NF1, BTRC and MAPK14 may play a vital role in OA synovial disease.
Conclusion: The Hub genes and key miRNAs discovered in this study may be potential biomarkers in the development of OA synovitis, and provide research methods and target basis for the pathogenesis and treatment of OA.
... It was initially postulated that the excess FGF-23 is produced by neurofibromas resulting in a tumor induced osteomalacia like syndrome. 3,8 However, recent literature has disputed this theory by showing absent FGF-23 staining in histopathological examination of neurofibroma of a NF1 patient with hypophosphatemic osteomalacia who had elevated circulating levels of FGF23. 2 It has been proposed that the bone may be source of excess FGF-23 in NF-1 patients. This was supported by a study done on mice, where the NF1 gene deficient osteocytes in conditional knock out mice (NF1cKO) produced excess FGF-23 in their bones and exhibited an osteomalacia-like bone phenotype. ...
Hypophosphatemic osteomalacia is a rare form of metabolic bone disorder in neurofibromatosis type 1 (NF1). The exact disease mechanism of this disorder in NF1 is yet to be established. We present a 44-year-old female known to have NF1, who presents with debilitating bone pain, weakness and multiple fractures. Laboratory investigations showed persistent hypophosphatemia with renal phosphate wasting suggestive of hypophosphatemic osteomalacia. She also had concomitant vitamin D deficiency which contributed to the disease severity. Medical therapy with oral phosphate and vitamin D improved her symptoms without significant changes in fracture healing or phosphate levels.
... MEKK2 deficiency rescues NF1-associated skeletal phenotypes. Previously, Nf1 fl/fl ;Dmp1-Cre mice have been reported as a model of skeletal NF1, finding that they display spontaneous fractures, accompanied by reduced mechanical strength, low bone mineral density, and high cortical porosity with an osteomalacia-like bone phenotype 25 . Use of the Dmp1-Cre here has the advantage that it avoids the severe growth and joint dysplasia phenotypes associated with the deletion of NF1 in early osteoprogenitors/skeletal stem cells as seen with Prx1 or Col2-Cre, which may complicate interpretation of genetic or pharmacologic rescue experiments 26,27 . ...
Neurofibromatosis type I (NF1) is characterized by prominent skeletal manifestations caused by NF1 loss. While inhibitors of the ERK activating kinases MEK1/2 are promising as a means to treat NF1, the broad blockade of the ERK pathway produced by this strategy is potentially associated with therapy limiting toxicities. Here, we have sought targets offering a more narrow inhibition of ERK activation downstream of NF1 loss in the skeleton, finding that MEKK2 is a novel component of a noncanonical ERK pathway in osteoblasts that mediates aberrant ERK activation after NF1 loss. Accordingly, despite mice with conditional deletion of Nf1 in mature osteoblasts (Nf1 fl/fl ;Dmp1-Cre) and Mekk2 −/− each displaying skeletal defects, Nf1 fl/fl ;Mekk2 −/− ;Dmp1-Cre mice show an amelioration of NF1-associated phenotypes. We also provide proof-of-principle that FDA-approved inhibitors with activity against MEKK2 can ameliorate NF1 skeletal pathology. Thus, MEKK2 functions as a MAP3K in the ERK pathway in osteoblasts, offering a potential new therapeutic strategy for the treatment of NF1.
... A second limitation is that the possibility of increased production of FGF23 from osteocytes cannot be denied. Kamiya et al. reported that serum FGF23 levels showed a four-fold increase in NF1 conditional knockout mice (cKO) compared with age-matched controls, and immunohistochemistry showed significantly increased FGF23 protein in the cKO bones [29]. Further evaluations about this should be conducted in future. ...
Background:
Neurofibromatosis type 1 is characterized by multiple café au lait spots and cutaneous and plexiform neurofibromas, and is one of the most common autosomal dominant hereditary disorders caused by mutations of the neurofibromatosis type 1 tumor suppressor gene. Osteomalacia in neurofibromatosis type 1 is very rare and is characterized by later onset in adulthood. In humans, fibroblast growth factor 23, which is a causative factor of tumor-induced osteomalacia, is not only a paracrine and autocrine factor, but is also a physiological regulator of phosphate balance in normal serum.
Case presentation:
Our patient was a 65-year-old Japanese woman whose neurofibromas began to appear when she was in elementary school. At age 28, she was diagnosed as having neurofibromatosis type 1. A spinal compression fracture and multiple rib fractures were identified in 2012 and 2017, respectively. Her laboratory findings revealed hypophosphatemia due to renal phosphate wasting and a high serum level of fibroblast growth factor 23. Neurofibromas located on the surface of her right forearm and left upper arm, in which a slight abnormal accumulation of tracers was observed on 111indium-pentetreotide scintigraphy, were surgically removed, but there was no improvement in hypophosphatemia or serum fibroblast growth factor 23 after surgery. Therefore, we administered eldecalcitol, which also failed to produce improvement in abnormal data. Subsequent combination with dibasic calcium phosphate hydrate led to improvement in some of the abnormalities, including hypophosphatemia. Immunohistochemical staining using anti-human fibroblast growth factor 23 antibody revealed slightly positive results, however, only one out of three amplifications of the fibroblast growth factor 23 gene was observed by real-time polymerase chain reaction, and no clear fibroblast growth factor 23 gene expression in the resected neurofibromas could be confirmed.
Conclusions:
We here describe a first rare case of a 65-year-old woman with neurofibromatosis type 1 associated with hypophosphatemic osteomalacia in which a high serum fibroblast growth factor 23 level was confirmed.
... Low BMD and higher rates of fractures have been described in patients with and without osseous defects [60]. The conditional knockout (Nf1 cKO) mice display significantly increased FGF23 with altered calcium-phosphorus metabolism and an osteomalacia-like bone phenotype implicating FGF-23 in the pathophysiology of the mineralization defect in NF1 [61]. Decreased bone mineral density has also been reported in Noonan syndrome [62] and Costello syndrome [63] caused by genes of the RAS-MAPK pathway. ...
Purpose of Review
To review the differential diagnosis of low bone mineral density (BMD).
Recent Findings
Osteoporosis is the most common cause of low BMD in adults; however, non-osteoporotic causes of low BMD should be considered in the differential diagnosis of patients with low BMD. Mild osteogenesis imperfecta, osteomalacia, and mineral and bone disorder of chronic kidney disease as well as several other rare diseases can be characterized by low BMD.
Summary
This review summarizes the differential diagnosis of low BMD. It is important to differentiate osteoporosis from other causes of low BMD since treatment regimens can vary tremendously between these different disease processes. In fact, some treatments for osteoporosis could worsen or exacerbate the mineral abnormalities in other causes of low BMD.
... This is important to notice because the progressive and long-term nature of tibia bowing and nonunion in NF1, and data from genetic mouse models related to this condition, all support the idea that the cell of origin for this condition is a proliferating, undifferentiated mesenchymal progenitor, prior to the expression of Col2 and Osx [10,11]. Hence, the traits and behavior of Nf1-deficient undifferentiated osteoprogenitors are likely to be more clinically relevant than the characteristics of Nf1-deficient mature osteoblasts or osteocytes for instance [60], that are unlikely to be ever generated based on the defective differentiation of Nf1-deficient osteoprogenitors. ...
Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by mutations in the NF1 gene. Recalcitrant bone healing following fracture (i.e. pseudarthrosis) is one of the most problematic skeletal complications associated with NF1. The etiology of this condition is still unclear; thus, pharmacological options for clinical management are limited. Multiple studies have shown the reduced osteogenic potential of Nf1-deficient osteoprogenitors. A recent transcriptome profiling investigation revealed that EREG and EGFR, encoding epiregulin and its receptor Epidermal Growth Factor Receptor 1, respectively, were among the top over-expressed genes in cells of the NF1 pseudarthrosis site. Because EGFR stimulation is known to inhibit osteogenic differentiation, we hypothesized that increased EREG and EGFR expression in NF1-deficient skeletal progenitors may contribute to their reduced osteogenic differentiation potential. In this study, we first confirmed via single-cell mRNA sequencing that EREG over-expression was associated with NF1 second hit somatic mutations in human bone cells, whereas Transforming Growth Factor beta 1 (TGFβ1) expression was unchanged. Second, using ex-vivo recombined Nf1-deficient mouse bone marrow stromal cells (mBMSCs), we show that this molecular signature is conserved between mice and humans, and that epiregulin generated by these cells is overexpressed and active, whereas soluble TGFβ1 expression and activity are not affected. However, blocking either epiregulin function or EGFR signaling by EGFR1 or pan EGFR inhibition (using AG-1478 and Poziotinib respectively) did not correct the differentiation defect of Nf1-deficient mBMSCs, as measured by the expression of Alpl, Ibsp and alkaline phosphatase activity. These results suggest that clinically available pharmacological strategies aimed at inhibiting EGFR signaling are unlikely to have a significant benefit for the management of bone non-union in children with NF1 PA.
