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Bone marrow contains resident cellular components that are not only involved in bone maintenance but also regulate hematopoiesis and immune responses. The immune system and bone interact with each other, coined osteoimmunology. Hashimoto's thyroiditis (HT) is one of the most common chronic autoimmune diseases which is accompanied by lymphocytic infiltration. It shows elevating thyroid autoantibody levels at an early stage and progresses to thyroid dysfunction ultimately. Different effects exert on bone metabolism during different phases of HT. In this review, we summarized the mechanisms of the long-term effects of HT on bone and the relationship between thyroid autoimmunity and osteoimmunology. For patients with HT, the bone is affected not only by thyroid function and the value of TSH, but also by the setting of the autoimmune background. The autoimmune background implies a breakdown of the mechanisms that control self-reactive system, featuring abnormal immune activation and presence of autoantibodies. The etiology of thyroid autoimmunity and osteoimmunology is complex and involves a number of immune cells, cytokines and chemokines, which regulate the pathogenesis of HT and osteoporosis at the same time, and have potential to affect each other. In addition, vitamin D works as a potent immunomodulator to influence both thyroid immunity and osteoimmunology. We conclude that HT affects bone metabolism at least through endocrine and immune pathways.

Objective: To clarify the role and mechanism of miR-17-92 cluster in islet beta-cell repair after streptozotocin intervention. Methods: Genetically engineered mice (miR-17-92βKO) and control RIP-Cre mice were intraperitoneally injected with multiple low dose streptozotocin. Body weight, random blood glucose (RBG), fasting blood glucose, and intraperitoneal glucose tolerance test (IPGTT) were monitored regularly. Mice were sacrificed for histological analysis 8 weeks later. Morphological changes of pancreas islets, quantity, quality, apoptosis, and proliferation of beta-cells were measured. Islets from four groups were isolated. MiRNA and mRNA were extracted and quantified. Results: MiR-17-92βKO mice showed dramatically elevated fasting blood glucose and impaired glucose tolerance after streptozotocin treatment in contrast to control mice, the reason of which is reduced beta-cell number and total mass resulting from reduced proliferation, enhanced apoptosis of beta-cells. Genes related to cell proliferation and insulin transcription repression were significantly elevated in miR-17-92βKO mice treated with streptozotocin. Furthermore, genes involved in DNA biosynthesis and damage repair were dramatically increased in miR-17-92βKO mice with streptozotocin treatment. Conclusion: Collectively, our results demonstrate that homozygous deletion of miR-17-92 cluster in mouse pancreatic beta-cells promotes the development of experimental diabetes, indicating that miR-17-92 cluster may be positively related to beta-cells restoration and adaptation after streptozotocin-induced damage.

Postmenopausal osteoporosis (PMOP) is a prevalent skeletal disorder associated with menopause-related estrogen withdrawal. PMOP is characterized by low bone mass, deterioration of the skeletal microarchitecture, and subsequent increased susceptibility to fragility fractures, thus contributing to disability and mortality. Accumulating evidence indicates that abnormal expansion of marrow adipose tissue (MAT) plays a crucial role in the onset and progression of PMOP, in part because both bone marrow adipocytes and osteoblasts share a common ancestor lineage. The cohabitation of MAT adipocytes, mesenchymal stromal cells, hematopoietic cells, osteoblasts and osteoclasts in the bone marrow creates a microenvironment that permits adipocytes to act directly on other cell types in the marrow. Furthermore, MAT, which is recognized as an endocrine organ, regulates bone remodeling through the secretion of adipokines and cytokines. Although an enhanced MAT volume is linked to low bone mass and fractures in PMOP, the detailed interactions between MAT and bone metabolism remain largely unknown. In this review, we examine the possible mechanisms of MAT expansion under estrogen withdrawal and further summarize emerging findings regarding the pathological roles of MAT in bone remodeling. We also discuss the current therapies targeting MAT in osteoporosis. A comprehensive understanding of the relationship between MAT expansion and bone metabolism in estrogen deficiency conditions will provide new insights into potential therapeutic targets for PMOP.

XLαs, the extra-large isoform of alpha-subunit of the stimulatory guanine nucleotide-binding protein (Gsα), is paternally expressed. The significance of XLαs in humans remains largely unknown. Here, we report a patient who presented with increased bone mass, hypophosphatemia, and elevated parathyroid hormone (PTH) levels. His serum calcium was in the lower limit of the normal range. Whole exome sequencing of this subject found a novel non-sense variant c.424G>T (p. G142*) in the first exon of XLαs, which was inherited from his father and transmitted to his daughter. This variant was predicted to exclusively influence the expression of XLαs, while possibly having no significant effects on other gene products of this locus. Ellsworth-Howard test revealed normal renal response to PTH in proband. Human SaOS2 cells transfected with mutant XLαs failed to generate cyclic adenosine monophosphate under PTH stimulation, indicating skeletal resistance to this hormone. This subject showed higher circulating sclerostin, dickkopf1, and osteoprotegerin (OPG) levels, while lower receptor activator of nuclear factor kappa-B ligand/OPG ratio, leading to reduced bone resorption. Our findings indicate that XLαs plays a critical role in bone metabolism and GNAS locus should be considered as a candidate gene for high bone mass.

