Bone resorption goes green.

In this issue of Cell, McDonald et al. show that giant multinucleated, bone-resorbing osteoclasts dissolve into smaller cells, termed "osteopmorhs," which re-form into osteoclasts at distal bone sites (McDonald et al., 2021). These findings overturn the long-standing premise that osteoclasts differentiate solely from hematopoietic precursors and undergo apoptosis after completing resorption.

FSH blockade improves cognition in mice with Alzheimer's disease.

Alzheimer's disease has a higher incidence in older women, with a spike in cognitive decline that tracks with visceral adiposity, dysregulated energy homeostasis and bone loss during the menopausal transition1,2. Inhibiting the action of follicle-stimulating hormone (FSH) reduces body fat, enhances thermogenesis, increases bone mass and lowers serum cholesterol in mice3-7. Here we show that FSH acts directly on hippocampal and cortical neurons to accelerate amyloid-ß and Tau deposition and impair cognition in mice displaying features of Alzheimer's disease. Blocking FSH action in these mice abrogates the Alzheimer's disease-like phenotype by inhibiting the neuronal C/EBPß-δ-secretase pathway. These data not only suggest a causal role for rising serum FSH levels in the exaggerated Alzheimer's disease pathophysiology during menopause, but also reveal an opportunity for treating Alzheimer's disease, obesity, osteoporosis and dyslipidaemia with a single FSH-blocking agent.

Abaloparatide prevents immobilization-induced cortical but not trabecular bone loss after spinal cord injury.

Spinal cord injury (SCI) causes severe and resistant sublesional disuse bone loss. Abaloparatide, a modified parathyroid hormone related peptide, is an FDA approved drug for treatment of severe osteoporosis with potent anabolic activity. The effects of abaloparatide on SCI-induced bone loss remain undefined. Thus, female mice underwent sham or severe contusion thoracic SCI causing hindlimb paralysis. Mice then received subcutaneous injection of vehicle or 20 µg/kg/day abaloparatide for 35 days. Micro-computed tomography (micro-CT) analysis of the distal and midshaft femoral regions of the SCI-vehicle mice revealed reduced trabecular fractional bone volume (56%), thickness (75%), and cortical thickness (80%) compared to sham-vehicle controls. Treatment with abaloparatide did not prevent SCI-induced changes in trabecular or cortical bone. However, histomorphometry evaluation of the SCI-abaloparatide mice demonstrated that abaloparatide treatment increased osteoblast (241%) and osteoclast (247%) numbers and the mineral apposition rate (131%) compared to SCI-vehicle animals. In another independent experiment, treatment with 80 µg/kg/day abaloparatide significantly attenuated SCI-induced loss in cortical bone thickness (93%) when compared to SCI-vehicle mice (79%) but did not prevent SCI-induced trabecular bone loss or elevation in cortical porosity. Biochemical analysis of the bone marrow supernatants of the femurs showed that SCI-abaloparatide animals had 2.3-fold increase in procollagen type I N-terminal propeptide, a bone formation marker than SCI-vehicle animals. SCI groups had 70% higher levels of cross-linked C-telopeptide of type I collagen, a bone resorption marker, than sham-vehicle mice. These findings suggest that abaloparatide protects the cortical bone against the deleterious effects of SCI by promoting bone formation.

Absent LH signaling rescues the anxiety phenotype in aging female mice.

Clinical studies and experimental data together support a role for pituitary gonadotropins, including luteinizing hormone (LH), otherwise considered solely as fertility hormones, in age-related cognitive decline. Furthermore, rising levels of LH in post-menopausal women have been implicated in the high prevalence of mood disorders. This study was designed to examine the effect of deficient LH signaling on both cognitive and emotional behavior in 12-month-old Lhcgr-/- mice. For this, we established and validated a battery of five tests, including Dark-Light Box (DLB), Y-Maze Spontaneous Alternation, Novel Object Recognition (NOR), and contextual and cued Fear Conditioning (FCT) tests. We found that 12-month-old female wild type mice display a prominent anxiety phenotype on DLB and FCT. This phenotype was not seen in 12-month-old female Lhcgr-/- mice, indicating full phenotypic rescue. Furthermore, there was no effect of LHCGR depletion on recognition memory or working spatial memory on NOR and Y-maze testing, respectively, in 12-month-old mice, notwithstanding the absence of a basal phenotype in wild type littermates. The latter data do not exclude an effect of LH on cognition documented in previous studies. Finally, 12-month-old male mice and 3-month-old male and female mice did not consistently display deficits on any test. The data collectively document, for the first time, that loss of LH signaling reverses age-related emotional disturbances, a prelude to future targeted therapies that block LH action.