Congenital pseudarthrosis of the tibia (CPT) is a severe pathology marked by spontaneous bone fractures that fail to heal, leading to fibrous nonunion. Half of patients with CPT are affected by the multisystemic genetic disorder neurofibromatosis type 1 (NF1) caused by mutations in the NF1 tumor suppressor gene, a negative regulator of RAS–mitogen-activated protein kinase (MAPK) signaling pathway. Here, we analyzed patients with CPT and Prss56-Nf1 knockout mice to elucidate the pathogenic mechanisms of CPT-related fibrous nonunion and explored a pharmacological approach to treat CPT. We identified NF1 -deficient Schwann cells and skeletal stem/progenitor cells (SSPCs) in pathological periosteum as affected cell types driving fibrosis. Whereas NF1 -deficient SSPCs adopted a fibrotic fate, NF1 -deficient Schwann cells produced critical paracrine factors including transforming growth factor–β and induced fibrotic differentiation of wild-type SSPCs. To counteract the elevated RAS-MAPK signaling in both NF1 -deficient Schwann cells and SSPCs, we used MAPK kinase (MEK) and Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) inhibitors. Combined MEK-SHP2 inhibition in vivo prevented fibrous nonunion in the Prss56-Nf1 knockout mouse model, providing a promising therapeutic strategy for the treatment of fibrous nonunion in CPT.
Congenital pseudarthrosis of the tibia (CPT, HP:0009736), commonly known as bowing of the tibia, is a rare congenital tibia malformation characterized by spontaneous tibial fractures and the difficulty of reunion after tibial fractures during early childhood, with a very low prevalence between 1/250,000 ~ 1/140,000. While 80% ~ 84% of CPT cases present with neurofibromatosis type 1, caused by the mutations in NF1, the underlying cause of CPT is still unclear. Considering the congenital nature and the low prevalence of CPT, we hypothesized that the rare genomic mutations may contribute to CPT. In this study, we conducted whole exome sequencing on 159 patients with CPT and full-length transcriptome sequencing on an additional 3 patients with CPT. The data analysis showed there were 179 significantly up-regulated genes which were enriched in 40 biological processes among which 21 biological processes hold their loss of function (LoF) excesses between 159 cases against 208 controls from 1000 Genomes Project. From those 21 biological processes with LoF excesses, there were 259 LoF-carried genes among which 40 genes with 56 LoF variations in 63 patients were enriched in osteoclast differentiation pathway (hsa04380) with its 3 directly regulated pathways including MAPK signaling pathway (hsa04010), calcium signaling pathway (hsa04020) and PI3K-Akt signaling pathway (hsa04151), as well as fluid shear stress and atherosclerosis pathway (hsa05418) while 12 patients carried 9 LoF variations in the NF1 gene. The rare LoF variations in these pathways accounted for ~39.6% of this CPT cohort. These findings shed light on the novel genetic mutations and molecular pathways involved in CPT, providing a new framework for understanding how the genetic variations regulate the biological processes in the pathology of CPT and indicating potential next directions to further elucidate the pathogenesis of CPT.
The RASopathies comprise an ever-growing number of clinical syndromes resulting from germline mutations in components of the RAS/MAPK signaling pathway. While multiple organs and tissues may be affected by these mutations, this review will focus on how these mutations specifically impact the musculoskeletal system. Herein, we review the genetics and musculoskeletal phenotypes of these syndromes in humans. We discuss how mutations in the RASopathy syndromes have been studied in translational mouse models. Finally, we discuss how signaling molecules within the RAS/MAPK pathway are involved in normal and abnormal bone biology in the context of osteoblasts, osteoclasts and chondrocytes.
Introduction:
After the onset of bone metastasis, tumor cells appear to modify surrounding microenvironments for their benefit, and particularly, the levels of circulating fibroblast growth factor (FGF) 23 in patients with tumors have been highlighted.
Materials and methods:
We have attempted to verify if human breast carcinoma MDA-MB-231 cells metastasized in the long bone of nu/nu mice would synthesize FGF23. Serum concentrations of calcium, phosphate (Pi) and FGF23 were measured in control nu/nu mice, bone-metastasized mice, and mice with mammary gland injected with MDA-MB-231 cells mimicking primary mammary tumors.
Results and conclusions:
MDA-MB-231 cells revealed intense FGF23 reactivity in metastasized lesions, whereas MDA-MB-231 cells cultured in vitro or when injected into the mammary glands (without bone metastasis) showed weak FGF23 immunoreactivity. Although the bone-metastasized MDA-MB-231 cells abundantly synthesized FGF23, osteocytes adjacent to the FGF23-immunopositive tumors, unlike intact osteocytes, showed no FGF23. Despite significantly elevated serum FGF23 levels in bone-metastasized mice, there was no significant decrease in the serum Pi concentration when compared with the intact mice and mice with a mass of MDA-MB-231 cells in mammary glands. The metastasized femora showed increased expression and FGFR1 immunoreactivity in fibroblastic stromal cells, whereas femora of control mice showed no obvious FGFR1 immunoreactivity. Taken together, it seems likely that MDA-MB-231 cells synthesize FGF23 when metastasized to a bone, and thus affect FGFR1-positive stromal cells in the metastasized tumor nest in a paracrine manner.
Biomineralization is regulated by inorganic pyrophosphate (PP i ), a potent physiological inhibitor of hydroxyapatite crystal growth. Progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) act to increase local extracellular levels of PP i , inhibiting mineralization. The periodontal complex includes 2 mineralized tissues, cementum and alveolar bone (AB), both essential for tooth attachment. Previous studies demonstrated that loss of function of ANK or ENPP1 (reducing PP i ) resulted in increased cementum formation, suggesting PP i metabolism may be a target for periodontal regenerative therapies. To compare the effects of genetic ablation of Ank, Enpp1, and both factors concurrently on cementum and AB regeneration, mandibular fenestration defects were created in Ank knockout ( Ank KO), Enpp1 mutant ( Enpp1 asj/asj ), and double KO (dKO) mice. Genetic ablation of Ank, Enpp1, or both factors increased cementum regeneration compared to controls at postoperative days (PODs) 15 and 30 ( Ank KO: 8-fold, 3-fold; Enpp1 asj/asj : 7-fold, 3-fold; dKO: 11-fold, 4-fold, respectively) associated with increased fluorochrome labeling and expression of mineralized tissue markers, dentin matrix protein 1 ( Dmp1/DMP1), osteopontin ( Spp1/OPN), and bone sialoprotein ( Ibsp/BSP). Furthermore, dKO mice featured increased cementum thickness compared to single KOs at POD15 and Ank KO at POD30. No differences were noted in AB volume between genotypes, but osteoblast/osteocyte markers were increased in all KOs, partially mineralized osteoid volume was increased in dKO versus controls at POD15 (3-fold), and mineral density was decreased in Enpp1 asj/asj and dKOs at POD30 (6% and 9%, respectively). Increased numbers of osteoclasts were present in regenerated AB of all KOs versus controls. These preclinical studies suggest PP i modulation as a potential and novel approach for cementum regeneration, particularly targeting ENPP1 and/or ANK. Differences in cementum and AB regeneration in response to reduced PP i conditions highlight the need to consider tissue-specific responses in strategies targeting regeneration of the entire periodontal complex.
Le syndrome de Noonan (SN) est une maladie génétique relativement fréquente (prévalence≈1/2000) associant de multiples défauts développementaux (cardiopathies congénitales, retard de croissance, dysmorphie et prédisposition tumorale). Le SN et des syndromes apparentés, regroupés au sein de famille des Rasopathies, sont causés par des mutations d'acteurs ou de régulateurs de la voie de signalisation Ras-MAPK et partagent l'hyperactivation de cette voie comme origine physiopathologique (Tajan et al., 2018). Au-delà des altérations développementales, de récentes études ont souligné que les Rasopathies pouvaient être associées à des anomalies du métabolisme énergétique (Dard et al., 2018). En effet, les patients atteints de Rasopathies, aussi bien que différents modèles murins de ces maladies, présentent une dépense énergétique augmentée et une réduction de leur indice de masse corporelle et de leur adiposité (Leoni, et al. 2016 ; Oba et al, 2018, Tajan et al, 2014). Cependant, les mécanismes sous-jacents à ces altérations métaboliques sont encore mal compris. Dans cette étude, nous avons tiré parti d'un modèle murin du SN, portant à l'état hétérozygote une mutation du gène codant SHP2 (SHP2D61G/+) fréquemment retrouvée chez les patients SN. Nous avons observé que les souris SN affichaient une augmentation de leur dépense énergétique sans modification majeure de leurs prises hydrique et alimentaire ou de leur activité locomotrice spontanée. De plus, les souris SN ont une réduction de tous leurs dépôts adipeux, sont résistantes à l'obésité lorsque mises en régime riche en graisse, et présentent des modifications fonctionnelles de leurs tissus adipeux. De façon intéressante, nous avons également documenté une augmentation de la densité mitochondriale, de la biogenèse et de la respiration dans le tissu adipeux inguinal des souris SN, suggérant un phénomène de " beigisation ". En accord avec ces résultats, UCP1 est spécifiquement surexprimé dans ce tissu et les souris SN présentent un phénotype de résistance au froid. D'un point de vue mécanistique, l'expression de mutants de SHP2 associés au SN augmente la transdifférenciation de cellules adipeuses souches multipotentes humaines (hMADS) vers un phénotype beige, révélant un processus cellule-autonome. De plus, un traitement chronique des souris SN par un inhibiteur pharmacologique de MEK1 normalise l'expression génique des marqueurs de mitochondriogenèse et d'UCP1, traduisant un phénotype Ras-MAPK dépendant. De manière surprenante, dans certains tissus non adipeux (foie, muscle) de même que dans une lignée cellulaire fibroblastique exprimant des mutants SN, nous avons mesuré une diminution de la fonction et/ou de la biogenèse mitochondriales, en accord avec des travaux antérieurs (Lee et al., 2009). Cette contradiction apparente pourrait être liée aux capacités oxydatives différentes des tissus/cellules considérés. A l'appui de cette hypothèse, les souris SN présentent un décalage plus rapide du quotient respiratoire vers l'utilisation du glucose, soulignant une gestion différentielle de ce substrat. Ainsi, cette étude révèle que le mutant de SHP2 associé au SN promeut des dysfonctions métaboliques complexes, ouvrant de nouvelles perspectives dans la compréhension de la physiopathologie de cette maladie. Au-delà des maladies rares, cela pourrait ouvrir de nouvelles pistes dans l'identification de mécanismes responsables de désordres métaboliques plus fréquents.
