Senescent Osteocytes Research Articles

Single-cell Transcriptomic Analysis Identifies Senescent Osteocytes that Trigger Bone Destruction in Breast Cancer Metastasis.

Breast cancer bone metastases increase fracture risk and are a major cause of morbidity and mortality among women. Upon colonization by tumor cells, the bone microenvironment undergoes profound reprogramming to support cancer progression, which disrupts the balance between osteoclasts and osteoblasts and leads to bone lesions. A deeper understanding of the processes mediating this reprogramming could help develop interventions for treating patients with bone metastases. Here, we demonstrated that osteocytes in established breast cancer bone metastasis develop premature senescence and a distinctive senescence-associated secretory phenotype (SASP) that favors bone destruction. Single-cell RNA sequencing identified osteocytes from mice with breast cancer bone metastasis enriched in senescence, SASP markers, and pro-osteoclastogenic genes. Multiplex in situ hybridization and AI-assisted analysis depicted osteocytes with senescence-associated satellite distension, telomere dysfunction, and p16Ink4a expression in mice and patients with breast cancer bone metastasis. Breast cancer cells promoted osteocyte senescence and enhanced their osteoclastogenic potential in in vitro and ex vivo organ cultures. Clearance of senescent cells with senolytics suppressed bone resorption and preserved bone mass in mice with breast cancer bone metastasis. These results demonstrate that osteocytes undergo pathological reprogramming by breast cancer cells and identify osteocyte senescence as an initiating event triggering lytic bone disease in breast cancer metastases.

Osteocyte ferroptosis induced by ATF3/TFR1 contributes to cortical bone loss during ageing.

Cortical bone loss is intricately associated with ageing and coincides with iron accumulation. The precise role of ferroptosis, characterized by iron overload and lipid peroxidation, in senescent osteocytes remains elusive. We found that ferroptosis was a crucial mode of osteocyte death in cortical bone during ageing. Using a single-cell transcriptome analysis, we identified activating transcription factor 3 (ATF3) as a critical driver of osteocyte ferroptosis. Elevated ATF3 expression in senescent osteocytes promotes iron uptake by upregulating transferrin receptor 1 while simultaneously inhibiting solute carrier family 7-member 11-mediated cystine import. This process leads to an iron overload and lipid peroxidation, culminating in ferroptosis. Importantly, ATF3 inhibition in aged mice effectively alleviated ferroptosis in the cortical bone and mitigated cortical bone mass loss. Taken together, our findings establish a pivotal role of ferroptosis in cortical bone loss in older adults, providing promising prevention and treatment strategies for osteoporosis and fractures.

Young osteocyte-derived extracellular vesicles facilitate osteogenesis by transferring tropomyosin-1

BackgroundBone marrow mesenchymal stem cells (BMSCs) can undergo inadequate osteogenesis or excessive adipogenesis as they age due to changes in the bone microenvironment, ultimately resulting in decreased bone density and elevated risk of fractures in senile osteoporosis. This study aims to investigate the effects of osteocyte senescence on the bone microenvironment and its influence on BMSCs during aging.ResultsPrimary osteocytes were isolated from 2-month-old and 16-month-old mice to obtain young osteocyte-derived extracellular vesicles (YO-EVs) and senescent osteocyte-derived EVs (SO-EVs), respectively. YO-EVs were found to significantly increase alkaline phosphatase activity, mineralization deposition, and the expression of osteogenesis-related genes in BMSCs, while SO-EVs promoted BMSC adipogenesis. Neither YO-EVs nor SO-EVs exerted an effect on the osteoclastogenesis of primary macrophages/monocytes. Our constructed transgenic mice, designed to trace osteocyte-derived EV distribution, revealed abundant osteocyte-derived EVs embedded in the bone matrix. Moreover, mature osteoclasts were found to release osteocyte-derived EVs from bone slices, playing a pivotal role in regulating the functions of the surrounding culture medium. Following intravenous injection into young and elderly mouse models, YO-EVs demonstrated a significant enhancement of bone mass and biomechanical strength compared to SO-EVs. Immunostaining of bone sections revealed that YO-EV treatment augmented the number of osteoblasts on the bone surface, while SO-EV treatment promoted adipocyte formation in the bone marrow. Proteomics analysis of YO-EVs and SO-EVs showed that tropomyosin-1 (TPM1) was enriched in YO-EVs, which increased the matrix stiffness of BMSCs, consequently promoting osteogenesis. Specifically, the siRNA-mediated depletion of Tpm1 eliminated pro-osteogenic activity of YO-EVs both in vitro and in vivo.ConclusionsOur findings suggested that YO-EVs played a crucial role in maintaining the balance between bone resorption and formation, and their pro-osteogenic activity declining with aging. Therefore, YO-EVs and the delivered TPM1 hold potential as therapeutic targets for senile osteoporosis.Graphical abstract