Blocking FSH induces thermogenic adipose tissue and reduces body fat.

Menopause is associated with bone loss and enhanced visceral adiposity. A polyclonal antibody that targets the ß-subunit of the pituitary hormone follicle-stimulating hormone (Fsh) increases bone mass in mice. Here, we report that this antibody sharply reduces adipose tissue in wild-type mice, phenocopying genetic haploinsufficiency for the Fsh receptor gene Fshr. The antibody also causes profound beiging, increases cellular mitochondrial density, activates brown adipose tissue and enhances thermogenesis. These actions result from the specific binding of the antibody to the ß-subunit of Fsh to block its action. Our studies uncover opportunities for simultaneously treating obesity and osteoporosis.

Repurposing erectile dysfunction drugs tadalafil and vardenafil to increase bone mass.

We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine ß-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.

First-in-class humanized FSH blocking antibody targets bone and fat.

Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a KD of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH-FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHß subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing.

Oxytocin regulates body composition.

The primitive neurohypophyseal nonapeptide oxytocin (OXT) has established functions in parturition, lactation, appetite, and social behavior. We have shown that OXT has direct actions on the mammalian skeleton, stimulating bone formation by osteoblasts and modulating the genesis and function of bone-resorbing osteoclasts. We deleted OXT receptors (OXTRs) selectively in osteoblasts and osteoclasts using Col2.3Cre and Acp5Cre mice, respectively. Both male and female Col2.3Cre+:Oxtrfl/fl mice recapitulate the low-bone mass phenotype of Oxtr+/- mice, suggesting that OXT has a prominent osteoblastic action in vivo. Furthermore, abolishment of the anabolic effect of estrogen in Col2.3Cre+:Oxtrfl/fl mice suggests that osteoblastic OXTRs are necessary for estrogen action. In addition, the high bone mass in Acp5Cre+:Oxtrfl/fl mice indicates a prominent action of OXT in stimulating osteoclastogenesis. In contrast, we found that in pregnant and lactating Col2.3Cre+:Oxtrfl/fl mice, elevated OXT inhibits bone resorption and rescues the bone loss otherwise noted during pregnancy and lactation. However, OXT does not contribute to ovariectomy-induced bone loss. Finally, we show that OXT acts directly on OXTRs on adipocytes to suppress the white-to-beige transition gene program. Despite this direct antibeiging action, injected OXT reduces total body fat, likely through an action on OXT-ergic neurons. Consistent with an antiobesity action of OXT, Oxt-/- and Oxtr-/- mice display increased total body fat. Overall, the actions of OXT on bone mass and body composition provide the framework for future therapies for osteoporosis and obesity.

Mechanism of glucocerebrosidase activation and dysfunction in Gaucher disease unraveled by molecular dynamics and deep learning.

The lysosomal enzyme glucocerebrosidase-1 (GCase) catalyzes the cleavage of a major glycolipid glucosylceramide into glucose and ceramide. The absence of fully functional GCase leads to the accumulation of its lipid substrates in lysosomes, causing Gaucher disease, an autosomal recessive disorder that displays profound genotype-phenotype nonconcordance. More than 250 disease-causing mutations in GBA1, the gene encoding GCase, have been discovered, although only one of these, N370S, causes 70% of disease. Here, we have used a knowledge-based docking protocol that considers experimental data of protein-protein binding to generate a complex between GCase and its known facilitator protein saposin C (SAPC). Multiscale molecular-dynamics simulations were used to study lipid self-assembly, membrane insertion, and the dynamics of the interactions between different components of the complex. Deep learning was applied to propose a model that explains the mechanism of GCase activation, which requires SAPC. Notably, we find that conformational changes in the loops at the entrance of the substrate-binding site are stabilized by direct interactions with SAPC and that the loss of such interactions induced by N370S and another common mutation, L444P, result in destabilization of the complex and reduced GCase activation. Our findings provide an atomistic-level explanation for GCase activation and the precise mechanism through which N370S and L444P cause Gaucher disease.

FSIP1 regulates autophagy in breast cancer.