Bone morphogenetic proteins (BMPs) were first described over 50 years ago as potent inducers of ectopic bone formation when administrated subcutaneously. Preclinical studies have extensively examined the osteoinductive properties of BMPs in vitro and new bone formation in vivo. BMPs (BMP-2, BMP-7) have been used in orthopedics over 15 years. While osteogenic function of BMPs has been widely accepted, our previous studies demonstrated that loss-of-function of BMP receptor type IA (BMPR1A), a potent receptor for BMP-2, increased net bone mass by significantly inhibiting bone resorption in mice, indicating a positive role of BMP signaling in bone resorption. The physiological role of BMPs (i.e. osteogenic vs. osteoclastogenic) is still largely unknown. The purpose of this study was to investigate the physiological role of BMP signaling in endogenous long bones during adult stages. For this purpose, we conditionally and constitutively activated the Smad-dependent canonical BMP signaling thorough BMPR1A in osteoblast lineage cells using the mutant mice (Col1CreER™:caBmpr1a). Because trabecular bones were largely increased in the loss-of-function mouse study for BMPR1A, we hypothesized that the augmented BMP signaling would affect endogenous trabecular bones. In the mutant bones, the Smad phosphorylation was enhanced within physiological level three-fold while the resulting gross morphology, bodyweights, bone mass/shape/length, serum calcium/phosphorus levels, collagen cross-link patterns, and healing capability were all unchanged. Interestingly, we found; 1) increased expressions of both bone formation and resorption markers in femoral bones, 2) increased osteoblast and osteoclast numbers together with dynamic bone formation parameters by trabecular bone histomorphometry, 3) modest bone architectural phenotype with reduced bone quality (i.e. reduced trabecular bone connectivity, larger diametric size but reduced cortical bone thickness, and reduced bone mechanical strength), and 4) increased expression of SOST, a downstream target of the Smad-dependent BMPR1A signaling, in the mutant bones. This study is clinically insightful because gain-of-function of BMP signaling within a physiological window does not increase bone mass while it alters molecular and cellular aspects of osteoblast and osteoclast functions as predicted. These findings help explain the high-doses of BMPs (i.e. pharmacological level) in clinical settings required to substantially induce a bone formation, concurrent with potential unexpected side effects (i.e. bone resorption, inflammation) presumably due to a broader population of cell-types exposed to the high-dose BMPs rather than osteoblastic lineage cells.
Objectives:
Changes in serum protein levels of fibroblast growth factor 23 (FGF23) and Klotho resulting from bone metabolism are still controversial. The purpose of this study was to observe the relationship between FGF23 and Klotho serum proteins and lumbar spine bone mineral density (LBMD) in northern Chinese postmenopausal women.
Methods:
This was a community-based cross-sectional study carried out in Shenyang, a northern Chinese city. The study included 355 postmenopausal women with an average age of 62.92 ± 8.78 years. FGF23 and Klotho serum proteins were measured using a sandwich enzyme immunoassay. LBMD was examined using dual-energy X-ray absorptiometry. Pearson's correlation and regression analyses were performed to investigate the associations among them.
Results:
The LgKlotho was positively correlated with LBMD (r = 0.105). There was a linear relationship between LgKlotho serum levels and LBMD (P = 0.007) after adjusting for BMI, and the relationship still existed after adjustments for many confounding variables (P = 0.045), including age, BMI, systolic blood pressure, diastolic blood pressure, total protein, total bilirubin, high-density lipoprotein cholesterol, fasting blood glucose, serum calcium, estimated glomerular filtration rate, serum uric acid, estradiol, cigarette smoking, alcohol consumption, milk intake, calcium and vitamin D supplements, physical exercise, and fracture history in postmenopausal women. FGF23 serum levels were, however, not significantly associated with LBMD.
Conclusions:
Klotho was positively correlated with LBMD, and there was a linear relationship between Klotho serum protein levels and LBMD; however, the levels of serum Klotho were not independently associated with reduced LBMD in northern Chinese postmenopausal women. Moreover, serum FGF23 levels were not significantly related to LBMD in this sample population.
Objective:
The aim of this study was to explore the correlation between GALNT3 gene and osteoporosis.
Patients and methods:
In this study, 184 cases of osteoporosis that were treated at our hospital from 2013 to 2014 were selected as research subjects in the observation group. In addition, 84 healthy people were selected as the control group from 2013 to 2014. The bone mineral density of the observation and control groups were detected by x-rays and the expression levels and differences of mRNA of the GALNT3 gene and protein in their body was detected using fluorescence quantitative polymerase chain reaction (qPCR), enzyme-linked immunoassay, and Western blotting.
Results:
X-ray results suggest that when compared to the healthy group, bone mineral density of patients in the observation group was significantly lower than that of research subjects in the control group, with significant differences. The fluorescence qPCR results suggest that the expression levels of mRNA of the GALNT3 gene in patients with osteoporosis were significantly lower than that in the healthy group (p<0.05). Enzyme-linked immunosorbent assay (ELISA) results suggest that the expression levels of the GALNT3 gene in patients with osteoporosis (1.26±0.32) μg/L was significantly lower than that in the healthy group (12.41±0.28) μg/L, with significant differences (p<0.05). The Western blotting results agreed with the ELISA results. We also found in our research that the bone mineral density of patients with osteoporosis significantly correlated with the expression levels of the GALNT3 gene (r=0.95).
Conclusions:
Therefore, the GALNT3 gene significantly correlated with osteoporosis and the low expression of GALNT3 gene can promote the occurrence and deterioration of osteoporosis.
Noonan syndrome (NS; Mendelian Inheritance in Men (MIM) ♯163950) and related syndromes (Noonan syndrome with multiple lentigines (NS-ML, formerly called LEOPARD syndrome; MIM ♯151100), Noonan-like syndrome with loose anagen hair (NS-LAH; MIM ♯607721), Costello syndrome (CS; MIM ♯218040), Cardio-Facio-Cutaneous syndrome (CFCS; MIM ♯115150), type I Neurofibromatosis (NF1; MIM ♯162200), and Legius syndrome (LS; MIM ♯611431)) are a group of related genetic disorders, associating distinctive facial features, cardiopathies, growth and skeletal abnormalities, developmental delay/mental retardation, and tumor predisposition. Clinically described more than 50 years ago, disease genes have been identified throughout the three last decades, providing a molecular basis to better understand their physiopathology and to identify targets for therapeutic strategies. Most of those genes encode proteins belonging to or regulating the so-called RAS/Mitogen-Activated Protein Kinase (MAPK) signaling pathway, so that these syndromes have been gathered under the naming Rasopathies. In this review, we will provide a clinical overview of Rasopathies and an update of their genetics. We will then focus on the functional and pathophysiological impacts of Rasopathies-causing mutations, and discuss therapeutic perspectives and future directions.
Osteomalacia in neurofibromatosis is a rare entity and distinct from more common dysplastic skeletal affections of this disease. As a rule, it is characterized by later onset in adulthood. There is renal phosphate loss with hypophosphatemia and multiple pseudofractures in the typical cases. The hypophosphatemic conditions that interfere in bone mineralization comprise many hereditary or acquired diseases, all of them sharing the same pathophysiological mechanism-reduction in phosphate reabsorption by the renal tubuli. This process leads to chronic hyperphosphaturia and hypophosphatemia, associated with inappropriately normal or low levels of calcitriol, causing rickets in children and osteomalacia in adults.
Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in NF1. Among the earliest manifestations is tibial pseudoarthrosis and persistent nonunion after fracture. To further understand the pathogenesis of pseudoarthrosis and the underlying bone remodeling defect, pseudoarthrosis tissue and cells cultured from surgically resected pseudoarthrosis tissue from NF1 individuals were analyzed using whole-exome and whole-transcriptome sequencing as well as genomewide microarray analysis. Genomewide analysis identified multiple genetic mechanisms resulting in somatic bi-allelic NF1 inactivation; no other genes with recurring somatic mutations were identified. Gene expression profiling identified dysregulated pathways associated with neurofibromin deficiency, including phosphoinosital-3-kinase (PI3K) and mitogenactivated protein kinase (MAPK) signaling pathways. Unlike aggressive NF1-associated malignancies, tibial pseudoarthrosis tissue does not harbor a high frequency of somatic mutations in oncogenes or other tumor-suppressor genes, such as p53. However, gene expression profiling indicates pseudoarthrosis tissue has a tumor-promoting transcriptional pattern, despite lacking tumorigenic somatic mutations. Significant overexpression of specific cancer-associated genes in pseudoarthrosis highlights a potential for receptor tyrosine kinase inhibitors to target neurofibromin-deficient pseudoarthrosis and promote proper bone remodeling and fracture healing. © 2014 American Society for Bone and Mineral Research.
The human skeleton is a miracle of engineering, combining both toughness and light weight. It does so because bones possess cellular mechanisms wherein external mechanical loads are sensed. These mechanical loads are transformed into biological signals, which ultimately direct bone formation and/or bone resorption. Osteocytes, since they are ubiquitous in the mineralized matrix, are the cells that sense mechanical loads and transduce the mechanical signals into a chemical response. The osteocytes then release signaling molecules, which orchestrate the recruitment and activity of osteoblasts or osteoclasts, resulting in the adaptation of bone mass and structure. In this review, we highlight current insights in bone adaptation to external mechanical loading, with an emphasis on how a mechanical load placed on whole bones is translated and amplified into a mechanical signal that is subsequently sensed by the osteocytes. This article is part of a Special Issue entitled Osteocyte.
