FSH, bone, belly and brain.

The pituitary gland, often called the "master gland", orchestrates multiple effector hormonal organs and other glands by secreting various tropic hormones, which play a significant role in a myriad of physiological processes including skeletal modeling and remodeling, fat and glucose metabolism, and cognitive and psychological processes. The findings of the expression of receptors for each pituitary hormone and the hormone itself in skeleton, fat and immune cells suggested that their role is much broader than the traditional or classic role. Follicle-stimulating hormone (FSH), once believed to regulate gonadal function - gonadal development and maturation at puberty and gamete production during the fertile phase - is also found to involve in fat and bone metabolism as well as cognition, which provides us a better understanding of complex physiology. This emerging understanding of the non-reproductive role of FSH opens potential therapeutic opportunity to address detrimental health burden during and after menopause, namely osteoporosis, obesity and dementia. In this Review, we outline the current understanding of crosstalk between the pituitary, bone, adipose tissue and brain through FSH. The pre-clinical evidence from genetic and pharmacologic intervention in rodent models, and human data from population-based observation, genetic studies, and a small number of studies with interventional nature support an independent skeletal, lipogenic and cognitive effect of FSH and more.

An Atlas of Brain-Bone Sympathetic Neural Circuits.

There is clear evidence that the sympathetic nervous system (SNS) mediates bone metabolism. Histological studies show abundant SNS innervation of the periosteum and bone marrow--these nerves consist of noradrenergic fibers that immunostain for tyrosine hydroxylase, dopamine beta hydroxylase, or neuropeptide Y. Nonetheless, the brain sites that send efferent SNS outflow to bone have not yet been characterized. Using pseudorabies (PRV) viral transneuronal tracing, we report, for the first time, the identification of central SNS outflow sites that innervate bone. We find that the central SNS outflow to bone originates from 87 brain nuclei, sub-nuclei and regions of six brain divisions, namely the midbrain and pons, hypothalamus, hindbrain medulla, forebrain, cerebral cortex, and thalamus. We also find that certain sites, such as the raphe magnus (RMg) of the medulla and periaqueductal gray (PAG) of the midbrain, display greater degrees of PRV152 infection, suggesting that there is considerable site-specific variation in the levels of central SNS outflow to bone. This comprehensive compendium illustrating the central coding and control of SNS efferent signals to bone should allow for a greater understanding of the neural regulation of bone metabolism, and importantly and of clinical relevance, mechanisms for central bone pain.

A review of nucleic acid-based detection methods for foodborne viruses: Sample pretreatment and detection techniques.

Viruses are major pathogens that cause food poisoning when ingested via contaminated food and water. Therefore, the development of foodborne virus detection technologies that can be applied throughout the food distribution chain is essential for food safety. A common nucleic acid-based detection method is polymerase chain reaction (PCR), which has become the gold standard for monitoring food contamination by viruses due to its high sensitivity, and availability of commercial kits. However, PCR-based methods are labor intensive and time consuming, and are vulnerable to inhibitors that may be present in food samples. In addition, the methods are restricted with regard to site of analysis due to the requirement of expensive and large equipment for sophisticated temperature regulation and signal analysis procedures. To overcome these limitations, optical and electrical readout biosensors based on nucleic acid isothermal amplification technology and nanomaterials have emerged as alternatives for nucleic acid-based detection of foodborne viruses. Biosensors are promising portable detection tools owing to their easy integration into compact platforms and ability to be operated on-site. However, the complexity of food components necessitates the inclusion of tedious preprocessing steps, and the lack of stability studies on residual food components further restricts the practical application of biosensors as a universal detection method. Here, we summarize the latest advances in nucleic acid-based strategies for the detection of foodborne viruses, including PCR-based and isothermal amplification-based methods, gene amplification-free methods, as well as food pretreatment methods. The principles, strengths/disadvantages, and performance of each method, problems to be solved, and future prospects for the development of a universal detection method are discussed.