We examined the effects of tumor necrosis factor-α (TNFα) and interleukin-6 (IL6) gene knockout in preserving the bone loss induced by ovariectomy (OVX) and the mechanisms involved in bone metabolism. Twenty female wild-type (WT), TNFα-knockout (TNFα−/−) or IL6-knockout (IL6−/−) mice aged 12 weeks were sham-operated (SHAM) or subjected to OVX and killed after 4 weeks. Bone mass and skeletal microarchitecture were determined using micro-CT. Bone marrow stromal cells (BMSCs) from all three groups (WT, TNFα−/− and IL6−/−) were induced to differentiate into osteoblasts or osteoclasts and treated with 17-β-estradiol. Bone metabolism was assessed by histological analysis, serum analyses and qRT-PCR. OVX successfully induced a high turnover in all mice, but a repair effect was observed in TNFα−/− and IL6−/− mice. The ratio of femoral trabecular bone volume to tissue volume, trabecular number and trabecular thickness were significantly decreased in WT mice subjected to OVX, but increased in TNFα−/− mice (1.62, 1.34, 0.27-fold respectively; P −/− mice (1.34, 0.80, 0.22-fold respectively; P −/− mice when compared to the IL6−/− mice. Both, TNFα−/− and IL6−/− BMSCs exhibited decreased numbers of TRAP-positive cells and an increase in ALP-positive cells, with or without E2 treatment (P TNFα or IL6 significantly upregulated mRNA expressions of osteoblast-related genes (Runx2 and Col1a1) and downregulated osteoclast-related mRNA for TRAP, MMP9 and CTSK in vivo and in vitro, TNFα knockout appeared to have roles beyond IL6 knockout in upregulating Col1a1 mRNA expression and downregulating mRNA expressions of WNT-related genes (DKK1 and Sost) and TNF-related activation-induced genes (TRAF6). TNFα seemed to be more potentially invasive in inhibiting bone formation and enhancing TRAF6-mediated osteoclastogenesis than IL6, implying that the regulatory mechanisms of TNFα and IL6 in bone metabolism may be different.

To compare the effects of high-fat diet (HFD) and high-fructose diet (HFrD) on bone metabolism at different time points, dynamically observe the bone histology and femur trabecular micro-architecture, and analyze the underlying mechanisms. Sixty–Five male 6- to 7-week-old C57BL/6J mice were given HFD, HFrD, or standard diets (SD) for 8, 16, and 24 weeks. Micro-computed tomography (μCT) and bone histology were used to measure bone mass and trabecular micro-structure. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expression of genes related to bone and lipid metabolisms. Compared to SD mice, femoral trabecular bone mass was significantly increased in both HFrD mice and HFD mice at 8 weeks, it continued to be higher in HFrD mice at 16 and 24 weeks with the highest level at 16 weeks, but it was significantly decreased in HFD mice at 16 and 24 weeks. HFD mice showed more epididymal fat accumulation than HFrD mice. mRNA expression of Runx2 was up-regulated at 8 and 16 weeks, but down-regulated at 24 weeks similarly in both HFrD mice and HFD mice. mRNA expression of MMP9 and CTSK was up-regulated at 8 and 16 weeks in HFD mice, but down-regulated at 24 weeks in both HFrD mice and HFD mice. Our data indicated that the HFrD and HFD had different modulating effects on bone mass. After short-term feeding, both HFrD and HFD showed positive effects on bone mass; however, after long-term feeding, bone mass was decreased in HFD mice. In contrast, the bone mass was first increased and then decreased in the HFrD mice. On the basis of these findings, we speculated that chronic consumption of fat and fructose would exert detrimental effects on bone mass which might a combination action of body mass, fat mass, and bone formation/bone resorption along with proinflammatory factor and bone marrow environment.

Bone marrow adipose tissue (MAT) is distinct from white adipose tissue (WAT) or brown adipose tissue (BAT) for its location, feature and function. As a largely ignored adipose depot, it is situated in bone marrow space and resided with bone tissue side-by-side. MAT is considered not only as a regulator of bone metabolism through paracrine, but also as a functionally particular adipose tissue that may contribute to global metabolism. Adipokines, inflammatory factors and other molecules derived from bone marrow adipocytes may exert systematic effects. In this review, we summary the evidence from several aspects including development, distribution, histological features and phenotype to elaborate the basic characteristics of MAT. We discuss the association between bone metabolism and MAT, and highlight our current understanding of this special adipose tissue. We further demonstrate the probable relationship between MAT and energy metabolism, as well as glucose metabolism. On the basis of preliminary results from animal model and clinical studies, we propose that MAT has its unique secretory and metabolic function, although there is no in-depth study at present.

GNAS is one of the most complex gene loci in the human genome and encodes multiple gene products. XLas, the extra-large isoform of alpha-subunit of the stimulatory guanine nucleotide-binding protein (Gas), is paternally inherited. Although XLas can mimic the action of Gas, its significance remains largely unknown in humans. Here we report a patient presented with increased bone mass, hypophosphatemia, and elevated parathyroid hormone levels. His serum calcium was in the lower limit of normal range. DEXA scan revealed progressive increase in the bone density of this patient. Whole exome sequencing of this subject found a novel nonsense mutation c.424G>T (p. G142X) in the first exon of XLas, which was inherited from his father and transmitted to his daughter. This mutation was predicted to exclusively influence the expression of XLas, while may have no significant effects on other gene products of this locus. SaOS2 cells transfected with mutant XLas failed to generate cAMP under parathyroid hormone stimulation, indicating skeletal resistance to this hormone. This subject showed higher circulating SOST, DKK1 and OPG levels, while lower RANKL levels and RANKL/OPG ratio, leading to reduced bone resorption. It is speculated that this patient may belong to a very rare type of pseudohypoparathyroidism with selective skeletal resistance but normal renal tubular response to parathyroid hormone. Our findings indicate that XLas plays a critical role in bone metabolism and GNAS locus should be considered as a candidate gene for high bone mass.