The last decade has provided a virtual explosion of data on the molecular biology and function of osteocytes. Far from being the passive placeholder in bone, this cell has been found to have numerous functions, such as acting as an orchestrator of bone remodeling through regulation of both osteoclast and osteoblast activity and also functioning as an endocrine cell. The osteocyte is a source of soluble factors not only to target cells on the bone surface but also to target distant organs, such as kidney, muscle, and other tissues. This cell plays a role in both phosphate metabolism and calcium availability and can remodel its perilacunar matrix. Osteocytes compose 90% to 95% of all bone cells in adult bone and are the longest lived bone cell, up to decades within their mineralized environment. As we age, these cells die, leaving behind empty lacunae that frequently micropetrose. In aged bone such as osteonecrotic bone, empty lacunae are associated with reduced remodeling. Inflammatory factors such as
Osteocytes embedded in bone have been postulated to orchestrate bone homeostasis by regulating both bone-forming osteoblasts and bone-resorbing osteoclasts. We find here that purified osteocytes express a much higher amount of receptor activator of nuclear factor-κB ligand (RANKL) and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts and bone marrow stromal cells. Furthermore, the severe osteopetrotic phenotype that we observe in mice lacking RANKL specifically in osteocytes indicates that osteocytes are the major source of RANKL in bone remodeling in vivo.
Osteoclasts resorb the mineralized matrices formed by chondrocytes or osteoblasts. The cytokine receptor activator of nuclear factor-κB ligand (RANKL) is essential for osteoclast formation and thought to be supplied by osteoblasts or their precursors, thereby linking bone formation to resorption. However, RANKL is expressed by a variety of cell types, and it is unclear which of them are essential sources for osteoclast formation. Here we have used a mouse strain in which RANKL can be conditionally deleted and a series of Cre-deleter strains to demonstrate that hypertrophic chondrocytes and osteocytes, both of which are embedded in matrix, are essential sources of the RANKL that controls mineralized cartilage resorption and bone remodeling, respectively. Moreover, osteocyte RANKL is responsible for the bone loss associated with unloading. Contrary to the current paradigm, RANKL produced by osteoblasts or their progenitors does not contribute to adult bone remodeling. These results suggest that the rate-limiting step of matrix resorption is controlled by cells embedded within the matrix itself.
Mutations in NF1 cause neurofibromatosis type I (NF1), a disorder characterized, among other clinical manifestations, by generalized and focal bony lesions. Dystrophic scoliosis and tibial pseudoarthrosis are the most severe skeletal manifestations for which treatment is not satisfactory, emphasizing the dearth of knowledge related to the biology of NF1 in bone cells. Using reporter mice, we report here that the mouse Col2α1-Cre promoter (collagen, type II, alpha 1) is active not only in chondrocytes but also in adult bone marrow osteoprogenitors giving rise to osteoblasts. Based on this finding, we crossed the Col2α1-Cre transgenic and Nf1(flox/flox) mice to determine whether loss of Nf1 in axial and appendicular osteochondroprogenitors recapitulates the skeletal abnormalities of NF1 patients. By microtomographic and X-rays studies, we show that Nf1(Col2)(-/-) mice display progressive scoliosis and kyphosis, tibial bowing and abnormalities in skull and anterior chest wall formation. These defects were accompanied by a low bone mass phenotype, high bone cortical porosity, osteoidosis, increased osteoclastogenesis and decreased osteoblast number, as quantified by histomorphometry and 3D-microtomography. Loss of Nf1 in osteochondroprogenitors also caused severe short stature and intervertebral disc defects. Blockade of the RAS/ERK activation characteristic of Nf1(-/-) osteoprogenitors by lovastatin during embryonic development could attenuate the increased cortical porosity observed in mutant pups. These data and the skeletal similarities between this mouse model and NF1 patients thus suggest that activation of the RAS/ERK pathway by Nf1 loss-of-function in osteochondroprogenitors is responsible for the vertebral and tibia lesions in NF1 patients, and that this molecular signature may represent a good therapeutic target.
Fibroblastic growth factor 23 (FGF23) is a circulating phosphaturic hormone. Inactivating mutations of the endopeptidase PHEX or the SIBLING protein DMP1 result in equivalent intrinsic bone mineralization defects and increased Fgf23 expression in osteocytes. The mechanisms whereby PHEX and DMP1 regulate Fgf23 expression are unknown. We examined the possibility that PHEX and DMP1 regulate Fgf23 through a common pathway by analyzing the phenotype of compound Phex and Dmp1 mutant mice (Hyp/Dmp1(-/-)). Compared to single-mutant littermates, compound-mutant Hyp/Dmp1(-/-) mice displayed nonadditive elevations of serum FGF23 (1912 ± 183, 1715 ± 178, and 1799 ± 181 pg/ml), hypophosphatemia (P(i): 6.0 ± 0.3, 5.8 ± 0.2, and 5.4 ± 0.1 mg/dl), and severity of rickets/osteomalacia (bone mineral density: -36, -36, and -30%). Microarray analysis of long bones identified gene expression profiles implicating common activation of the FGFR pathway in all the mutant groups. Furthermore, inhibiting FGFR signaling using SU5402 in Hyp- and Dmp1(-/-)-derived bone marrow stromal cells prevented the increase in Fgf23 mRNA expression (129- and 124-fold increase in Hyp and Dmp1(-/-) vs. 1.3-fold in Hyp+SU5402 and 2.5-fold in Dmp1(-/-)+SU5402, P<0.05). For all analyses, samples collected from nonmutant wild-type littermates served as controls. These findings indicate that PHEX and DMP1 control a common pathway regulating bone mineralization and FGF23 production, the latter involving activation of the FGFR signaling in osteocytes.
Tumor-induced osteomalacia (TIO) is a rare and fascinating paraneoplastic syndrome in which patients present with bone pain, fractures, and muscle weakness. The cause is high blood levels of the recently identified phosphate and vitamin D-regulating hormone, fibroblast growth factor 23 (FGF23). In TIO, FGF23 is secreted by mesenchymal tumors that are usually benign, but are typically very small and difficult to locate. FGF23 acts primarily at the renal tubule and impairs phosphate reabsorption and 1α-hydroxylation of 25-hydroxyvitamin D, leading to hypophosphatemia and low levels of 1,25-dihydroxy vitamin D. A step-wise approach utilizing functional imaging (F-18 fluorodeoxyglucose positron emission tomography and octreotide scintigraphy) followed by anatomical imaging (computed tomography and/or magnetic resonance imaging), and, if needed, selective venous sampling with measurement of FGF23 is usually successful in locating the tumors. For tumors that cannot be located, medical treatment with phosphate supplements and active vitamin D (calcitriol or alphacalcidiol) is usually successful; however, the medical regimen can be cumbersome and associated with complications. This review summarizes the current understanding of the pathophysiology of the disease and provides guidance in evaluating and treating these patients. Novel imaging modalities and medical treatments, which hold promise for the future, are also reviewed.
Oncogenic Osteomalacia syndrome is associated with mesenchymal tumours, caused by a protein secreted from tumours which inhibits tubular renal phosphate absorption and reduces 1,25 dihydroxy vitamin-D renal conversion. It manifests as osteomalacia with hypophosphataemia and hyperphosphaturia. Association of neurofibromatosis with oncogenic osteomalacia is unusual. We report a rare case of oncogenic osteomalacia with generalized neurofibromatosis which presented to us as pathological fracture.
The bone morphogenetic protein (BMP) and Wnt signaling pathways both contribute essential roles in regulating bone mass. However, the molecular interactions between these pathways in osteoblasts are poorly understood. We recently reported that osteoblast-targeted conditional knockout (cKO) of BMP receptor type IA (BMPRIA) resulted in increased bone mass during embryonic development, where diminished expression of Sost as a downstream effector of BMPRIA resulted in increased Wnt/beta-catenin signaling. Here, we report that Bmpr1a cKO mice exhibit increased bone mass during weanling stages, again with evidence of enhanced Wnt/beta-catenin signaling as assessed by Wnt reporter TOPGAL mice and TOPFLASH luciferase. Consistent with negative regulation of the Wnt pathway by BMPRIA signaling, treatment of osteoblasts with dorsomorphin, an inhibitor of Smad-dependent BMP signaling, enhanced Wnt signaling. In addition to Sost, Wnt inhibitor Dkk1 also was downregulated in cKO bone. Expression levels of Dkk1and Sost were upregulated by BMP2 treatment and downregulated by Noggin. Moreover, expression of a constitutively active Bmpr1a transgene in mice resulted in the upregulation of both Dkk1 and Sost and partially rescued the Bmpr1a cKO bone phenotype. These effectors are differentially regulated by mitogen-activated protein kinase (MAPK) p38 because pretreatment of osteoblasts with SB202190 blocked BMP2-induced Dkk1 expression but not Sost. These results demonstrate that BMPRIA in osteoblasts negatively regulates endogenous bone mass and Wnt/beta-catenin signaling and that this regulation may be mediated by the activities of Sost and Dkk1. This study highlights several interactions between BMP and Wnt signaling cascades in osteoblasts that may be amenable to therapeutic intervention for the modification of bone mass density.