Senolytics deplete senescent osteocytes and improve bone health in metastatic breast cancer

Senolytics deplete senescent osteocytes and improve bone health in metastatic breast cancer

Eldecalcitol protected osteocytes against ferroptosis of D-gal-induced senescent MLO-Y4 cells and ovariectomized mice

BackgroundActive vitamin D analog eldecalcitol is clinically applied in treatment of postmenopausal osteoporosis. This study aims to determine the role of eldecalcitol in the protection of osteocytes from senescence and the associated ferroptosis. MethodsThe MLO-Y4 osteocytes were exposed to D-gal inducing senescence. The ovariectomized (OVX) mice treated with D-gal using as an aging inducer were intraperitoneally injected with eldecalcitol. The multiplexed confocal imaging, fluorescence in situ hybridization and transmission electron microscopy were applied in assessing osteocytic properties. Immunochemical staining and immunoblotting were carried out to detect abundance and expression of molecules. ResultsThe ablation of vitamin D receptor led to a reduction in amounts of osteocytes, a loss of dendrites, an increase in mRNA expression of SASP factors and in protein expression of senescent factors, as well as changes in mRNA expression of ferroptosis-related genes (PTGS2 & RGS4). Eldecalcitol reversed senescent phenotypes of MLO-Y4 cells shown by improving cell morphology and density, decreasing β-gal-positive cell accumulation, and down-regulating protein expression (P16, P21 & P53). Eldecalcitol reduced intracellular ROS and MDA productions, elevated JC-1 aggregates, and up-regulated expression of Nrf2 and GPX4. Eldecalcitol exhibited osteopreserve effects in D-gal-induced aging OVX mice. The confocal imaging displayed its improvement on osteocytic network organization. Eldecalcitol decreased the numbers of senescent osteocytes at tibial diaphysis by SADS assay and attenuated mRNA expression of SASP factors as well as down-regulated protein expression of senescence-related factors and restored levels of ferroptotic biomarkers in osteocytes-enriched bone fraction. It reduced 4-HNE staining area, stimulated Nrf2-positive staining, and promoted nuclear translocation of Nrf2 in osteocytes of mice as well as inhibited and promoted protein expression of 4-HNE and Nrf2, respectively, in osteocytes-enriched bone fraction. ConclusionsThe present study revealed the ameliorative effects of eldecalcitol on senescence and the associated ferroptosis of osteocytes, contributing to its preservation against osteoporosis of D-gal-induced senescent ovariectomized mice.

Down-expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenesis and accelerates age-related bone loss via PTEN/PI3K/AKT pathway.

To investigate the effects of senescent osteocytes on bone homeostasis in the progress of age-related osteoporosis and explore the underlying mechanism. In a series of in vitro experiments, we used tert-Butyl hydroperoxide (TBHP) to induce senescence of MLO-Y4 cells successfully, and collected conditioned medium (CM) and senescent MLO-Y4 cell-derived exosomes, which were then applied to MC3T3-E1 cells, separately, to evaluate their effects on osteogenic differentiation. Furthermore, we identified differentially expressed microRNAs (miRNAs) between exosomes from senescent and normal MLO-Y4 cells by high-throughput RNA sequencing. Based on the key miRNAs that were discovered, the underlying mechanism by which senescent osteocytes regulate osteogenic differentiation was explored. Lastly, in the in vivo experiments, the effects of senescent MLO-Y4 cell-derived exosomes on age-related bone loss were evaluated in male SAMP6 mice, which excluded the effects of oestrogen, and the underlying mechanism was confirmed. The CM and exosomes collected from senescent MLO-Y4 cells inhibited osteogenic differentiation of MC3T3-E1 cells. RNA sequencing detected significantly lower expression of miR-494-3p in senescent MLO-Y4 cell-derived exosomes compared with normal exosomes. The upregulation of exosomal miR-494-3p by miRNA mimics attenuated the effects of senescent MLO-Y4 cell-derived exosomes on osteogenic differentiation. Luciferase reporter assay demonstrated that miR-494-3p targeted phosphatase and tensin homolog (PTEN), which is a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overexpression of PTEN or inhibition of the PI3K/AKT pathway blocked the functions of exosomal miR-494-3p. In SAMP6 mice, senescent MLO-Y4 cell-derived exosomes accelerated bone loss, which was rescued by upregulation of exosomal miR-494-3p. Reduced expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenic differentiation and accelerates age-related bone loss via PTEN/PI3K/AKT pathway.