The high incidences of bone metastasis in patients with breast cancer, prostate cancer and lung cancer still remains a puzzling issue. The “seeds and soil” hypothesis suggested that bone marrow (soil) may provide a favorable “niche” for tumor cells (seed). When seeking for effective ways to prevent and treat tumor bone metastasis, most researchers focus on tumor cells (seed) but not the bone marrow microenvironment (soil). In reality, only a fraction of circulating tumor cells (CTCs) could survive and colonize in bone. Thus, the bone marrow microenvironment could ultimately determine the fate of tumor cells that have migrated to bone. Bone marrow adipocytes (BMAs) are abundant in the bone marrow microenvironment. Mounting evidence suggests that BMAs may play a dominant role in bone metastasis. BMAs could directly provide energy for tumor cells, enhance the tumor cell proliferation, and resistance to chemotherapy and radiotherapy. BMAs are also known for releasing some inflammatory factors and adipocytokines to promote or inhibit bone metastasis. In this review, we made a comprehensive summary for the interaction between BMAs and bone metastasis. More importantly, we discussed the potentially promising methods for the prevention and treatment of bone metastasis. Genetic disruption and pharmaceutical inhibition may be effective in inhibiting the formation and pro-tumor functions of BMAs.

Background Research on the characteristics and mechanisms of diabetes in Tibetans is scant. Especially, there is no study on the relationship between osteocalcin and glucose metabolism. The objective of this study was to investigate the associations of serum total osteocalcin (tOC) and undercarboxylated osteocalcin (ucOC) with glucose and lipid metabolism in Chinese indigenous Tibetans with different degrees of glucose tolerance. Methods In this study, 160 middle-aged Tibetan men were involved, including 46 subjects with normal glucose tolerance (NGT), 52 subjects with impaired glucose regulation (IGR) and 62 subjects with type 2 diabetes. The homeostasis model assessment (HOMA) parameters, including HOMA-IR and HOMA-B, were used to estimate insulin resistance and β-cell function, respectively. Adiponectin, leptin, testosterone, 1,25-dihydroxyvitamin D, tOC and ucOC were measured using ELISA kits. Results After adjustment for age and body mass index, plasma tOC level was correlated negatively with fasting and 30-min post-OGTT glucose, HOMA-IR, leptin and testosterone; plasma ucOC level was correlated negatively with 30-min post-OGTT glucose, total cholesterol and 1,25-dihydroxyvitamin D; ucOC : tOC was correlated positively with leptin. The negative association between HOMA-IR and tOC remained significant after correcting for adiponectin; however, the association disappeared after correcting for leptin. HOMA-IR was correlated negatively with age, adiponectin and tOC, and positively with total cholesterol, triglyceride and leptin. Stepwise linear regression analysis revealed that total cholesterol, leptin and adiponectin were independent predictors for HOMA-IR in all subjects. Conclusions Our data support a link between osteocalcin and glucose metabolism in middle-aged Tibetan men. The improved glucose tolerance exerted by tOC may be related to improved insulin sensitivity rather than improved β-cell function. Copyright © 2013 John Wiley & Sons, Ltd.

Rationale Currently, the relationship between heterozygous mutations in SLC34A1 and hypophosphatemia is controversial. Here we report an autosomal dominant hypophosphatemia pedigree carrying a novel heterozygous mutation in SLC34A1. Patient concerns The proband is a 32-year old young man, presented with progressive pain and weakness in his lower extremities for more than 5 years. The proband showed persistent hypophosphatemia and low TmPO4/GFR values, indicating renal phosphate leak. His grandfather, father, and one of his uncles showed the similar symptoms. Diagnoses Autosomal dominant hypophosphatemia. Interventions and outcomes Phosphorus supplement was prescribed to the proband and his affected uncle. Both their serum phosphorus levels recovered to normal and their symptoms such as back pain and lower extremity weakness were completely relieved. Whole exome sequencing was performed to identify disease-causing mutations in proband. Lessons A novel heterozygous missense mutation c.680A>G (p. N227S) in exon 7 of SLC34A1 was found in proband by whole exome sequencing, which was also found in other 4 family members of this pedigree. Our report of an autosomal dominant hypophosphatemia pedigree with 5 mutant carriers enriches the clinical phenotype caused by the SLC34A1 mutations and further affirms the heterozygous mutations are causative for hypophosphatemia.

MicroRNAs (miRNAs) have been identified to play important functions during osteoblast proliferation, differentiation, and apoptosis. The miR-17~92 cluster is highly conserved in all vertebrates. Loss-of-function of the miR-17-92 cluster results in smaller embryos and immediate postnatal death of all animals. Germline hemizygous deletions of MIR17HG are accounted for microcephaly, short stature, and digital abnormalities in a few cases of Feingold syndrome. These reports indicate that miR-17~92 may play important function in skeletal development and mature. To determine the functional roles of miR-17~92 in bone metabolism as well as osteoblast proliferation and differentiation. Murine embryonic stem cells D3 and osteoprogenitor cell line MC3T3-E1 were induced to differentiate into osteoblasts; the expression of miR-17-92 was assayed by quantitative real-time RT-PCR. The skeletal phenotypes were assayed in mice heterozygous for miR-17~92 (miR-17~92 +/Δ ). To determine the possibly direct function of miR-17~92 in bone cells, osteoblasts from miR-17~92 +/Δ mice were investigated by ex vivo cell culture. miR-17, miR-92a, and miR-20a within miR-17-92 cluster were expressed at high level in bone tissue and osteoblasts. The expression of miR-17-92 was down-regulated along with osteoblast differentiation, the lowest level was found in mature osteoblasts. Compared to wildtype controls, miR-17-92 +/Δ mice showed significantly lower trabecular and cortical bone mineral density, bone volume and trabecular number at 10 weeks old. mRNA expression of Runx2 and type I collagen was significantly lower in bone from miR-17-92 +/Δ mice. Osteoblasts from miR-17-92 +/Δ mice showed lower proliferation rate, ALP activity and less calcification. Our research suggests that the miR-17-92 cluster critically regulates bone metabolism, and this regulation is mostly through its function in osteoblasts.