Given the dramatic increase in skeletal size during growth, the need to preserve skeletal mass during adulthood, and the large capacity of bone to store calcium and phosphate, juxtaposed with the essential role of phosphate in energy metabolism and the adverse effects of hyperphosphatemia, it is not surprising that a complex systems biology has evolved that permits cross-talk between bone and other organs to adjust phosphate balance and bone mineralization in response to changing physiological requirements. This review examines the newly discovered signaling pathways involved in the endocrine functions of bone, such as those mediated by the phosphaturic and 1,25(OH)2D-regulating hormone FGF23, and the broader systemic effects associated with abnormalities of calcium and phosphate homeostasis.
The neurofibromatosis (NF1) gene shows significant homology to mammalian GAP and is an important regulator of the ras signal transduction pathway. To study the function of NF1 in normal development and to try and develop a mouse model of NF1 disease, we have used gene targeting in ES cells to generate mice carrying a null mutation at the mouse Nf1 locus. Although heterozygous mutant mice, aged up to 10 months, likely attributable to a severe malformation of the heart. Interestingly, mutant embryos also display hyperplasia of neural crest-derived sympathetic ganglia. These results identify new roles for NF1 in development and indicate that some of the abnormal growth phenomena observed in NF1 patients can be recapitulated in neurofibromin-deficient mice.
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Tumor-induced osteomalacia (TIO) is one of the paraneoplastic diseases characterized by hypophosphatemia caused by renal phosphate wasting. Because removal of responsible tumors normalizes phosphate metabolism, an unidentified humoral phosphaturic factor is believed to be responsible for this syndrome. To identify the causative factor of TIO, we obtained cDNA clones that were abundantly expressed only in a tumor causing TIO and constructed tumor-specific cDNA contigs. Based on the sequence of one major contig, we cloned 2,270-bp cDNA, which turned out to encode fibroblast growth factor 23 (FGF23). Administration of recombinant FGF23 decreased serum phosphate in mice within 12 h. When Chinese hamster ovary cells stably expressing FGF23 were s.c. implanted into nude mice, hypophosphatemia with increased renal phosphate clearance was observed. In addition, a high level of serum alkaline phosphatase, low 1,25-dihydroxyvitamin D, deformity of bone, and impairment of body weight gain became evident. Histological examination showed marked increase of osteoid and widening of growth plate. Thus, continuous production of FGF23 reproduced clinical, biochemical, and histological features of TIO in vivo. Analyses for recombinant FGF23 products produced by Chinese hamster ovary cells indicated proteolytic cleavage of FGF23 at the RXXR motif. Recent genetic study indicates that missense mutations in this RXXR motif of FGF23 are responsible for autosomal dominant hypophosphatemic rickets, another hypophosphatemic disease with similar features to TIO. We conclude that overproduction of FGF23 causes TIO, whereas mutations in the FGF23 gene result in autosomal dominant hypophosphatemic rickets possibly by preventing proteolytic cleavage and enhancing biological activity of FGF23.
Sclerosteosis, a skeletal disorder characterized by high bone mass due to increased osteoblast activity, is caused by loss of the SOST gene product, sclerostin. The localization in bone and the mechanism of action of sclerostin are not yet known, but it has been hypothesized that it may act as a bone morphogenetic protein (BMP) antagonist. We show here that SOST/sclerostin is expressed exclusively by osteocytes in mouse and human bone and inhibits the differentiation and mineralization of murine preosteoblastic cells (KS483). Although sclerostin shares some of the actions of the BMP antagonist noggin, we show here that it also has actions distinctly different from it. In contrast to noggin, sclerostin did not inhibit basal alkaline phosphatase (ALP) activity in KS483 cells, nor did it antagonize BMP-stimulated ALP activity in mouse C2C12 cells. In addition, sclerostin had no effect on BMP-stimulated Smad phosphorylation and direct transcriptional activation of MSX-2 and BMP response element reporter constructs in KS483 cells. Its unique localization and action on osteoblasts suggest that sclerostin may be the previously proposed osteocyte-derived factor that is transported to osteoblasts at the bone surface and inhibits bone formation.
The osteocyte, a terminally differentiated cell comprising 90%-95% of all bone cells, may have multiple functions, including acting as a mechanosensor in bone (re)modeling. Dentin matrix protein 1 (encoded by DMP1) is highly expressed in osteocytes and, when deleted in mice, results in a hypomineralized bone phenotype. We investigated the potential for this gene not only to direct skeletal mineralization but also to regulate phosphate (P(i)) homeostasis. Both Dmp1-null mice and individuals with a newly identified disorder, autosomal recessive hypophosphatemic rickets, manifest rickets and osteomalacia with isolated renal phosphate-wasting associated with elevated fibroblast growth factor 23 (FGF23) levels and normocalciuria. Mutational analyses showed that autosomal recessive hypophosphatemic rickets family carried a mutation affecting the DMP1 start codon, and a second family carried a 7-bp deletion disrupting the highly conserved DMP1 C terminus. Mechanistic studies using Dmp1-null mice demonstrated that absence of DMP1 results in defective osteocyte maturation and increased FGF23 expression, leading to pathological changes in bone mineralization. Our findings suggest a bone-renal axis that is central to guiding proper mineral metabolism.
Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder primarily characterized by the formation of neurofibromas,
café-au-lait spots and freckling. Skeletal abnormalities such as short stature or bowing/pseudarthrosis of the tibia are relatively
common. To investigate the role of the neurofibromin in skeletal development, we crossed Nf1flox mice with Prx1Cre mice to
inactivate Nf1 in undifferentiated mesenchymal cells of the developing limbs. Similar to NF1 affected individuals, Nf1Prx1 mice show bowing of the tibia and diminished growth. Tibial bowing is caused by decreased stability of the cortical bone
due to a high degree of porosity, decreased stiffness and reduction in the mineral content as well as hyperosteoidosis. Accordingly,
osteoblasts show an increase in proliferation and a decreased ability to differentiate and mineralize in vitro. The reduction in growth is due to lower proliferation rates and a differentiation defect of chondrocytes. Abnormal vascularization
of skeletal tissues is likely to contribute to this pathology as it exerts a negative effect on cortical bone stability. Furthermore,
Nf1 has an important role in the development of joints, as shown by fusion of the hip joints and other joint abnormalities,
which are not observed in neurofibromatosis type I. Thus, neurofibromin has multiple essential roles in skeletal development
and growth.
Recent studies suggest a critical role of osteocytes in controlling skeletal development and bone remodeling although the molecular mechanism is largely unknown. This study investigated BMP signaling in osteocytes by disrupting Bmpr1a under the Dmp1-promoter. The conditional knockout (cKO) mice displayed a striking osteosclerotic phenotype with increased trabecular bone volume, thickness, number, and mineral density as assessed by X-ray and micro-CT. The bone histomorphometry, H&E, and TRAP staining revealed a dramatic increase in trabecular and cortical bone masses but a sharp reduction in osteoclast number. Moreover, there was an increase in BrdU positive osteocytes (2~5-fold) and osteoid volume (~4-fold) but a decrease in the bone formation rate (~85%) in the cKO bones, indicating a defective mineralization. The SEM analysis revealed poorly formed osteocytes: a sharp increase in cell numbers, a great reduction in cell dendrites, and a remarkable change in the cell distribution pattern. Molecular studies demonstrated a significant decrease in the Sost mRNA levels in bone (>95%), and the SOST protein levels in serum (~85%) and bone matrices. There was a significant increase in the β-catenin (>3-fold) mRNA levels as well as its target genes Tcf1 (>6-fold) and Tcf3 (~2-fold) in the cKO bones. We also showed a significant decrease in the RANKL levels of serum proteins (~65%) and bone mRNA (~57%), and a significant increase in the Opg mRNA levels (>20-fold) together with a significant reduction in the Rankl/Opg ratio (>95%), which are responsible for a sharp reduction in the cKO osteoclasts. The values of mechanical strength were higher in cKO femora (i.e. max force, displacement, and work failure). These results suggest that loss of BMP signaling specifically in osteocytes dramatically increases bone mass presumably through simultaneous inhibition of RANKL and SOST, leading to osteoclast inhibition and Wnt activation together. Finally, a working hypothesis is proposed to explain how BMPR1A controls bone remodeling by inhibiting cell proliferation and stimulating differentiation. It is reported that RANKL and SOST are abundantly expressed by osteocytes. Thus, BMP signaling through BMPR1A plays important roles in osteocytes.
Traditionally, control of phosphorus in the body has been considered secondary to the tighter control of calcium by parathyroid hormone and vitamin D. However, over the past decade, substantial advances have been made in understanding the control of phosphorus by the so-called phosphatonin system, the lynchpin of which is fibroblast growth factor 23 (FGF23). FGF23 binds to the klotho/FGFR1c receptor complex in renal tubular epithelial cells, leading to upregulation of Na/Pi cotransporters and subsequent excretion of phosphorus from the body. In addition, FGF23 inhibits parathyroid hormone and the renal 1α-hydroxylase enzyme, while it stimulates 24-hydroxylase, leading to decreased 1,25-dihydroxyvitamin D3. FGF23 is intimately involved in the pathogenesis of a number of diseases, particularly the hereditary hypophosphatemic rickets group and chronic kidney disease, and is a target for the development of new treatments in human medicine. Little work has been done on FGF23 or the other phosphatonins in veterinary medicine, but increases in FGF23 are seen with chronic kidney disease in cats, and increased FGF23 expression has been found in soft tissue sarcomas in dogs.
© The Author(s) 2015.