Extracellular matrix-degrading enzymes as a biofilm control strategy for food-related microorganisms.

Biofilm is one of the major problems in food industries and is difficult to be removed or prevented by conventional sanitizers. In this review, we discussed the extracellular matrix-degrading enzymes as a strategy to control biofilms of foodborne pathogenic and food-contaminating bacteria. The biofilms can be degraded by using the enzymes targeting proteins, polysaccharides, extracellular DNA, or lipids which mainly constitute the extracellular polymeric substances of biofilms. However, the efficacy of enzymes varies by the growth medium, bacterial species, strains, or counterpart microorganisms due to a high variation in the composition of extracellular polymeric substances. Several studies demonstrated that the combined treatment using conventional sanitizers or multiple enzymes can synergistically enhance the biofilm removal efficacies. In this review, the application of the immobilized enzymes on solid substrates is also discussed as a potential strategy to prevent biofilm formation on food contact surfaces.

Recent Advances in Biosensor Development for the Detection of Viral Particles in Foods: A Comprehensive Review.

Viral foodborne diseases cause serious harm to human health and the economy. Rapid, accurate, and convenient approaches for detecting foodborne viruses are crucial for preventing diseases. Biosensors integrating electrochemical and optical properties of nanomaterials have emerged as effective tools for the detection of viruses in foods. However, they still face several challenges, including substantial sample preparation and relatively poor sensitivity due to complex food matrices, which limit their field applications. Hence, the purpose of this review is to provide an overview of recent advances in biosensing techniques, including electrochemical, SERS-based, and colorimetric biosensors, for detecting viral particles in food samples, with emerging techniques for extraction/concentration of virus particles from food samples. Moreover, the principle, design, and advantages/disadvantages of each biosensing method are comprehensively described. This review covers the recent development of rapid and sensitive biosensors that can be used as new standards for monitoring food safety and food quality in the food industry.

Pituitary crosstalk with bone, adipose tissue and brain.

Traditional textbook physiology has ascribed unitary functions to hormones from the anterior and posterior pituitary gland, mainly in the regulation of effector hormone secretion from endocrine organs. However, the evolutionary biology of pituitary hormones and their receptors provides evidence for a broad range of functions in vertebrate physiology. Over the past decade, we and others have discovered that thyroid-stimulating hormone, follicle-stimulating hormone, adrenocorticotropic hormone, prolactin, oxytocin and arginine vasopressin act directly on somatic organs, including bone, adipose tissue and liver. New evidence also indicates that pituitary hormone receptors are expressed in brain regions, nuclei and subnuclei. These studies have prompted us to attribute the pathophysiology of certain human diseases, including osteoporosis, obesity and neurodegeneration, at least in part, to changes in pituitary hormone levels. This new information has identified actionable therapeutic targets for drug discovery.

Biosynthesis of Silver Nanoparticles from Duchesnea indica Extracts Using Different Solvents and Their Antibacterial Activity.

Silver nanoparticles (AgNPs) were synthesized using the whole plant of Duchesnea indica (DI) which was extracted in different solvents; the antimicrobial effects of the extract were investigated in this study. The extraction of DI was performed using three different solvents: water, pure ethanol (EtOH), and pure dimethyl sulfoxide (DMSO). AgNP formation was monitored by measuring the UV-Vis spectrum of each reaction solution. After synthesis for 48 h, the AgNPs were collected and the negative surface charge and size distribution of the synthesized AgNPs were measured using dynamic light scattering (DLS). The AgNP structure was determined by high-resolution powder X-ray diffraction (XRD) and the AgNP morphology was investigated using transmission electron microscopy (TEM). AgNP antibacterial activities were evaluated against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, and Pseudomonas aeruginosa using the disc diffusion method. Additionally, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were also determined. Biosynthesized AgNPs showed enhanced antibacterial activity against B. cereus, S. aureus, E. coli, S. enteritidis, and P. aeruginosa compared with that of pristine solvent extract. These results suggest that AgNPs synthesized from extracts of DI are promising antibacterial agents against pathogenic bacteria and can be further applied in the food industry.