Studies suggest interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) may be beneficial in obesity-related disorders with inconsistent results. This study was designed to investigate and compare their pathophysiological roles in lipid metabolism with gene knockout approach. Male wild-type (WT), IL-6 knockout (IL-6-/-), and TNF-α knockout (TNF-α-/-) mice were maintained on either a chow diet or a high-fat diet (HFD) for 12 weeks. Indices of lipid metabolism in blood, adipose, and liver were determined. Our data showed that IL-6-/- and TNF-α-/- mice were more pronounced in body weight gains, hypercholesterolemia, fasting and post-load hyperglycemia on a chow diet or a HFD compared with WT mice. In WT mice feeding on a HFD, lipolysis and glyceroneogenesis were enhanced, lipogenesis was inhibited in adipose tissue; lipogenesis was increased in liver tissue. IL-6-/- mice on a chow diet or a HFD showed similar metabolic phenotypes as WT mice on a HFD, since those mice had similar expression profiles of lipid-related genes in adipose tissue and liver. However, TNF-α-/- mice were different. Therefore, IL-6-/- and TNF-α-/- mice showed different hepatic triglyceride infiltration in response to different diets. Our study suggests that complete blockage of IL-6 and TNF-α is unbeneficial in obesity and obesity-related metabolic disorders in mice.

The purpose of the study was to determine the roles of interleukin-6 (IL6) in fat and bone communication. Male wild-type (WT) mice and IL6 knockout (IL6−/−) mice were fed with either regular diet (RD) or high-fat diet (HFD) for 12 weeks. Bone mass and bone microstructure were evaluated by micro-computed tomography. Gene expression related to lipid and bone metabolisms was assayed with real-time quantitative polymerase chain reaction. Bone marrow cells from both genotypes were induced to differentiate into osteoblasts or osteoclasts, and treated with palmitic acid (PA). HFD increased the body weight and fat pad weight, and impaired lipid metabolism in both WT and IL6−/− mice. The dysregulation of lipid metabolism was more serious in IL6−/− mice. Trabecular bone volume fraction, trabecular bone number and trabecular bone thickness were significantly downregulated in WT mice after HFD than those in the RD (P −/− mice than those in WT mice on the HFD (P −/− osteoblasts displayed higher alkaline phosphatase (ALP) activity and higher mRNA levels of Runx2 and Colla1 than those in WT osteoblasts both in the control and PA treatment group (P −/− mice showed significantly lower mRNA levels of PPARγ and leptin and higher mRNA levels of adiponectin in comparison with WT mice on HFD. In conclusion, these findings suggested that IL6 gene deficiency antagonized HFD-induced bone loss. IL6 might bridge lipid and bone metabolisms and could be a new potential therapeutic target for lipid metabolism disturbance-related bone loss.

BACKGROUND Obesity was traditionally considered as a positive regulator on the strength of bone. With the in-depth study, obesity is considered as a major risk factor for osteoporosis. Some proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) are the factors that fat uses to negatively regulate bone metabolism. OBJECTIVE This review was aimed to summarize and critically discuss the convincing evidence for the therapeutic effectiveness of pro-inflammatory cytokines for the treatment of obesity-related bone disorders. RESULTS Obese people and animals show a higher level of serum TNF-α and IL-6, which are produced by macrophages derived from adipose tissue. These pro-inflammatory cytokines regulate the proliferation and apoptosis of adipocyte, promote lipolysis, inhibit lipid synthesis and decrease blood lipids through autocrine and paracrine way. On the other hand, TNF-α and IL-6 regulate bone metabolism through the endocrine way. Several reports suggest that TNF-α is a negative regulator of osteoblast at some stages of differentiation and positively regulates osteoclast proliferation and differentiation. In contrast, IL-6 influences osteoblasts and osteoclasts through complex mechanisms, which reflect dual effects. In addition, TNF-α and IL-6 may regulate bone metabolism indirectly by regulating adiponectin and leptin released from adipocytes. CONCLUSION In this review, we first summarize the role of TNF-α and IL-6 in lipid and bone metabolisms. We further discuss how TNF-α and IL-6 regulate the communication between fat and bone, and their pathological roles in obesity-related bone disorders. Lastly, we discuss the possibility of using pro-inflammatory signaling pathway as a therapeutic target to develop drug for obesity-related bone disorders.