Introduction:
Phosphoinositide 3-kinases (PI3Ks) constitute one of the most important signaling pathways, playing a vital role in cellular differentiation and proliferation with a key function in cellular receptor triggered signal transduction downstream of tyrosine kinase receptors and/or G-protein coupled receptors. PI3K promotes cell survival proliferation, protein synthesis and glucose metabolism by generating secondary messengers phospholipid phosphatidyl 3,4,5-triphosphate and signaling via AKT/mTOR regulation. Deregulation of PI3K pathways have been observed in cancer, diabetes, neurological and inflammatory diseases and is an attractive target for pharmaceutical industries.
Areas covered:
In this review, the authors explain different PI3K assay methodologies. Furthermore, the authors summarize the techno-scientific principles and their utility in profiling novel chemical entities against PI3Ks. Specifically, the authors compare different PI3K assay formats explaining their mode of detection as well as their advantages and limitations for drug discovery efforts.
Expert opinion:
Developing lipid (PI3K) kinase assays involves significant effort and a rational understanding is needed due to the intrinsic lipidic nature of phospholipid phosphatidyl 4,5-biphosphate, which is used as an in vitro substrate for assays with PI3K isoforms. The assay of choice should be versatile, homogenous and definitely adaptable for high-throughput screening campaigns. Additionally, these assays are expected to dissect the mechanism of action of novel compounds (inhibitor characterization) against PI3K. Existing methods provide the versatility to medicinal chemists such that they can choose one or more assay platform to progress their compounds while profiling and/or inhibitor characterization.
Body phosphate homeostasis is regulated by a hormonal counter-balanced intestine-bone-kidney axis. The major systemic hormones involved in this axis are parathyroid hormone (PTH), 1,25-dihydroxyvitamin-D, and fibroblast growth factor-23 (FGF23). FGF23, produced almost exclusively by the osteocytes, is a phosphaturic hormone that plays a major role in regulation of the bone remodeling process. Remodeling composite components, bone mineralization and resorption cycles create a continuous influx-efflux loop of the inorganic phosphate (Pi) through the skeleton. This “bone Pi loop,” which is formed, is controlled by local and systemic factors according to phosphate homeostasis demands. Although FGF23 systemic actions in the kidney, and for the production of PTH and 1,25-dihydroxyvitamin-D are well established, its direct involvement in bone metabolism is currently poorly understood. This review presents the latest available evidence suggesting two aspects of FGF23 bone local activity: (a) Regulation of FGF23 production by both local and systemic factors. The suggested local factors include extracellular levels of Pi and pyrophosphate (PPi), (the Pi/PPi ratio), and another osteocyte-derived protein, sclerostin. In addition, 1,25-dihydroxyvitamin-D, synthesized locally by bone cells, may contribute to regulation of FGF23 production. The systemic control is achieved via PTH and 1,25-dihydroxyvitamin-D endocrine functions. (b) FGF23 acts as a local agent, directly affecting bone mineralization. We support the assumption that under balanced physiological conditions, sclerostin, by para- autocrine signaling, upregulates FGF23 production by the osteocyte. FGF23, in turn, acts as a mineralization inhibitor, by stimulating the generation of the major mineralization antagonist—PPi. © 2014 BioFactors, 2014
Taller women are at increased risk for fracture despite having wider bones that better tolerate bending. As wider bones require less material to achieve a given bending strength, we hypothesized that taller women assemble bones with relatively thinner and more porous cortices because excavation of a larger medullary canal may be accompanied by excavation of more intracortical canals. Three-dimensional images of distal tibia, fibula and radius were obtained in vivo using high-resolution peripheral quantitative computed tomography in a twin study of 345 females aged 40 to 61 years, 93 with at least one fracture. Cortical porosity below as well as above 100 microns, and microarchitecture were quantified using Strax1.0, a new algorithm. Multivariable linear and logistic regression using generalized estimating equation methods quantified associations between height and microarchitecture and estimated the associations with fracture risk. Each standard deviation (SD) greater height was associated with a 0.69 SD larger tibia total cross sectional area (CSA), 0.66 SD larger medullary CSA, 0.50 SD higher medullary CSA/total CSA (i.e., thinner cortices relative to the total CSA due to a proportionally larger medullary area) and 0.42 SD higher porosity (all p < 0.001). Cortical area was 0.45 SD larger in absolute terms but 0.50 SD smaller in relative terms. These observations were confirmed by examining trait correlations in twin pairs. Fracture risk was associated with height, total CSA, medullary CSA/total CSA and porosity in univariate analyses. In multivariable analyses, distal tibia, medullary CSA/total CSA and porosity predicted fracture independently; height was no longer significant. Each SD greater porosity was associated with fracture; odds ratios; distal tibia 1.55; 95% CI: 1.11-2.15, distal fibula 1.47; 95% CI 1.14-1.88, distal radius 1.22; 95% CI 0.96-1.55. Taller women assemble wider bones with relatively thinner and more porous cortices predisposing to fracture. © 2013 American Society for Bone and Mineral Research.
Background:
Neurofibromatosis 1 (NF1) is an autosomal dominant disorder with various skeletal abnormalities occurring as part of a complex phenotype. Tibial dysplasia, which typically presents as anterolateral bowing of the leg with subsequent fracture and nonunion (pseudarthrosis), is a serious but infrequent osseous manifestation of NF1. Over the past several years, results from clinical and experimental studies have advanced our knowledge of the role of NF1 in bone. On the basis of current knowledge, we propose a number of concepts to consider as a theoretical approach to the optimal management of tibial pseudarthrosis.
Methods:
A literature review for both clinical treatment and preclinical models for tibial dysplasia in NF1 was performed. Concepts were discussed and developed by experts who participated in the Children's Tumor Foundation sponsored International Bone Abnormalities Consortium meeting in 2011.
Results:
Concepts for a theoretical approach to treating tibial pseudarthrosis include: bone fixation appropriate to achieve stability in any given case; debridement of the "fibrous pseudarthrosis tissue" between the bone segments associated with the pseudarthrosis; creating a healthy vascular bed for bone repair; promoting osteogenesis; controlling overactive bone resorption (catabolism); prevention of recurrence of the "fibrous pseudarthrosis tissue"; and achievement of long-term bone health to prevent recurrence.
Conclusions:
Clinical trials are needed to assess effectiveness of the wide variation of surgical and pharmacologic approaches currently in practice for the treatment of tibial pseudarthrosis in NF1.
Level of evidence:
Level V, expert opinion.
Although recent studies have established that osteocytes function as secretory cells that regulate phosphate metabolism, the biomolecular mechanism(s) underlying these effects remain incompletely defined. However, investigations focusing on the pathogenesis of X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR), and autosomal recessive hypophosphatemic rickets (ARHR), heritable disorders characterized by abnormal renal phosphate wasting and bone mineralization, have clearly implicated FGF23 as a central factor in osteocytes underlying renal phosphate wasting, documented new molecular pathways regulating FGF23 production, and revealed complementary abnormalities in osteocytes that regulate bone mineralization. The seminal observations leading to these discoveries were the following: 1) mutations in FGF23 cause ADHR by limiting cleavage of the bioactive intact molecule, at a subtilisin-like protein convertase (SPC) site, resulting in increased circulating FGF23 levels and hypophosphatemia; 2) mutations in DMP1 cause ARHR, not only by increasing serum FGF23, albeit by enhanced production and not limited cleavage, but also by limiting production of the active DMP1 component, the C-terminal fragment, resulting in dysregulated production of DKK1 and β-catenin, which contributes to impaired bone mineralization; and 3) mutations in PHEX cause XLH both by altering FGF23 proteolysis and production and causing dysregulated production of DKK1 and β-catenin, similar to abnormalities in ADHR and ARHR, but secondary to different central pathophysiological events. These discoveries indicate that ADHR, XLH, and ARHR represent three related heritable hypophosphatemic diseases that arise from mutations in, or dysregulation of, a single common gene product, FGF23 and, in ARHR and XLH, complimentary DMP1 and PHEX directed events that contribute to abnormal bone mineralization. This article is part of a Special Issue entitled Special Issue: Osteocyte.
The onset of manifestations of the common, autosomal dominantly inherited disease type 1 neurofibromatosis (NF1) is usually in childhood. To begin to understand the pathogenesis of NF1, we analyzed the developmental pattern of expression of the protein product of the NF1 gene, neurofibromin, by Western blotting and immunohistochemistry using the rat as a model system. Neurofibromin is uniformly distributed throughout embyronic day 10 and 12 rat embryos. By embryonic day 16, neurofibromin immunore-activity is enriched in neurons of the cortical plate, in peripheral ganglia, and in developing CNS and PNS fiber tracts, but remains detectable outside the nervous system. Expression decreases in nonneural tissues by postnatal day 6, and neurofibromin is greatly decreased (lung, adrenal cortex, skin) or absent (skeletal muscle, cartilage) in adult tissues except for brain, spinal cord, peripheral nerve, and adrenal medulla. Transient expression of neurofibromin during development in many tissues suggests the importance of this GTPase-activating protein in morphogenesis and organ growth. A separate role is proposed for neurofibromin in growing axons and in the mature nervous system. © 1993 Wiley-Liss, Inc.
