Orchiectomy sensitizes cortical bone in male mice to the harmful effects of kynurenine.

Kynurenine (Kyn) is a tryptophan metabolite that increases with age and promotes musculoskeletal dysfunction. We previously found a sexually dimorphic pattern in how Kyn affects bone, with harmful effects more prevalent in females than males. This raises the possibility that male sex steroids might exert a protective effect that blunts the effects of Kyn in males. To test this, orchiectomy (ORX) or sham surgeries were performed on 6-month-old C57BL/6 mice, after which mice received Kyn (10 mg/kg) or vehicle via intraperitoneal injection, once daily, 5×/week, for four weeks. Bone histomorphometry, DXA, microCT, and serum marker analyses were performed after sacrifice. In vitro studies were performed to specifically test the effect of testosterone on activation of aryl hydrocarbon receptor (AhR)-mediated signaling by Kyn in mesenchymal-lineage cells. Kyn treatment reduced cortical bone mass in ORX- but not sham-operated mice. Trabecular bone was unaffected. Kyn's effects on cortical bone in ORX mice were attributed primarily to enhanced endosteal bone resorption activity. Bone marrow adipose tissue was increased in Kyn-treated ORX animals but was unchanged by Kyn in sham-operated mice. ORX surgery increased mRNA expression of the aryl hydrocarbon receptor (AhR) and its target gene Cyp1a1 in the bone, suggesting a priming and/or amplification of AhR signaling pathways. Mechanistic in vitro studies revealed that testosterone blunted Kyn-stimulated AhR transcriptional activity and Cyp1a1 expression in mesenchymal-linage cells. These data suggest a protective role for male sex steroids in blunting the harmful effects of Kyn in cortical bone. Therefore, testosterone may play an important role in regulating Kyn/AhR signaling in musculoskeletal tissues, suggesting crosstalk between male sex steroids and Kyn signaling may influence age-associated musculoskeletal frailty.

Immune Reconstitution Bone Loss Exacerbates Bone Degeneration Due to Natural Aging in a Mouse Model.

BACKGROUND: Immune reconstitution bone loss (IRBL) is a common side-effect of antiretroviral therapy (ART) in people with human immunodeficiency virus (PWH). Immune reconstitution bone loss acts through CD4+ T-cell/immune reconstitution-induced inflammation and is independent of antiviral regimen. Immune reconstitution bone loss may contribute to the high rate of bone fracture in PWH, a cause of significant morbidity and mortality. Although IRBL is transient, it remains unclear whether bone recovers, or whether it is permanently denuded and further compounds bone loss associated with natural aging. METHODS: We used a validated IRBL mouse model involving T-cell reconstitution of immunocompromised mice. Mice underwent cross-sectional bone phenotyping of femur and/or vertebrae between 6 and 20 months of age by microcomputed tomography (µCT) and quantitative bone histomorphometry. CD4+ T cells were purified at 20 months to quantify osteoclastogenic/inflammatory cytokine expression. RESULTS: Although cortical IRBL in young animals recovered with time, trabecular bone loss was permanent and exacerbated skeletal decline associated with natural aging. At 20 months of age, reconstituted CD4+ T cells express enhanced osteoclastogenic cytokines including RANKL, interleukin (IL)-1ß, IL-17A, and tumor necrosis factor-α, consistent with elevated osteoclast numbers. CONCLUSIONS: Immune reconstitution bone loss in the trabecular compartment is permanent and further exacerbates bone loss due to natural aging. If validated in humans, interventions to limit IRBL may be important to prevent fractures in aging PWH.

The Glucocorticoid Receptor in Osterix-Expressing Cells Regulates Bone Mass, Bone Marrow Adipose Tissue, and Systemic Metabolism in Female Mice During Aging.