MiR-17-92 cluster contributes to the regulation of mammalian development, aging and tumorigenesis. The functional roles of miR-17-92 in pancreatic beta-cells are largely unknown. In this study, we found that conditional deletion of miR-17-92 in mouse pancreatic beta-cells (miR-17-92βKO) significantly reduces glucose tolerance and the first phase of insulin secretion, despite normal ad libitum fed and fasting glucose levels. Proliferation is down-regulated in pancreatic beta-cells after deleting miR-17-92. MiR-17-92βKO mice show higher phosphatase and tensin homologue (PTEN) and lower phosphorylated AKT in islets. Under high fat diet challenge for 16 weeks, miR-17-92βKO mice lose compensation and exhibit higher glucose levels, and lower insulin secretion. Collectively, these data suggest that miR-17-92 is a critical contributor to molecular mechanisms regulating glucose-stimulated insulin secretion and pancreatic beta-cell adaptation under metabolic stress.

Bone has emerged as a novel endocrine organ for its ability to produce hormones and involvement in several regulatory feedback loops. Osteocalcin (OCN) is released into bloodstream during bone resorption and has been demonstrated to exert endocrine regulation on islets, fat and male testis to form feedback loops. We hypothesize that bone delivers its energy metabolism signals to related energy-regulating organs through OCN based on the following evidence: First, OCN has close interactions with islets and fat, and it shows ability to stimulate islets and fat to secret insulin and adiponectin, respectively. Islets and fat are important organs involved in energy metabolism. Second, OCN undergoes physiological fluctuations during a lifetime. In children and adolescents, during the development of osteoporosis or after bone fracture, OCN level increases significantly. The elevated OCN at these stages represents enhanced bone turnover and metabolic activity, which require more energy supply. Therefore, the metabolic activity of bone and the energy-related organs like fat and islets are closely linked by circulating OCN. Through systemic release of OCN, bone delivers its energy-demanding information to other organs to satisfy its energy requirement.

Individuals with nerofibromatosis Type 1 (NF1) frequently suffer a spectrum of bone pathologies, such as abnormal skeletal development (scoliosis, congenital bowing, and congenital pseudoarthroses, etc), lower bone mineral density with increased fracture risk. These skeletal problems may result, in part, from abnormal osteoclastogenesis. Enhanced RAS/PI3K activity has been reported to contribute to abnormal osteoclastogenesis in Nf1 heterozygous (Nf1+/-) mice. However, the specific downstream pathways linked to NF1 abnormal osteoclastogenesis have not been defined. Our aim was to determine whether mammalian target of rapamycin (mTOR) was a key effector responsible for abnormal osteoclastogenesis in NF1. Primary osteoclast-like cells (OCLs) were cultured from Nf1 wild-type (Nf1+/+) and Nf1+/- mice. Compared to Nf1+/+ controls, there were 20% more OCLs induced from Nf1+/- mice. Nf1+/- OCLs were larger and contained more nuclei. Hyperactive mTOR signaling was detected in Nf1+/- OCLs. Inhibition of mTOR signaling by rapamycin in Nf1+/- OCLs abrogated abnormalities in cellular size and number. Moreover, we found that hyperactive mTOR signaling induced abnormal osteoclastogenesis major through hyper-proliferation. Our research suggests that neurofibromin directly regulates osteoclastogenesis through mTOR signaling pathway. Inhibiting mTOR may represent a viable strategy to treat NF1 bone diseases.

SHARPIN is a novel protein thought to interact with SHANK family and is widely expressed in multiple tissues/cells, including osteoblasts and osteoclasts. Loss-of-function of Sharpin develops the chronic proliferative dermatitis mutation (CPDM) in mice as well as a severe inflammation in other organs. The actual function of SHARPIN is poorly understood. Our aim was to determine the functional roles of SHARPIN in bone metabolism by using CPDM mice. The skeletal phenotypes were determined by peripheral quantitative computed tomography, micro-computed tomography, and quantitative real-time RT-PCR, the cellular functions of osteoblasts and osteoclasts were investigated by ex vivo cell culture. Compared to wild-type controls, CPDM mice demonstrated significantly lower total and cortical bone mineral content and bone mineral density, trabecular and cortical bone volume, and trabecular number. The mRNA expression of Runx2, osterix, type I collagen, and osteocalcin was significantly lower in the bone from CPDM mice. Osteoclasts and osteoblasts from CPDM mice were functionally defective. Our result suggests that SHARPIN plays important regulating roles in bone metabolism. These functional roles may either come from systemic chronic inflammatory or directly signaling pathway within bone cells.

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.

Individuals with neurofibromatosis type 1 (NF1) have a high incidence of osteoporosis and osteopenia. However, understanding of the cellular and molecular basis of these sequelae is incomplete. Osteoclasts are specialized myeloid cells that are the principal bone-resorbing cells of the skeleton. We found that Nf1(+/-) mice contain elevated numbers of multinucleated osteoclasts. Both osteoclasts and osteoclast progenitors from Nf1(+/-) mice were hyperresponsive to limiting concentrations of M-CSF and receptor activator of NF-kappaB ligand (RANKL) levels. M-CSF-stimulated p21(ras)-GTP and Akt phosphorylation was elevated in Nf1(+/-) osteoclasts associated with gains of function in survival, proliferation, migration, adhesion, and lytic activity. These gains of function are associated with more severe bone loss following ovariectomy as compared with that in syngeneic WT mice. Intercrossing Nf1(+/-) mice and mice deficient in class 1(A) PI3K (p85alpha) restored elevated PI3K activity and Nf1(+/-) osteoclast functions to WT levels. Furthermore, in vitro-differentiated osteoclasts from NF1 patients also displayed elevated Ras/PI3K activity and increased lytic activity analogous to those in murine Nf1(+/-) osteoclasts. Collectively, our results identify a what we believe to be a novel cellular and biochemical NF1-haploinsufficient phenotype in osteoclasts that has potential implications for the pathogenesis of NF1 bone disease.