Development and biophysical characterization of a humanized FSH-blocking monoclonal antibody therapeutic formulated at an ultra-high concentration.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the development of a unique formulation for our first-in-class FSH-blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer's disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L-methionine and chelating agent disodium EDTA improved the formulation's long-term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, confirmed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) Spectroscopy. Three rapid freeze-thaw cycles at -80 °C/25 °C or -80 °C/37 °C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (Tm) for formulated MS-Hu6 increased by >4.80 °C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra-high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.

Development and Biophysical Characterization of a Humanized FSH-Blocking Monoclonal Antibody Therapeutic Formulated at an Ultra-High Concentration.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the creation of a unique formulation for our first-in- class FSH-blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer's disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L-methionine and chelating agent disodium EDTA improved the formulation's long-term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, conformed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. Three rapid freeze-thaw cycles at -80°C/25°C or -80°C/37°C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (T m ) for formulated MS-Hu6 increased by >4.80°C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra-high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.

Bone circuitry and interorgan skeletal crosstalk.

The past decade has seen significant advances in our understanding of skeletal homeostasis and the mechanisms that mediate the loss of bone integrity in disease. Recent breakthroughs have arisen mainly from identifying disease-causing mutations and modeling human bone disease in rodents, in essence, highlighting the integrative nature of skeletal physiology. It has become increasingly clear that bone cells, osteoblasts, osteoclasts, and osteocytes, communicate and regulate the fate of each other through RANK/RANKL/OPG, liver X receptors (LXRs), EphirinB2-EphB4 signaling, sphingolipids, and other membrane-associated proteins, such as semaphorins. Mounting evidence also showed that critical developmental pathways, namely, bone morphogenetic protein (BMP), NOTCH, and WNT, interact each other and play an important role in postnatal bone remodeling. The skeleton communicates not only with closely situated organs, such as bone marrow, muscle, and fat, but also with remote vital organs, such as the kidney, liver, and brain. The metabolic effect of bone-derived osteocalcin highlights a possible role of skeleton in energy homeostasis. Furthermore, studies using genetically modified rodent models disrupting the reciprocal relationship with tropic pituitary hormone and effector hormone have unraveled an independent role of pituitary hormone in skeletal remodeling beyond the role of regulating target endocrine glands. The cytokine-mediated skeletal actions and the evidence of local production of certain pituitary hormones by bone marrow-derived cells displays a unique endocrine-immune-skeletal connection. Here, we discuss recently elucidated mechanisms controlling the remodeling of bone, communication of bone cells with cells of other lineages, crosstalk between bone and vital organs, as well as opportunities for treating diseases of the skeleton.

Optimizing a therapeutic humanized follicle-stimulating hormone-blocking antibody formulation by protein thermal shift assay.

Biopharmaceutical products are formulated using several Food and Drug Administration (FDA) approved excipients within the inactive ingredient limit to maintain their storage stability and shelf life. Here, we have screened and optimized different sets of excipient combinations to yield a thermally stable formulation for the humanized follicle-stimulating hormone (FSH)-blocking antibody, MS-Hu6. We used a protein thermal shift assay in which rising temperatures resulted in the maximal unfolding of the protein at the melting temperature (Tm ). To determine the buffer and pH for a stable solution, four different buffers with a pH range from 3 to 8 were screened. This resulted in maximal Tm s at pH 5.62 for Fab in phosphate buffer and at pH 6.85 for Fc in histidine buffer. Upon testing a range of salt concentrations, MS-Hu6 was found to be more stable at lower concentrations, likely due to reduced hydrophobic effects. Molecular dynamics simulations revealed a higher root-mean-square deviation with 1 mM than with 100 mM salt, indicating enhanced stability, as noted experimentally. Among the stabilizers tested, Tween 20 was found to yield the highest Tm and reversed the salt effect. Among several polyols/sugars, trehalose and sucrose were found to produce higher thermal stabilities. Finally, binding of recombinant human FSH to MS-Hu6 in a final formulation (20 mM phosphate buffer, 1 mM NaCl, 0.001% w/v Tween 20, and 260 mM trehalose) resulted in a thermal shift (increase in Tm ) for the Fab, but expectedly not in the Fc domain. Given that we used a low dose of MS-Hu6 (1 µM), the next challenge would be to determine whether 100-fold higher, industry-standard concentrations are equally stable.