Hallmarks of aging-associated osteoporosis include bone loss, bone marrow adipose tissue (BMAT) expansion, and impaired osteoblast function. Endogenous glucocorticoid levels increase with age, and elevated glucocorticoid signaling, associated with chronic stress and dysregulated metabolism, can have a deleterious effect on bone mass. Canonical glucocorticoid signaling through the glucocorticoid receptor (GR) was recently investigated as a mediator of osteoporosis during the stress of chronic caloric restriction. To address the role of the GR in an aging-associated osteoporotic phenotype, the current study utilized female GR conditional knockout (GR-CKO; GRfl/fl :Osx-Cre+) mice and control littermates on the C57BL/6 background aged to 21 months and studied in comparison to young (3- and 6-month-old) mice. GR deficiency in Osx-expressing cells led to low bone mass and BMAT accumulation that persisted with aging. Surprisingly, however, GR-CKO mice also exhibited alterations in muscle mass (reduced % lean mass and soleus fiber size), accompanied by reduced voluntary physical activity, and also exhibited higher whole-body metabolic rate and elevated blood pressure. Moreover, increased lipid storage was observed in GR-CKO osteoblastic cultures in a glucocorticoid-dependent fashion despite genetic deletion of the GR, and could be reversed via pharmacological inhibition of the mineralocorticoid receptor (MR). These findings provide evidence of a role for the GR (and possibly the MR) in facilitating healthy bone maintenance with aging in females. The effects of GR-deficient bone on whole-body physiology also demonstrate the importance of bone as an endocrine organ and suggest evidence for compensatory mechanisms that facilitate glucocorticoid signaling in the absence of osteoblastic GR function; these represent new avenues of research that may improve understanding of glucocorticoid signaling in bone toward the development of novel osteogenic agents. © 2021 American Society for Bone and Mineral Research (ASBMR).

The Senolytic Drug Navitoclax (ABT-263) Causes Trabecular Bone Loss and Impaired Osteoprogenitor Function in Aged Mice.

Senescence is a cellular defense mechanism that helps cells prevent acquired damage, but chronic senescence, as in aging, can contribute to the development of age-related tissue dysfunction and disease. Previous studies clearly show that removal of senescent cells can help prevent tissue dysfunction and extend healthspan during aging. Senescence increases with age in the skeletal system, and selective depletion of senescent cells or inhibition of their senescence-associated secretory phenotype (SASP) has been reported to maintain or improve bone mass in aged mice. This suggests that promoting the selective removal of senescent cells, via the use of senolytic agents, can be beneficial in the treatment of aging-related bone loss and osteoporosis. Navitoclax (also known as ABT-263) is a chemotherapeutic drug reported to effectively clear senescent hematopoietic stem cells, muscle stem cells, and mesenchymal stromal cells in previous studies, but its in vivo effects on bone mass had not yet been reported. Therefore, the purpose of this study was to assess the effects of short-term navitoclax treatment on bone mass and osteoprogenitor function in old mice. Aged (24 month old) male and female mice were treated with navitoclax (50 mg/kg body mass daily) for 2 weeks. Surprisingly, despite decreasing senescent cell burden, navitoclax treatment decreased trabecular bone volume fraction in aged female and male mice (-60.1% females, -45.6% males), and BMSC-derived osteoblasts from the navitoclax treated mice were impaired in their ability to produce a mineralized matrix (-88% females, -83% males). Moreover, in vitro administration of navitoclax decreased BMSC colony formation and calcified matrix production by aged BMSC-derived osteoblasts, similar to effects seen with the primary BMSC from the animals treated in vivo. Navitoclax also significantly increased metrics of cytotoxicity in both male and female osteogenic cultures (+1.0 to +11.3 fold). Taken together, these results suggest a potentially harmful effect of navitoclax on skeletal-lineage cells that should be explored further to definitively assess navitoclax's potential (or risk) as a therapeutic agent for combatting age-related musculoskeletal dysfunction and bone loss.

Neutralization of CD40 ligand costimulation promotes bone formation and accretion of vertebral bone mass in mice.