The osteocyte, a terminally differentiated cell comprising 90%–95% of all bone cells1,2, may have multiple functions, including acting as a mechanosensor in bone (re)modeling3. Dentin matrix protein 1 (encoded by DMP1) is highly expressed in osteocytes4 and, when deleted in mice, results in a hypomineralized bone phenotype5. We investigated the potential for this gene not only to direct skeletal mineralization but also to regulate phosphate (Pi) 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.

Obesity and osteoporosis are two common chronic diseases, however, the basis for the correlation between them remains largely unknown. The pro-inflammation cytokine tumor necrosis factor-alpha (TNF-α) plays important roles in lipid and bone metabolisms, which may be a good candidate in the correlation between obesity and osteoporosis. We investigated the pathological roles of TNF-α in high-fat diet (HFD)-induced bone loss. Wild-type (WT) mice and TNF-α knockout (TNF-α(-/-)) mice were fed with the standard diet or the HFD for 12 weeks. Bone marrow stromal cells (BMSCs) from both genotypes were induced to differentiate into osteoblasts and treated with palmitic acid (PA). Bone mass and microstructure of femurs were evaluated by micro-CT. Lipid and bone metabolisms were investigated by histological and plasma analyses, and real-time PCR. On the HFD, both TNF-α(-/-) and WT mice presented notable visceral obesity, dyslipidemia. Adipogenesis and osteoclastogenesis were enhanced, while osteoblastogenesis was reduced in both genotypes. However, the changes were significantly different between TNF-α(-/-) and WT mice after the HFD. The gain of body and fat-pad weight was less and adipocyte area was smaller by 22 % in TNF-α(-/-) mice. Osteoclast numbers and plasma CTX level were lower by 40 % and by 23 % in TNF-α(-/-) mice. There were more ALP positive cells in the PA-treated TNF-α(-/-) BMSCs. mRNA expression of PPAR-γ was lower while that of Runx2 was higher in the bone from TNF-α(-/-) HFD group and in the PA-treated TNF-α(-/-) BMSCs, compared to WT on the same treatment. Furthermore, femoral trabecular bone mass and trabecular bone number were significantly decreased in WT mice on the HFD, whereas they were increased by 1.56-fold and 1.53-fold, respectively, in TNF-α(-/-) mice on the same diet (P0.05). Our results demonstrated that TNF-α gene knockout retained HFD-induced femoral trabecular bone loss mainly by suppressing adipogenesis and osteoclastogenesis, and enhancing osteoblastogenesis, which suggests that TNF-α plays a critical role in the development of HFD-related bone metabolic disorders and it may be a new potential therapeutic target for obesity-related bone loss.

Fibroblast growth factor (FGF)-23 has emerged as an endocrine regulator of phosphate and of vitamin D metabolism. It is produced in bone and, unlike other FGFs, circulates in the bloodstream to ultimately regulate phosphate handling and vitamin D production in the kidney. Presently, it is unknown which of the seven principal FGF receptors (FGFRs) transmits FGF23 biological activity. Furthermore, the molecular basis for the endocrine mode of FGF23 action is unclear. Herein, we performed surface plasmon resonance and mitogenesis experiments to comprehensively characterize receptor binding specificity. Our data demonstrate that FGF23 binds and activates the c splice isoforms of FGFR1–3, as well as FGFR4, but not the b splice isoforms of FGFR1–3. Interestingly, highly sulfated and longer glycosaminoglycan (GAG) species were capable of promoting FGF23 mitogenic activity. We also show that FGF23 induces tyrosine phosphorylation and inhibits sodium-phosphate cotransporter Npt2a mRNA expression using opossum kidney cells, a model kidney proximal tubule cell line. Removal of cell surface GAGs abolishes the effects of FGF23, and exogenous highly sulfated GAG is capable of restoring FGF23 activity, suggesting that proximal tubule cells naturally express GAGs that are permissive for FGF23 action. We propose that FGF23 signals through multiple FGFRs and that the unique endocrine actions of FGF23 involve escape from FGF23-producing cells and circulation to the kidney, where highly sulfated GAGs most likely act as cofactors for FGF23 activity. Our biochemical findings provide important insights into the molecular mechanisms by which dysregulated FGF23 signaling leads to disorders of hyper- and hypophosphatemia. (Endocrinology 146: 4647–4656, 2005)

Fibroblast growth factor-23 (FGF23) is a circulating factor that plays critical roles in phosphate and vitamin D metabolism. The goal of our studies was to dissect the pathways directing the vitamin D-phosphate-FGF23 homeostatic axis. To test the role of diet in the regulation of Fgf23, wild-type (WT) mice were fed either a standard (0.44% phosphorus) or a low-phosphate (0.02%) diet. WT mice on standard diet had a serum phosphate of 9.5 +/- 0.3 mg/dl and an Fgf23 concentration of 99.0 +/- 10.6 pg/ml; mice on the low-phosphate diet had a phosphate of 5.0 +/- 0.2 mg/dl (P0.01) and an Fgf23 of 10.6 +/- 3.7 pg/ml (P0.01). To genetically separate the effects of phosphate and vitamin D on Fgf23, we examined vitamin D receptor null (VDR(-/-)) mice, which are hypocalcemic and hypophosphatemic secondary to hyperparathyroidism. On standard diets, WT and VDR(+/-) mice had Fgf23 levels of 106.0 +/- 30.7 and 90.6 +/- 17.3 pg/ml, respectively, whereas Fgf23 was undetectable in the VDR(-/-). Animals were then placed on a diet that normalizes serum calcium and phosphorus. This 'rescue' increased Fgf23 in WT to 192.3 +/- 32.5 pg/ml and in VDR(+/-) to 388.2 +/- 89.6pg/ml, and importantly, in VDR(-/-) to 476.9 +/- 60.1 pg/ml (P0.01 vs. WT). In addition, renal vitamin D 1-alpha hydroxylase (1alpha-OHase) mRNA levels were corrected to WT levels in the VDR(-/-) mice. In summary, Fgf23 is suppressed in diet-induced hypophosphatemia and in hypophosphatemia associated with secondary hyperparathyroidism. Normalization of serum phosphate by diet in VDR(-/-) mice increases Fgf23. Thus, our results demonstrate that Fgf23 is independently regulated by phosphate and by vitamin D.