Fibrous sheath interacting protein 1 (FSIP1) is a cancer antigen expressed in the majority of breast cancer tissues and is associated with poor prognosis. However, the role of FSIP1 in the progression and drug sensitivity of triple-negative breast cancer (TNBC) has not been explored. Here, we show that FSIP1 deficiency by shRNA-mediated knockdown or CRISPR-Cas9-mediated knockout significantly inhibits the proliferation and invasion of TNBC cells and impairs chemotherapy-induced growth inhibition in vivo. Computational modeling predicted that FSIP1 binds to ULK1, and this was established by coimmunoprecipitation. FSIP1 deficiency promoted autophagy, enhanced AMP-activated protein kinase (AMPK) signaling, and decreased mechanistic target of rapamycin (mTOR) and Wnt/ß-catenin activity. In contrast, knockdown of AMPK or inhibition of autophagy restored the sensitivity to chemotherapy drugs in TNBC cells. Our findings uncover a role of FSIP1 as well as mechanisms underlying FSIP1 action in drug sensitivity and may, therefore, aid in design of TNBC therapies.

Epitope-specific monoclonal antibodies to FSHß increase bone mass.

Pituitary hormones have long been thought solely to regulate single targets. Challenging this paradigm, we discovered that both anterior and posterior pituitary hormones, including FSH, had other functions in physiology. We have shown that FSH regulates skeletal integrity, and, more recently, find that FSH inhibition reduces body fat and induces thermogenic adipose tissue. A polyclonal antibody raised against a short, receptor-binding epitope of FSHß was found not only to rescue bone loss postovariectomy, but also to display marked antiobesity and probeiging actions. Questioning whether a single agent could be used to treat two medical conditions of public health importance--osteoporosis and obesity--we developed two further monoclonal antibodies, Hf2 and Mf4, against computationally defined receptor-binding epitopes of FSHß. Hf2 has already been shown to reduce body weight and fat mass and cause beiging in mice on a high-fat diet. Here, we show that Hf2, which binds mouse Fsh in immunoprecipitation assays, also increases cortical thickness and trabecular bone volume, and microstructural parameters, in sham-operated and ovariectomized mice, noted on microcomputed tomography. This effect was largely recapitulated with Mf4, which inhibited bone resorption by osteoclasts and stimulated new bone formation by osteoblasts. These effects were exerted in the absence of alterations in serum estrogen in wild-type mice. We also reconfirm the existence of Fshrs in bone by documenting the specific binding of fluorescently labeled FSH, FSH-CH, in vivo. Our study provides the framework for the future development of an FSH-based therapeutic that could potentially target both bone and fat.

Correction: The high-density lipoprotein receptor Scarb1 is required for normal bone differentiation in vivo and in vitro.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cytochrome c oxidase dysfunction enhances phagocytic function and osteoclast formation in macrophages.

The mitochondria-to-nucleus retrograde signaling (MtRS) pathway aids in cellular adaptation to stress. We earlier reported that the Ca2+- and calcineurin-dependent MtRS induces macrophage differentiation to bone-resorbing osteoclasts. However, mechanisms through which macrophages sense and respond to cellular stress remain unclear. Here, we induced mitochondrial stress in macrophages by knockdown (KD) of subunits IVi1 or Vb of cytochrome c oxidase (CcO). Whereas both IVi1 and Vb KD impair CcO activity, IVi1 KD cells produced higher levels of cellular and mitochondrial reactive oxygen species with increased glycolysis. Additionally, IVi1 KD induced the activation of MtRS factors NF-κB, NFAT2, and C/EBPδ as well as inflammatory cytokines, NOS 2, increased phagocytic activity, and a greater osteoclast differentiation potential at suboptimal RANK-L concentrations. The osteoclastogenesis in IVi1 KD cells was reversed fully with an IL-6 inhibitor LMT-28, whereas there was minimal rescue of the enhanced phagocytosis in these cells. In agreement with our findings in cultured macrophages, primary bone marrow-derived macrophages from MPV17-/- mice, a model for mitochondrial dysfunction, also showed higher propensity for osteoclast formation. This is the first report showing that CcO dysfunction affects inflammatory pathways, phagocytic function, and osteoclastogenesis.-Angireddy, R., Kazmi, H. R., Srinivasan, S., Sun, L., Iqbal, J., Fuchs, S. Y., Guha, M., Kijima, T., Yuen, T., Zaidi, M., Avadhani, N. G. Cytochrome c oxidase dysfunction enhances phagocytic function and osteoclast formation in macrophages.