Objective: Immunosuppressive biologics are used in the management of RA and additional immunomodulators are under investigation including modulators of the CD40/CD40 ligand (CD40L) costimulation pathway. Tampering with immune function can have unanticipated skeletal consequences due to disruption of the immuno-skeletal interface, a nexus of shared cells and cytokine effectors serving discrete functions in both immune and skeletal systems. In this study, we examined the effect of MR1, a CD40L neutralizing antibody, on physiological bone remodelling in healthy mice. Methods: Female C57BL6 mice were treated with MR1 and BMD was quantified by dual energy X-ray absorptiometry and indices of trabecular bone structure were quantified by micro-CT. Serum biochemical markers were used to evaluate bone turnover and formation indices by histomorphometry. Results: Unexpectedly, MR1 stimulated significant accretion of BMD and trabecular bone mass in the spine, but not in long bones. Surprisingly, bone accretion was accompanied by a significant increase in bone formation, rather than suppression of bone resorption. Mechanistically, MR1-induced bone accrual was associated with increased Treg development and elevated production of cytotoxic T lymphocyte antigen 4, a costimulation inhibitor that promotes T cell anergy and CD8+ T cell expression of the bone anabolic ligand Wnt-10b. Conclusion: Our studies reveal an unexpected bone anabolic activity of pharmacological CD40L suppression. Therapeutic targeting of the CD40L pathway may indeed have unforeseen consequences for the skeleton, but may also constitute a novel strategy to promote bone formation to ameliorate osteoporotic bone loss and reduce fracture risk in the axial skeleton.

Protein kinase D1 conditional null mice show minimal bone loss following ovariectomy.

We previously found that 3- and 6-month-old male mice with conditional ablation of protein kinase D1 (PRKD1) in osteoprogenitor cells (expressing Osterix) exhibited reduced bone mass. Others have demonstrated similar effects in young female PRKD1-deficient mice. Here we examined the bone resorptive response of adult female floxed control and conditional knockout (cKO) mice undergoing sham surgery or ovariectomy (OVX). Femoral and tibial bone mineral density (BMD) values were significantly reduced upon OVX in control, but not cKO, females compared to the respective sham-operated mice. Micro-CT analysis showed that OVX significantly increased trabecular number and decreased trabecular spacing in cKO but not control mice. Finally, in control mice serum levels of a marker of bone resorption (pyridinoline crosslinks) and the osteoclast activator RANKL significantly increased upon OVX; however, no such OVX-induced increase was observed in cKO mice. Our results suggest the potential importance of PRKD1 in response to estrogen loss in bone.

Deletion of protein kinase D1 in osteoprogenitor cells results in decreased osteogenesis in vitro and reduced bone mineral density in vivo.

Protein kinase D1 (PRKD1) is thought to play a role in a number of cellular functions, including proliferation and differentiation. We hypothesized that PRKD1 in bone marrow-derived mesenchymal stem cells (BMMSC) could modulate osteogenesis. In BMMSCs from floxed PRKD1 mice, PRKD1 ablation with adenovirus-mediated Cre-recombinase expression inhibited BMMSC differentiation in vitro. In 3- and 6-month-old conditional knockout mice (cKO), in which PRKD1 was ablated in osteoprogenitor cells by osterix promoter-driven Cre-recombinase, bone mineral density (BMD) was significantly reduced compared with floxed control littermates. Microcomputed tomography analysis also demonstrated a decrease in trabecular thickness and bone volume fraction in cKO mice at these ages. Dynamic bone histomorphometry suggested a mineralization defect in the cKO mice. However, by 9 months of age, the bone appeared to compensate for the lack of PRKD1, and BMD was not different. Taken together, these results suggest a potentially important role for PRKD1 in bone formation.

Loss of Hdac3 in osteoprogenitors increases bone expression of osteoprotegerin, improving systemic insulin sensitivity.