Gain-of-function mutations in fibroblast growth factor-23 (FGF23) are responsible for autosomal dominant hypophosphatemic rickets, a disorder of isolated renal phosphate wasting. Patients with the disorder display hypophosphatemia with normocalcemia as well as inappropriately normal 1,25-dihydroxyvitamin D [1,25(OH)2D3] concentrations. Reciprocally tumoral calcinosis (TC) patients are often hyperphosphatemic with inappropriately normal or elevated serum 1,25(OH)2D3 levels and have ectopic and vascular calcifications, a phenotype similar to that of Fgf23 null mice. Therefore, the goal of the present studies was to test whether FGF23 was a candidate gene for TC. Two sisters in a consanguineous TC family had hyperphosphatemia and normal 1,25(OH)2D3 levels with characteristic ectopic and vascular calcifications. Interestingly, these patients had low-normal intact serum FGF23 levels but demonstrated FGF23 concentrations approximately 40 times normal when assessed with a C-terminal FGF23 serum assay. Mutational analyses identified a homozygous S71G mutation in FGF23 in the TC patients, which was not found in control alleles. Finally, modeling demonstrated that the S71G mutation most likely destabilizes full-length FGF23. In summary, recessive FGF23 mutations can lead to TC. Additionally, our findings indicate that FGF23 may adopt an unstable conformation in some TC patients, possibly leading to nonfunctional FGF23 protein.

The association between lipid and bone metabolism has become an increasing focus of interest in recent years, and accumulating evidence has shown that atherosclerosis (AS) and osteoporosis (OP), a disorder of bone metabolism, frequently co-exist. Fat and bone are known to share a common progenitor cell: Multipotent mesenchymal stem cells (MSC) in the bone marrow (BM), which are able to differentiate into various cell phenotypes, including osteoblasts, adipocytes and chondrocytes. Laboratory-based and clinical trials have shown that increasing adipocytes are accompanied by a decrease in bone mineral density (BMD) and bone mass. Statins, lipid-lowering drugs used to treat hyperlipidemia, also provide benefit in the treatment of OP. There is thus evidence that the metabolism of lipids is correlated with that of bone, and that the two are mutually regulated. The present review primarily focuses on the potential association between lipid metabolism disturbance and OP, based on biological metabolism, pathophysiological processes, results from clinical and experimental animal studies, processes involved in the differentiation of adipocytes and osteoblasts, as well as pharmacological treatments of these diseases.

Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 176 RXXR 179 /S 180 proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene–environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR.

Fibroblast growth factor 23 (FGF23) is a hormone required for normal renal phosphate reabsorption. FGF23 gain-of-function mutations result in autosomal dominant hypophosphatemic rickets (ADHR), and FGF23 loss-of-function mutations cause familial hyperphosphatemic tumoral calcinosis (TC). In this study, we identified a novel recessive FGF23 TC mutation, a lysine (K) substitution for glutamine (Q) (160 C > A) at residue 54 (Q54K). To understand the molecular consequences of all known FGF23-TC mutants (H41Q, S71G, M96T, S129F, and Q54K), these proteins were stably expressed in vitro. Western analyses revealed minimal amounts of secreted intact protein for all mutants, and ELISA analyses demonstrated high levels of secreted COOH-terminal FGF23 fragments but low amounts of intact protein, consistent with TC patients' FGF23 serum profiles. Mutant protein function was tested and showed residual, yet decreased, bioactivity compared with wild-type protein. In examining the role of the FGF23 COOH-terminal tail (residues 180–251) in protein processing and activity, truncated mutants revealed that the majority of the residues downstream from the known FGF23 SPC protease site (176RXXR179/S180) were not required for protein secretion. However, residues adjacent to the RXXR site (between residues 188 and 202) were required for full bioactivity. In summary, we report a novel TC mutation and demonstrate a common defect of reduced FGF23 stability for all known FGF23-TC mutants. Finally, the majority of the COOH-terminal tail of FGF23 is not required for protein secretion but is required for full bioactivity.

Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia due to inactivating mutations in FGF23 or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). Herein we report a homozygous missense mutation (H193R) in the KLOTHO (KL) gene of a 13-year-old girl who presented with severe tumoral calcinosis with dural and carotid artery calcifications. This patient exhibited defects in mineral ion homeostasis with marked hyperphosphatemia and hypercalcemia as well as elevated serum levels of parathyroid hormone and FGF23. Mapping of H193R mutation onto the crystal structure of myrosinase, a plant homolog of KL, revealed that this histidine residue was at the base of the deep catalytic cleft and mutation of this histidine to arginine should destabilize the putative glycosidase domain (KL1) of KL, thereby attenuating production of membrane-bound and secreted KL. Indeed, compared with wild-type KL, expression and secretion of H193R KL were markedly reduced in vitro, resulting in diminished ability of FGF23 to signal via its cognate FGF receptors. Taken together, our findings provide what we believe to be the first evidence that loss-of-function mutations in human KL impair FGF23 bioactivity, underscoring the essential role of KL in FGF23-mediated phosphate and vitamin D homeostasis in humans.