Musculoskeletal and neurocognitive clinical significance of adult hypophosphatasia.

Hypophosphatasia (HPP), also called Rathbun disease, is a rare genetic disorder that is caused by the loss-of-function mutation in the ALPL gene encoding tissue non-specific alkaline phosphatase. Doctor Rathbun first described the case of a 3-week-old infant who presented with severe osteopenia, rickets, and multiple radiographic fractures, and died shortly after of epileptic seizure and respiratory distress. The term "hypophosphatasia" was coined as the patients' alkaline phosphatase levels were significantly low. Since then, our understanding of HPP has evolved, and now we appreciate causative genetic mutation and the broad spectrum of clinical presentation depending on the age of onset, severity, and skeletal involvement: perinatal, infantile, childhood, adult and odontohypophosphatasia. The new development of enzyme replacement with asfostase alfa has saved the lives of severe form of hypophosphatasia. However, it is still unclear and remains challenging how to manage adult HPP that often presents with mild and non-specific symptoms such as muscle pain, joint stiffness, fatigue, anxiety, or low bone mass, which are common in the general population and not necessarily attributed to HPP. In this review, we will present 3 unique cases of adult HPP and discuss the pathophysiology, clinical presentation particularly neuromuscular and neurocognitive symptoms and management of adult HPP.

FSH-blocking therapeutic for osteoporosis.

Pharmacological and genetic studies over the past decade have established the follicle-stimulating hormone (FSH) as an actionable target for diseases affecting millions, namely osteoporosis, obesity, and Alzheimer's disease. Blocking FSH action prevents bone loss, fat gain, and neurodegeneration in mice. We recently developed a first-in-class, humanized, epitope-specific FSH-blocking antibody, MS-Hu6, with a KD of 7.52 nM. Using a Good Laboratory Practice (GLP)-compliant platform, we now report the efficacy of MS-Hu6 in preventing and treating osteoporosis in mice and parameters of acute safety in monkeys. Biodistribution studies using 89Zr-labeled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone and bone marrow. The MS-Hu6 displayed a ß phase t½ of 7.5 days (180 hr) in humanized Tg32 mice. We tested 217 variations of excipients using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze-thaw and at different temperatures, with minimal aggregation, and without self-, cross-, or hydrophobic interactions or appreciable binding to relevant human antigens. The MS-Hu6 showed the same level of "humanness" as human IgG1 in silico and was non-immunogenic in ELISpot assays for IL-2 and IFN-γ in human peripheral blood mononuclear cell cultures. We conclude that MS-Hu6 is efficacious, durable, and manufacturable, and is therefore poised for future human testing.

Independent Skeletal Actions of Pituitary Hormones.

Over the past years, pituitary hormones and their receptors have been shown to have non-traditional actions that allow them to bypass the hypothalamus-pituitary-effector glands axis. Bone cells-osteoblasts and osteoclasts-express receptors for growth hormone, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), adrenocorticotrophic hormone (ACTH), prolactin, oxytocin, and vasopressin. Independent skeletal actions of pituitary hormones on bone have been studied using genetically modified mice with haploinsufficiency and by activating or inactivating the receptors pharmacologically, without altering systemic effector hormone levels. On another front, the discovery of a TSH variant (TSH-ßv) in immune cells in the bone marrow and skeletal action of FSHß through tumor necrosis factor α provides new insights underscoring the integrated physiology of bone-immune-endocrine axis. Here we discuss the interaction of each pituitary hormone with bone and the potential it holds in understanding bone physiology and as a therapeutic target.