Type 2 diabetes is an emerging global health epidemic. Foundations for new therapies are arising from understanding interactions between body systems. Bone-derived factors that reduce RANKL (receptor activator of NF-kappa B ligand) signaling in the liver may prevent insulin resistance and the onset of type 2 diabetes. Here we demonstrate that deletion of the epigenetic regulator, Hdac3, in Osx1-expressing osteoprogenitors prevents insulin resistance induced by high fat diet by increasing serum and skeletal gene expression levels of osteoprotegerin (Opg), a natural inhibitor of RANKL signaling. Removal of one Opg allele in mice lacking Hdac3 in Osx1+ osteoprogenitors increases the insulin resistance of the Hdac3-deficient mice on a high fat diet. Thus, Hdac3-depletion in osteoblasts increases expression of Opg, subsequently preserving insulin sensitivity. The Hdac inhibitor vorinostat also increased Opg transcription and histone acetylation of the Opg locus. These results define a new mechanism by which bone regulates systemic insulin sensitivity.

Mimicking the effects of spaceflight on bone: Combined effects of disuse and chronic low-dose rate radiation exposure on bone mass in mice.

During spaceflight, crewmembers are subjected to biomechanical and biological challenges including microgravity and radiation. In the skeleton, spaceflight leads to bone loss, increasing the risk of fracture. Studies utilizing hindlimb suspension (HLS) as a ground-based model of spaceflight often neglect the concomitant effects of radiation exposure, and even when radiation is accounted for, it is often delivered at a high-dose rate over a very short period of time, which does not faithfully mimic spaceflight conditions. This study was designed to investigate the skeletal effects of low-dose rate gamma irradiation (8.5 cGy gamma radiation per day for 20 days, amounting to a total dose of 1.7 Gy) when administered simultaneously to disuse from HLS. The goal was to determine whether continuous, low-dose rate radiation administered during disuse would exacerbate bone loss in a murine HLS model. Four groups of 16 week old female C57BL/6 mice were studied: weight bearing + no radiation (WB+NR), HLS + NR, WB + radiation exposure (WB+RAD), and HLS+RAD. Surprisingly, although HLS led to cortical and trabecular bone loss, concurrent radiation exposure did not exacerbate these effects. Our results raise the possibility that mechanical unloading has larger effects on the bone loss that occurs during spaceflight than low-dose rate radiation.

Kynurenine, a Tryptophan Metabolite That Accumulates With Age, Induces Bone Loss.

Age-dependent bone loss occurs in humans and in several animal species, including rodents. The underlying causal mechanisms are probably multifactorial, although an age-associated increase in the generation of reactive oxygen species has been frequently implicated. We previously reported that aromatic amino acids function as antioxidants, are anabolic for bone, and that they may potentially play a protective role in an aging environment. We hypothesized that upon oxidation the aromatic amino acids would not only lose their anabolic effects but also potentially become a catabolic byproduct. When measured in vivo in C57BL/6 mice, the tryptophan oxidation product and kynurenine precursor, N-formylkynurenine (NFK), was found to increase with age. We tested the direct effects of feeding kynurenine (kyn) on bone mass and also tested the short-term effects of intraperitoneal kyn injection on bone turnover in CD-1 mice. µCT analyses showed kyn-induced bone loss. Levels of serum markers of osteoclastic activity (pyridinoline [PYD] and RANKL) increased significantly with kyn treatment. In addition, histological and histomorphometric studies showed an increase in osteoclastic activity in the kyn-treated groups in both dietary and injection-based studies. Further, kyn treatment significantly increased bone marrow adiposity, and BMSCs isolated from the kyn-injected mice exhibited decreased mRNA expression of Hdac3 and its cofactor NCoR1 and increased expression of lipid storage genes Cidec and Plin1. A similar pattern of gene expression is observed with aging. In summary, our data show that increasing kyn levels results in accelerated skeletal aging by impairing osteoblastic differentiation and increasing osteoclastic resorption. These data would suggest that kyn could play a role in age-induced bone loss. © 2017 American Society for Bone and Mineral Research.