FSIP1 binds HER2 directly to regulate breast cancer growth and invasiveness.

Fibrous sheath interacting protein 1 (FSIP1), a spermatogenesis-related testicular antigen, is expressed in abundance in breast cancers, particularly in those overexpressing human epidermal growth factor receptor 2 (HER2); however, little is known about its role in regulating the growth and metastasis of breast cancer cells. We and others have shown previously that FSIP1 expression in breast cancer correlates positively with HER2-positivity, recurrence, and metastases and negatively with survival. Here, using coimmunoprecipitation and microscale thermophoresis, we find that FSIP1 binds to the intracellular domain of HER2 directly. We further show that shRNA-induced FSIP1 knockdown in SKBR3 and MCF-7 breast cancer cells inhibits proliferation, stimulates apoptosis, attenuates epithelial-mesenchymal transition, and impairs migration and invasiveness. Consistent with reduced proliferation and enhanced apoptosis, xenotransplantation of SKBR3 cells stably transfected with sh-FSIP1 into nu/nu mice results in reduced tumor volumes compared with sh-NC transplants. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping using sh-FSIP1 gene signature yielded associations with extracellular matrix protein pathways, and a reduction in SNAI2 protein expression was confirmed on Western blot analysis. Complementarily, interrogation of the Connectivity Map using the same gene signature yielded, as top hits, chemicals known to inhibit epithelial-mesenchymal transition, including rapamycin, 17-N-allylamino-17-demethoxygeldanamycin, and LY294002. These compounds phenocopy the effects of sh-FSIP1 on SKBR3 cell viability. Thus, FSIP1 suppression limits oncogenesis and invasiveness in breast cancer cells and, considering its absence in most other tissues, including normal breast, may become a potential target for breast cancer therapy.

Clinical, genetic, and structural basis of congenital adrenal hyperplasia due to 11ß-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH), resulting from mutations in CYP11B1, a gene encoding 11ß-hydroxylase, represents a rare autosomal recessive Mendelian disorder of aberrant sex steroid production. Unlike CAH caused by 21-hydroxylase deficiency, the disease is far more common in the Middle East and North Africa, where consanguinity is common often resulting in identical mutations. Clinically, affected female newborns are profoundly virilized (Prader score of 4/5), and both genders display significantly advanced bone ages and are oftentimes hypertensive. We find that 11-deoxycortisol, not frequently measured, is the most robust biochemical marker for diagnosing 11ß-hydroxylase deficiency. Finally, computational modeling of 25 missense mutations of CYP11B1 revealed that specific modifications in the heme-binding (R374W and R448C) or substrate-binding (W116C) site of 11ß-hydroxylase, or alterations in its stability (L299P and G267S), may predict severe disease. Thus, we report clinical, genetic, hormonal, and structural effects of CYP11B1 gene mutations in the largest international cohort of 108 patients with steroid 11ß-hydroxylase deficiency CAH.

Clinical, genetic, and structural basis of apparent mineralocorticoid excess due to 11ß-hydroxysteroid dehydrogenase type 2 deficiency.

Mutations in 11ß-hydroxysteroid dehydrogenase type 2 gene (HSD11B2) cause an extraordinarily rare autosomal recessive disorder, apparent mineralocorticoid excess (AME). AME is a form of low renin hypertension that is potentially fatal if untreated. Mutations in the HSD11B2 gene result either in severe AME or a milder phenotype (type 2 AME). To date, ∼40 causative mutations have been identified. As part of the International Consortium for Rare Steroid Disorders, we have diagnosed and followed the largest single worldwide cohort of 36 AME patients. Here, we present the genotype and clinical phenotype of these patients, prominently from consanguineous marriages in the Middle East, who display profound hypertension and hypokalemic alkalosis. To correlate mutations with phenotypic severity, we constructed a computational model of the HSD11B2 protein. Having used a similar strategy for the in silico evaluation of 150 mutations of CYP21A2, the disease-causing gene in congenital adrenal hyperplasia, we now provide a full structural explanation for the clinical severity of AME resulting from each known HSD11B2 missense mutation. We find that mutations that allow the formation of an inactive dimer, alter substrate/coenzyme binding, or impair structural stability of HSD11B2 yield severe AME. In contrast, mutations that cause an indirect disruption of substrate binding or mildly alter intramolecular interactions result in type 2 AME. A simple in silico evaluation of novel missense mutations could help predict the often-diverse phenotypes of an extremely rare monogenic disorder.