The discovery of fibroblast growth factor 23 (FGF-23) has expanded our understanding of phosphate and vitamin D homeostasis and provided new insights into the pathogenesis of hereditary hypophosphatemic and hyperphosphatemic disorders, as well as acquired disorders of phosphate metabolism, such as chronic kidney disease. FGF-23 is secreted by osteoblasts and osteocytes in bone and principally targets the kidney to regulate the reabsorption of phosphate, the production and catabolism of 1,25-dihydroxyvitamin D and the expression of α-Klotho, an anti-ageing hormone. Secreted FGF-23 plays a central role in complex endocrine networks involving local bone-derived factors that regulate mineralization of extracellular matrix and systemic hormones involved in mineral metabolism. Inactivating mutations of PHEX, DMP1 and ENPP1, which cause hereditary hypophosphatemic disorders and primary defects in bone mineralization, stimulate FGF23 gene transcription in osteoblasts and osteocytes, at least in part, through canonical and intracrine FGF receptor pathways. These FGF-23 regulatory pathways may enable systemic phosphate and vitamin D homeostasis to be coordinated with bone mineralization. FGF-23 also functions as a counter-regulatory hormone for 1,25-dihydroxyvitamin D in a bone-kidney endocrine loop. FGF-23, through regulation of additional genes in the kidney and extrarenal tissues, probably has broader physiological functions beyond regulation of mineral metabolism that account for the association between FGF-23 and increased mortality and morbidity in chronic kidney disease.
The skeleton is frequently affected in individuals with neurofibromatosis type 1, and some of these bone manifestations can result in significant morbidity. The natural history and pathogenesis of the skeletal abnormalities of this disorder are poorly understood and consequently therapeutic options for these manifestations are currently limited. The Children's Tumor Foundation convened an International Neurofibromatosis Type 1 Bone Abnormalities Consortium to address future directions for clinical trials in skeletal abnormalities associated with this disorder. This report reviews the clinical skeletal manifestations and available preclinical mouse models and summarizes key issues that present barriers to optimal clinical management of skeletal abnormalities in neurofibromatosis type 1. These concepts should help advance optimal clinical management of the skeletal abnormalities in this disease and address major difficulties encountered for the design of clinical trials.
Although it is known that neurofibromatosis 1 (NF1) patients suffer from vitamin D deficiency and display decreased bone mineral density (BMD), a systematic clinical and histomorphometrical analysis is absent. Our data demonstrate that NF1 patients display high bone turnover and accumulation of osteoid and that supplementation of vitamin D has a beneficial effect on their BMD.
Neurofibromatosis 1 results in a wide range of clinical manifestations, including decreased BMD. Although it has been reported that NF1 patients have decreased vitamin D serum levels, the manifestation of the disease at the bone tissue level has rarely been analyzed.
Thus, we performed a clinical evaluation of 14 NF1 patients in comparison to age- and sex-matched control individuals. The analysis included dual X-ray absorptiometry osteodensitometry, laboratory parameters, histomorphometric and quantitative backscattered electron imaging (qBEI) analyses of undecalcified bone biopsies.
NF1 patients display significantly lower 25-(OH)-cholecalciferol serum levels and decreased BMD compared to control individuals. Histomorphometric analysis did not only reveal a reduced trabecular bone volume in biopsies from NF1 patients, but also a significantly increased osteoid volume and increased numbers of osteoblasts and osteoclasts. Moreover, qBEI analysis revealed a significant decrease of the calcium content in biopsies from NF1 patients. To address the question whether a normalization of calcium homeostasis improves BMD in NF1 patients, we treated four patients with cholecalciferol for 1 year, which resulted in a significant increase of BMD.
Taken together, our data provide the first complete histomorphometric analysis from NF1 patients. Moreover, they suggest that low vitamin D levels significantly contribute to the skeletal defects associated with the disease.
Bone morphogenetic proteins (BMPs) are known as ectopic bone inducers. The FDA approved BMPs (BMP2 and BMP7) for clinical use. However, direct effects of BMPs on endogenous bone metabolism are not yet well known. We conditionally disrupted BMP receptor type IA (BMPRIA) in osteoblasts during weanling and adult stages to show the impact of BMP signaling on endogenous bone modeling and remodeling. Cre recombination was detected in immature osteoblasts in the periosteum, osteoblasts, and osteocytes but not in chondrocytes and osteoclasts after tamoxifen administration. Bmpr1a conditional knockout mice (cKO) showed increased bone mass primarily in trabecular bone at P21 and 22 wk as determined by H&E staining. Vertebrae, tails, and ribs showed increased radiodensity at 22 wk, consistent with a significant increase in BMD. Both muCT and histomorphometry showed an increase in trabecular BV/TV and thickness of cKO adult bones, whereas osteoclast number, bone formation rate, and mineral apposition rate were decreased. Expression levels of bone formation markers (Runx2 and Bsp), resorption markers (Mmp9, Ctsk, and Tracp), and Rankl were decreased, and Opg was increased in adult bones, resulting in a reduction in the ratio of Rankl to osteoprotegerin (Opg). The reduction in osteoclastogenesis through the RANKL-OPG pathway was also observed in weanling stages and reproduced in newborn calvaria culture. These results suggest that Bmpr1a cKO increased endogenous bone mass primarily in trabecular bone with decreased osteoclastogenesis through the RANKL-OPG pathway. We conclude that BMPRIA signaling in osteoblasts affects both bone formation and resorption to reduce endogenous bone mass in vivo.
von Recklinghausen's neurofibromatosis (NF1) is a common inherited human disease. The events leading to patient symptoms from inheritance of a defective NF1 gene are unknown. Since knowledge of the distribution of the normal NF1 gene product should improve understanding of the pathogenesis of the disease, we raised antibodies against peptides coded by portions of the recently cloned human NF1 cDNA. These antibodies specifically recognize a 220 kd protein (neurofibromin) in both human and rat spinal cord. Neurofibromin is most abundant in the nervous system. Immunostaining of tissue sections indicates that neurons, oligodendrocytes, and nonmyelinating Schwann cells contain neurofibromin while astrocytes and myelinating Schwann cells do not. These results suggest a function for neurofibromin in the normal nervous system. Some NF1 disease manifestations, such as Schwann cell tumors and learning disabilities, may result from abnormalities in the cells that express neurofibromin.
Skeletal lesions are not uncommon in von Recklinghausen neurofibromatosis. Most of them are considered to be dysplastic in nature. Association of osteomalacia or rickets with neurofibromatosis has been documented only rarely. Reported herein is a 40-year-old woman with known von Recklinghausen neurofibromatosis who presented with bone pain, multiple pseudofractures, marked increase in osteoid by bone biopsy, and hypophosphatemia with renal phosphate wasting. Treatment with oral phosphate and vitamin D was effective. A survey of the literature revealed that 34 similar cases have been reported in the past. Although the exact pathogenetic mechanism remains to be determined, osteomalacia in neurofibromatosis appears to be distinct from more common dysplastic skeletal affections of this disease, being characterized by later onset in adulthood as a rule, renal phosphate loss with hypophosphatemia, multiple pseudofractures in typical cases, and response to treatment with pharmacological dose of vitamin D with or without phosphate supplement.
We performed iliac bone histomorphometry after in vivo double tetracycline labeling 3-14 years after intestinal bypass surgery for obesity in 21 patients, selected because of clinical suspicion of metabolic bone disease, and compared the results with those of 40 age-matched normal control subjects. Osteomalacia defined by rigorous kinetic criteria was found in six cases, histologic features of secondary hyperparathyroidism without significantly impaired mineralization in one case, and possible osteomalacia masked by impaired matrix synthesis in one case. In the patients with definite osteomalacia, nonfracture bone pain was more frequent, corrected plasma calcium lower, plasma alkaline phosphatase and magnesium higher, and secondary hyperparathyroidism more severe than in the other patients. In the patients without osteomalacia there was a 24.5% reduction in trabecular bone volume compared to the controls; in contrast to age-related bone loss and post-menopausal osteoporosis, this was due mainly to reduction in the thickness rather than the density of trabecular plates. About two-thirds of the reduction in trabecular thickness was due to reduction in interstitial bone thickness, representing the cumulative effect of increased depth of osteoclastic resorption cavities, probably due in part to secondary hyperparathyroidism. About one-third of the reduction in trabecular thickness was the result of reduced mean wall thickness, representing insufficient osteoblastic matrix synthesis, probably due in part to malabsorption of an unidentified nutrient necessary for normal bone health. Resorption indices were not increased at the time of the biopsy, but there were persistent defects in the recruitment and activity of osteoblasts. Clinically significant bone loss after intestinal shunt surgery, as in several other clinical situations, results from the combined effects of an unsustained increase in bone resorption and a sustained decrease in bone formation.
Human neurofibromatosis type 1 is a dominant disease caused by the inheritance of a mutant allele of the NF1 gene. In order to study NF1 function, we have constructed a mouse strain carrying a germline mutation in the murine homologue. Heterozygous animals do not exhibit the classical symptoms of the human disease, but are highly predisposed to the formation of various tumour types, notably phaeochomocytoma, a tumour of the neural crest-derived adrenal medulla, and myeloid leukaemia, both of which occur with increased frequency in human NF1 patients. The wild-type Nf1 allele is lost in approximately half of the tumours from heterozygous animals. In addition, homozygosity for the Nf1 mutation leads to abnormal cardiac development and mid-gestational embryonic lethality.
Although bone densitometry is often used as a surrogate to evaluate bone fragility, direct biomechanical testing of bone undoubtedly provides more information about mechanical integrity. Like any other specialized field, biomechanics contains its own techniques and vocabulary. This article serves as a guide to biomechanical principles and testing techniques for bone specimens.