Brain atlas for glycoprotein hormone receptors at single-transcript level.

There is increasing evidence that anterior pituitary hormones, traditionally thought to have unitary functions in regulating single endocrine targets, act on multiple somatic tissues, such as bone, fat, and liver. There is also emerging evidence for anterior pituitary hormone action on brain receptors in mediating central neural and peripheral somatic functions. Here, we have created the most comprehensive neuroanatomical atlas on the expression of TSHR, LHCGR, and FSHR. We have used RNAscope, a technology that allows the detection of mRNA at single-transcript level, together with protein level validation, to document Tshr expression in 173 and Fshr expression in 353 brain regions, nuclei and subnuclei identified using the Atlas for the Mouse Brain in Stereotaxic Coordinates. We also identified Lhcgr transcripts in 401 brain regions, nuclei and subnuclei. Complementarily, we used ViewRNA, another single-transcript detection technology, to establish the expression of FSHR in human brain samples, where transcripts were co-localized in MALAT1-positive neurons. In addition, we show high expression for all three receptors in the ventricular region-with yet unknown functions. Intriguingly, Tshr and Fshr expression in the ependymal layer of the third ventricle was similar to that of the thyroid follicular cells and testicular Sertoli cells, respectively. In contrast, Fshr was localized to NeuN-positive neurons in the granular layer of the dentate gyrus in murine and human brain-both are Alzheimer's disease-vulnerable regions. Our atlas thus provides a vital resource for scientists to explore the link between the stimulation or inactivation of brain glycoprotein hormone receptors on somatic function. New actionable pathways for human disease may be unmasked through further studies.

Bone-modifying agents for bone loss in patients with prostate cancer receiving androgen deprivation therapy; insights from a network meta-analysis.

BACKGROUND: The data of head-to-head comparisons of the effect of bone-modifying agents (BMAs) in patients with androgen deprivation therapy (ADT) for prostate cancer without skeletal metastasis is limited. Thus, we conducted a network meta-analysis to compare each BMA for the efficacy of bone mineral densities (BMDs) and the risk of fracture. METHODS: We performed a network meta-analysis to compare the change of BMDs and the risk of vertebral fracture in the studies included using a random-effect model. The primary outcomes are the change of BMD of the lumbar spine (LS) and the total hip (TH) from the baseline at 1 year from the initiation of the BMA and the risk of vertebral fracture. RESULTS: We identified and included 15 studies in this analysis. All BMAs except risedronate showed a significant increase of BMD of the LS compared with groups without BMA, among which zoledronate showed the most BMD gain. At TH, bisphosphonates (alendronate, pamidronate, and zoledronate) and denosumab showed significant elevation compared with the no-BMA group. Denosumab was associated with the most BMD gain at the TH. Only denosumab reduced the risk of vertebral fracture (relative risk [95% confidence interval]: 0.40 [0.20-0.81]). Although zoledronate showed the highest BMD gain at the LS, it did not reduce the risk of vertebral fracture in this analysis. CONCLUSION: Most bisphosphonates and denosumab significantly increased BMD at the LS and the TH in patients receiving ADT for prostate cancer without skeletal metastasis. In particular, zoledronate and denosumab were the most potent BMAs in terms of BMD increment at the LS and the TH, respectively. However, denosumab, not zoledronate, was the only BMA that showed a significant risk reduction of vertebral fracture. We need further studies to examine the change of bone quality and the effect on the risk of non-vertebral and hip fractures.