The high-density lipoprotein receptor Scarb1 is required for normal bone differentiation in vivo and in vitro.

We examined bone formation and turnover in high-density lipoprotein (HDL) receptor, scavenger receptor type I (Scarb1), knockout animals relative to wild-type (WT) controls. Scarb1-/- animals have elevated serum adrenocorticotropic hormone (ACTH) due to the role of Scarb1 in glucocorticoid production, which might cause increased bone mass. However, this was not observed: Scarb1-/- mice, with ACTH, over 1000 pg/ml relative to wild-type ACTH ~ 25 pg/ml, bone of the knockout animals was osteopenic relative to the wild type at 16 weeks, including bone volume/total volume and trabecular thickness. Other serum parameters of WT and Scarb1-/- animals in cortisol or calcium were unaffected, although Scarb1-/- animals had significantly elevated PTH and decreased phosphate. Osteoblast and osteoclast-related mRNAs extracted from bone were greatly decreased at 8 or 16 weeks. Importantly, in normal ACTH, osteogenic differentiation in vitro from mesenchymal stem cells showed reduced alkaline phosphatase and mineralization. In Scarb1-/- cells relative to WT, mRNAs for RunX2, alkaline phosphatase, type I collagen, and osteocalcin were reduced 40-90%, all p < 0.01, indicating a role of Scarb1 in osteoblast differentiation independent of ACTH. Additionally, in vitro osteoblast differentiation at variable ACTH in WT cells confirmed ACTH increasing bone differentiation, mineralization, alkaline phosphatase, and osteocalcin mRNA at 0-10 nM ACTH, but reduced bone differentiation at 100-1000 nM ACTH. Overall Scarb1-/- animals show inhibited bone formation with age. This may be a mixed effect on direct bone formation and of very high ACTH. Further, this work shows that both ACTH concentration and the HDL receptor Scarb1 play important independent roles in osteoblast differentiation.

Anabolic actions of Notch on mature bone.

Notch controls skeletogenesis, but its role in the remodeling of adult bone remains conflicting. In mature mice, the skeleton can become osteopenic or osteosclerotic depending on the time point at which Notch is activated or inactivated. Using adult EGFP reporter mice, we find that Notch expression is localized to osteocytes embedded within bone matrix. Conditional activation of Notch signaling in osteocytes triggers profound bone formation, mainly due to increased mineralization, which rescues both age-associated and ovariectomy-induced bone loss and promotes bone healing following osteotomy. In parallel, mice rendered haploinsufficient in γ-secretase presenilin-1 (Psen1), which inhibits downstream Notch activation, display almost-absent terminal osteoblast differentiation. Consistent with this finding, pharmacologic or genetic disruption of Notch or its ligand Jagged1 inhibits mineralization. We suggest that stimulation of Notch signaling in osteocytes initiates a profound, therapeutically relevant, anabolic response.

Functions of vasopressin and oxytocin in bone mass regulation.

Prior studies show that oxytocin (Oxt) and vasopressin (Avp) have opposing actions on the skeleton exerted through high-affinity G protein-coupled receptors. We explored whether Avp and Oxtr can share their receptors in the regulation of bone formation by osteoblasts. We show that the Avp receptor 1α (Avpr1α) and the Oxt receptor (Oxtr) have opposing effects on bone mass: Oxtr(-/-) mice have osteopenia, and Avpr1α(-/-) mice display a high bone mass phenotype. More notably, this high bone mass phenotype is reversed by the deletion of Oxtr in Oxtr(-/-):Avpr1α(-/-) double-mutant mice. However, although Oxtr is not indispensable for Avp action in inhibiting osteoblastogenesis and gene expression, Avp-stimulated gene expression is inhibited when the Oxtr is deleted in Avpr1α(-/-) cells. In contrast, Oxt does not interact with Avprs in vivo in a model of lactation-induced bone loss in which Oxt levels are high. Immunofluorescence microscopy of isolated nucleoplasts and Western blotting and MALDI-TOF of nuclear extracts show that Avp triggers Avpr1α localization to the nucleus. Finally, a specific Avpr2 inhibitor, tolvaptan, does not affect bone formation or bone mass, suggesting that Avpr2, which primarily functions in the kidney, does not have a significant role in bone remodeling.