Approximately 50% of neurofibromatosis type 1 (NF1) patients exhibit skeletal pathology, such as premature osteoporosis or pseudoarthroses. Loss of neurofibromin deregulates Ras signal transduction to affect generation of mitogen-activated protein kinase and Akt, both of which have been implicated in parathyroid hormone (PTH) anabolic mechanisms. Our aim was to determine if loss of neurofibromin impaired the anabolic effect of PTH on bone mass. Nf1 heterozygote (Nf1+/−) and wild type (Nf1 +/+ ) mice were treated with recombinant human PTH1–34 or vehicle once daily for 3–28 days. PTH enhanced mRNA expression of c-fos, junB, and fra2 in the distal femur metaphyses of both genotypes; expression of these transcripts was consistently lower in PTH-treated Nf1+/− mice. Despite lowered c-fos expression in Nf1+/− mice, PTH increased bone mass equivalently in both genotypes by 28 days. Ex vivo, Nf1 heterozygosity was associated with increased inducible osteoclasts in PTH-treated bone marrow cells and impairment of the actin stress fiber and cyclic adenosine monophosphate response to PTH in osteoprogenitors. Lower c-fos expression was previously thought to abrogate PTH responsiveness. Our results suggest crosstalk might occur between Ras signal transduction and the protein kinase A pathway in Nf1+/− mice. Ras signal transduction does not appear to be essential for the anabolic actions of PTH on bone. Because PTH was effective in the absence of Nf1, it may offer a useful approach to treat osteoporosis in NF1 patients.

Fibroblast growth factor 23 (FGF23) is a circulating factor that plays critical roles in phosphate and vitamin D metabolism, as evidenced by the fact that FGF23 missense mutations cause autosomal dominant hypophosphatemic rickets (ADHR). Autosomal dominant hypophosphatemic rickets is characterized by hypophosphatemia with inappropriately normal 1,25-dihydroxyvitamin D concentrations, as well as bone pain, fracture and rickets. This phenotype parallels that of patients with tumor induced osteomalacia (TIO), X-linked hypophosphatemic rickets (XLH), and fibrous dysplasia (FD), in whom elevated serum FGF23 levels are often observed. The fibroblast growth factor receptors (FGFR1-4) play key roles in skeletal development, as well as in normal metabolic processes. Several FGFR isoforms that potentially mediate the activity of FGF23 have been implicated. In the short term, these findings will lead to further understanding of FGF23 function, and potentially in the long term, to targeted therapies in disorders of hypo- and hyperphosphatemia that involve FGF23.

It is well known that fibroblast growth factor (FGF) family members are associated with embryonic development and are critical for basic metabolic functions. This review will focus upon fibroblast growth factor-23 (FGF23) and its roles in disorders associated with phosphate handling. The discovery that mutations in FGF23 were responsible for the isolated renal phosphate wasting disorder autosomal dominant hypophosphatemic rickets (ADHR) has ascribed novel functions to the FGF family. FGF23 circulates in the bloodstream, and animal models demonstrate that FGF23 controls phosphate and Vitamin D homeostasis through the regulation of specific renal proteins. The ADHR mutations in FGF23 produce a protein species less susceptible to proteolytic processing. X-linked hypophosphatemic rickets (XLH), tumor-induced osteomalacia (TIO), and fibrous dysplasia of bone (FD) are disorders involving phosphate homeostasis that share phenotypes with ADHR, indicating that FGF23 may be a common denominator for the pathophysiology of these syndromes. Our understanding of FGF23 will help to develop novel therapies for phosphate wasting disorders, as well as for disorders of increased serum phosphate, such as tumoral calcinosis, a rare disorder, and renal failure, a common disorder.

Skeletal problems and osteoporosis occur in up to 50% affected neurofibromatosis type 1 (NF1) humans. Inactivation of neurofibromin results in deregulation of Ras signal transduction. Little is known of bone biology in humans with NF1. The goal of our work was to determine if loss-of-function of Nf1 gene was associated with altered bone homeostasis and Ras signal transduction. Because homozygous Nf1 mice are embryonically lethal, heterozygote Nf1 (Nf1+/-) male mice were used to investigate skeletal phenotypes and osteoprogenitor functions, using standard in vivo and in vitro assays. We found that bone mass and geometry of Nf1+/- mice did not differ from wild type controls, despite a trend to less bone formation. Nf1+/- committed osteoprogenitors from femur metaphysis exhibited premature apoptosis and higher proliferation. Ras signaling was activated in primary Nf1+/- bone marrow-inducible osteoprogenitors. Inducible osteoprogenitors exhibited lower induction of osteoblast differentiation, assessed as alkaline phosphatase positive CFU-f. A screen of osteoblast marker genes showed a selective increase in osteopontin (OPN) mRNA and protein expression in these cells. OPN protein was increased in Nf1+/- bone, especially in cortical bone matrix. Because bone cell abnormalities in Nf1 haploinsufficiency were detected in vitro, redundant pathways must compensate for the deregulation of Ras signaling in vivo to maintain normal bone mass and function in vivo. Our in vitro data revealed that neurofibromin and its control of Ras signaling are required for osteoprogenitor homeostasis.