Mutations in the NF1 gene may cause developmental abnormalities and the formation of a variety of tumors of neural crest origin in humans. The NF1 gene codes for a large protein, neurofibromin (nf), which is structurally and functionally related to yeast and human ras-GTPase-activating proteins (ras-GAPs). Recently, two transcripts coding for type I and type II nf with different ras-GAP activity have been identified. Since ras proteins do not appear to be significantly regulated during mouse development, we examined if differential expression of neurofibromins may provide evidence for a role of nfs in regulating ras-mediated cell proliferation and differentiation. Nfs were expressed as early as E8. At E11 a marked increase of NF1 transcripts occurred and was associated with expression of nfs in all tissues. Type I and type II nfs each showed a different time course of expression and tissue localization, with type II nf present mainly from E8 through E10, although in the heart type II nf was present at E12. In some tissues such as heart and dorsal root ganglia rapid increases and decreases of nfs were detected related to differentiation of these tissues. These results are consistent with a role of nfs in regulating ras-mediated cell proliferation and differentiation during development and support distinct functional roles for type I and type II nfs.
NF1 is a heritable disease with multiple osseous lesions. The expression of the NF1 gene was studied in embryonic and adult rodent skeleton and in NF1-deficient embryos. The NF1 gene was expressed intensely in the cartilage and the periosteum. Impaired NF1 expression may lead to inappropriate development and dynamics of bones and ultimately to the osseous manifestations of the disease.
Neurofibromatosis type 1 is caused by mutations in the NF1 gene encoding the Ras GTPase activating protein (Ras-GAP) neurofibromin. Skeletal ailments such as short stature, kyphoscoliosis, and tibial bowing and pseudarthrosis are common osseous manifestations of NF1. These symptoms are congenital, implying a role for neurofibromin in proper bone growth. However, little is known about its expression in skeletal tissues during their development.
The expression of the NF1 gene was studied in normal and NF1+/- mouse fetuses at embryonic days 12.5-15.5 and in skeletal tissues of adult mice and rats. In situ hybridization, immunohistochemistry, and Western blot analysis were used to identify the NF1 gene expression profile.
NF1 mRNA and protein were elevated in resting, maturation, and hypertrophic chondrocytes at the growth plate. Parallel studies on NF1+/- embryos showed expression patterns identical to wildtype. The periosteum, including osteoblasts and osteoclasts, and osteocytes of the cortical bone of adult mice were also intensely labeled for NF1 protein and mRNA. Western transfer analysis detected NF1 protein in the respective rat tissues. Phosphorylation of p42 and p44 MAP kinases, the downstream consequence of Ras activation, was elevated in hypertrophic chondrocytes of NF1+/- embryos.
The results suggest that neurofibromin may act as a Ras-GAP in skeletal cells to attenuate Ras transduced growth signals and thus play a role during ossification and dynamics of bone. Loss of NF1 function may therefore lead to dysplastic bone growth, thereby causing the debilitating osseous symptoms of NF1.
Inactivating mutations of the PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) endopeptidase, the disease-causing gene in X-linked hypophosphatemia (XLH), results in increased circulating levels of fibroblastic growth factor-23 (FGF23), a bone-derived phosphaturic factor. To determine the causal role of FGF23 in XLH, we generated a combined Fgf23-deficient enhanced green fluorescent protein (eGFP) reporter and Phex-deficient Hyp mouse model (Fgf23(+/-)/Hyp). eGFP expression was expressed in osteocytes embedded in bone that exhibited marked upregulation of eGFP in response to Phex deficiency and in CD31-positive cells in bone marrow venules that expressed low eGFP levels independently of Phex. In bone marrow stromal cells (BMSCs) derived from Fgf23(-/-)/Hyp mice, eGFP expression was also selectively increased in osteocyte-like cells within mineralization nodules and detected in low levels in CD31-positive cells. Surprisingly, eGFP expression was not increased in cell surface osteoblasts, indicating that Phex deficiency is necessary but not sufficient for increased Fgf23 expression in the osteoblast lineage. Additional factors, associated with either osteocyte differentiation and/or extracellular matrix, are necessary for Phex deficiency to stimulate Fgf23 gene transcription in bone. Regardless, the deletion of Fgf23 from Hyp mice reversed the hypophosphatemia, abnormal 1,25(OH)(2)D(3) levels, rickets, and osteomalacia associated with Phex deficiency. These results suggest that Fgf23 acts downstream of Phex to cause both the renal and bone phenotypes in Hyp mice.
The transcription factor ATF4 enhances bone formation by favoring amino acid import and collagen synthesis in osteoblasts, a function requiring its phosphorylation by RSK2, the kinase inactivated in Coffin-Lowry Syndrome. Here, we show that in contrast, RSK2 activity, ATF4-dependent collagen synthesis, and bone formation are increased in mice lacking neurofibromin in osteoblasts (Nf1(ob)(-/-) mice). Independently of RSK2, ATF4 phosphorylation by PKA is enhanced in Nf1(ob)(-/-) mice, thereby increasing Rankl expression, osteoclast differentiation, and bone resorption. In agreement with ATF4 function in amino acid transport, a low-protein diet decreased bone protein synthesis and normalized bone formation and bone mass in Nf1(ob)(-/-) mice without affecting other organ weight, while a high-protein diet overcame Atf4(-/-) and Rsk2(-/-) mice developmental defects, perinatal lethality, and low bone mass. By showing that ATF4-dependent skeletal dysplasiae are treatable by dietary manipulations, this study reveals a molecular connection between nutrition and skeletal development.
Odontoblasts in dentin and osteocytes in bone contain dendritic processes. To test if their dendrites share a common feature, we compared their cellular morphology as visualized using scanning electron microscopy. Analysis of our data showed that both cells share an identical dendritic canalicular system and express extensive processes forming a complex network within the mineralized matrix. Because dentin matrix protein 1 (DMP1), an extracellular matrix protein, is highly expressed in both types of cells, we next tested, using a transgenic approach, whether a 9.6-kb Dmp1 promoter-4-kb 1st intron would be able to target Cre cDNA in these cells for expression/deletion of other genes in odontoblasts and osteocytes. We determined the specificity and efficiency of Cre activity by crossing Dmp1-Cre mice with ROSA26 reporter mice. Results showed that odontoblasts and osteocytes were specifically targeted, suggesting that this animal model will be useful for the preferential study of gene functions in both types of cells.
The majority of bone cell biology focuses on activity on the surface of the bone with little attention paid to the activity that occurs below the surface. However, with recent new discoveries, osteocytes, cells embedded within the mineralized matrix of bone, are becoming the target of intensive investigation. In this article, the distinctions between osteoblasts and their descendants, osteocytes, are reviewed. Osteoblasts are defined as cells that make bone matrix and osteocytes are thought to translate mechanical loading into biochemical signals that affect bone (re)modeling. Osteoblasts and osteocytes should have similarities as would be expected of cells of the same lineage, yet these cells also have distinct differences, particularly in their responses to mechanical loading and utilization of the various biochemical pathways to accomplish their respective functions. For example, the Wnt/beta-catenin signaling pathway is now recognized as an important regulator of bone mass and bone cell functions. This pathway is important in osteoblasts for differentiation, proliferation and the synthesis bone matrix, whereas osteocytes appear to use the Wnt/beta-catenin pathway to transmit signals of mechanical loading to cells on the bone surface. New emerging evidence suggests that the Wnt/beta-catenin pathway in osteocytes may be triggered by crosstalk with the prostaglandin pathway in response to loading which then leads to a decrease in expression of negative regulators of the pathway such as Sost and Dkk1. The study of osteocyte biology is becoming an intense area of research interest and this review will examine some of the recent findings that are reshaping our understanding of bone/bone cell biology.
Autosomal recessive hypophosphatemic rickets (ARHR), which is characterized by renal phosphate wasting, aberrant regulation of 1alpha-hydroxylase activity, and rickets/osteomalacia, is caused by inactivating mutations of dentin matrix protein 1 (DMP1). ARHR resembles autosomal dominant hypophosphatemic rickets (ADHR) and X-linked hypophosphatemia (XLH), hereditary disorders respectively caused by cleavage-resistant mutations of the phosphaturic factor FGF23 and inactivating mutations of PHEX that lead to increased production of FGF23 by osteocytes in bone. Circulating levels of FGF23 are increased in ARHR and its Dmp1-null mouse homologue. To determine the causal role of FGF23 in ARHR, we transferred Fgf23 deficient/enhanced green fluorescent protein (eGFP) reporter mice onto Dmp1-null mice to create mice lacking both Fgf23 and Dmp1. Dmp1(-/-) mice displayed decreased serum phosphate concentrations, inappropriately normal 1,25(OH)(2)D levels, severe rickets, and a diffuse form of osteomalacia in association with elevated Fgf23 serum levels and expression in osteocytes. In contrast, Fgf23(-/-) mice had undetectable serum Fgf23 and elevated serum phosphate and 1,25(OH)(2)D levels along with severe growth retardation and focal form of osteomalacia. In combined Dmp1(-/-)/Fgf23(-/-), circulating Fgf23 levels were also undetectable, and the serum levels of phosphate and 1,25(OH)(2)D levels were identical to Fgf23(-/-) mice. Rickets and diffuse osteomalacia in Dmp1-null mice were transformed to severe growth retardation and focal osteomalacia characteristic of Fgf23-null mice. These data suggest that the regulation of extracellular matrix mineralization by DMP1 is coupled to renal phosphate handling and vitamin D metabolism through a DMP1-dependent regulation of FGF23 production by osteocytes.
Fibroblast Growth Factor 23: a new dimension to diseases of calcium-phosphorus metabolism
- Hardcastle
Matrix-embedded cells control osteoclast formation
- J Xiong
- M Onal
- R L Jilka
- R S Weinstein
- S C Manolagas
- O 'brien
Xiong J, Onal M, Jilka RL, Weinstein RS, Manolagas SC, O'Brien CA.
Matrix-embedded cells control osteoclast formation. Nat Med.
2011;17(10):1235-41.