PLGA Core-Shell Nano/Microparticle Delivery System for Biomedical Application.

Core-shell particles are very well known for their unique features. Their distinctive inner core and outer shell structure allowed promising biomedical applications at both nanometer and micrometer scales. The primary role of core-shell particles is to deliver the loaded drugs as they are capable of sequence-controlled release and provide protection of drugs. Among other biomedical polymers, poly (lactic-co-glycolic acid) (PLGA), a food and drug administration (FDA)-approved polymer, has been recognized for the vehicle material. This review introduces PLGA core-shell nano/microparticles and summarizes various drug-delivery systems based on these particles for cancer therapy and tissue regeneration. Tissue regeneration mainly includes bone, cartilage, and periodontal regeneration.

Thyrotropin, Hyperthyroidism, and Bone Mass.

Thyrotropin (TSH), traditionally seen as a pituitary hormone that regulates thyroid glands, has additional roles in physiology including skeletal remodeling. Population-based observations in people with euthyroidism or subclinical hyperthyroidism indicated a negative association between bone mass and low-normal TSH. The findings of correlative studies were supported by small intervention trials using recombinant human TSH (rhTSH) injection, and genetic and case-based evidence. Genetically modified mouse models, which disrupt the reciprocal relationship between TSH and thyroid hormone, have allowed us to examine an independent role of TSH. Since the first description of osteoporotic phenotype in haploinsufficient Tshr +/- mice with normal thyroid hormone levels, the antiosteoclastic effect of TSH has been documented in both in vitro and in vivo studies. Further studies showed that increased osteoclastogenesis in Tshr-deficient mice was mediated by tumor necrosis factor α. Low TSH not only increased osteoclastogenesis, but also decreased osteoblastogenesis in bone marrow-derived primary osteoblast cultures. However, later in vivo studies using small and intermittent doses of rhTSH showed a proanabolic effect, which suggests that its action might be dose and frequency dependent. TSHR was shown to interact with insulin-like growth factor 1 receptor, and vascular endothelial growth factor and Wnt pathway might play a role in TSH's effect on osteoblasts. The expression and direct skeletal effect of a biologically active splice variant of the TSHß subunit (TSHßv) in bone marrow-derived macrophage and other immune cells suggest a local skeletal effect of TSHR. Further studies of how locally secreted TSHßv and systemic TSHß interact in skeletal remodeling through the endocrine, immune, and skeletal systems will help us better understand the hyperthyroidism-induced bone disease.

The hepcidin regulator erythroferrone is a new member of the erythropoiesis-iron-bone circuitry.

Background: Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. Methods: To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe-/- mouse model as well as ß-thalassemic (Hbbth3/+) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone. Results: We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low-bone-mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP-mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in Hbbth3/+mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Conclusions: Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in ß-thalassemia. Funding: YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania.

The NO-cGMP-PKG pathway in skeletal remodeling.

The nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway plays a critical role in skeletal homeostasis. Preclinical data using NO and its donors and genetically modified mice demonstrated that NO was required in bone remodeling and partly mediated the anabolic effects of mechanical stimuli and estrogen. However, the off-target effects and tachyphylaxis of NO limit its long-term use, and previous clinical trials using organic nitrates for osteoporosis have been disappointing. Among the other components in the downstream pathway, targeting cGMP-specific phosphodiesterase to promote the NO-cGMP-PKG signal is a viable option. There are growing in vitro and in vivo data that, among many other PDE families, PDE5A is highly expressed in skeletal tissue, and inhibiting PDE5A using currently available PDE5A inhibitors might increase the osteoanabolic signal and protect the skeleton. These preclinical data open the possibility of repurposing PDE5A inhibitors for treating osteoporosis. Further research is needed to address the primary target bone cell of PDE5A inhibition, the contribution of direct and indirect effects of PDE5A inhibition, and the pathophysiological changes in skeletal PDE5A expression in aging and hypogonadal animal models.