Elimination of Senescent Osteocytes by Bone-Targeting Delivery of β-Galactose-Modified Maytansinoid Prevents Age-Related Bone Loss.

The accumulation of senescent cells in bone during aging contributes to senile osteoporosis, and clearance of senescent cells by senolytics could effectively alleviate bone loss. However, the applications of senolytics are limited due to their potential toxicities. Herein, small extracellular vesicles (sEVs) have been modified by incorporating bone-targeting peptide, specifically (AspSerSer)6, to encapsulate galactose-modified Maytansinoids (DM1). These modified vesicles are referred to as (AspSerSer)6-sEVs/DM1-Gal, and they have been designed to specifically clear the senescent osteocytes in bone tissue. In addition, the elevated activity of lysosomal β-galactosidase in senescent osteocytes, but not normal cells in bone tissue, could break down DM1-Gal to release free DM1 for selective elimination of senescent osteocytes. Mechanically, DM1 could disrupt tubulin polymerization, subsequently inducing senescent osteocytes apoptosis. Further, administration of bone-targeting senolytics to aged mice could alleviate aged-related bone loss without non-obvious toxicity. Overall, this bone-targeting senolytics could act as a novel candidate for specific clearance of senescent osteocytes, ameliorating age-related bone loss, with a promising therapeutic potential for senile osteoporosis.

Lycopene Improves Bone Quality in SAMP6 Mice by Inhibiting Oxidative Stress, Cellular Senescence, and the SASP.

Cellular senescence (CS) is closely related to tissue ageing including bone ageing. CS and the senescence-associated secretory phenotype (SASP) have emerged as critical pathogenesis elements of senile osteoporosis. This study aims to investigate the effect of lycopene on senile osteoporosis. The senescence-accelerated mouse prone 6 (SAMP6) strain of mice is used as the senile osteoporosis model. Daily ingestion of lycopene for 8 weeks preserves the bone mass, density, strength, and microarchitecture in the SAMP6 mice. Moreover, these alterations are associated with a decrease in oxidative stress in the senile osteoporosis model. In addition, there is a reduction in osteoblast and osteocyte senescence and the SASP in the bone tissues of the SAMP6 mice. Lycopene improves bone health likely due to its antioxidant properties that may be linked with the regulation of CS and SASP in the SAMP6 mice. These results suggest that lycopene may be beneficial for the management of senile osteoporosis by inhibiting oxidative stress, CS, and the SASP.

Pyrroloquinoline quinone alleviates natural aging-related osteoporosis via a novel MCM3-Keap1-Nrf2 axis-mediated stress response and Fbn1 upregulation.

Age-related osteoporosis is associated with increased oxidative stress and cellular senescence. Pyrroloquinoline quinone (PQQ) is a water-soluble vitamin-like compound that has strong antioxidant capacity; however, the effect and underlying mechanism of PQQ on aging-related osteoporosis remain unclear. The purpose of this study was to investigate whether dietary PQQ supplementation can prevent osteoporosis caused by natural aging, and the potential mechanism underlying PQQ antioxidant activity. Here, we found that when 6-month-old or 12-month-old wild-type mice were supplemented with PQQ for 12 months or 6 months, respectively, PQQ could prevent age-related osteoporosis in mice by inhibiting osteoclastic bone resorption and stimulating osteoblastic bone formation. Mechanistically, pharmmapper screening and molecular docking studies revealed that PQQ appears to bind to MCM3 and reduces its ubiquitination-mediated degradation; stabilized MCM3 then competes with Nrf2 for binding to Keap1, thus activating Nrf2-antioxidant response element (ARE) signaling. PQQ-induced Nrf2 activation inhibited bone resorption through increasing stress response capacity and transcriptionally upregulating fibrillin-1 (Fbn1), thus reducing Rankl production in osteoblast-lineage cells and decreasing osteoclast activation; as well, bone formation was stimulated by inhibiting osteoblastic DNA damage and osteocyte senescence. Furthermore, Nrf2 knockout significantly blunted the inhibitory effects of PQQ on oxidative stress, on increased osteoclast activity and on the development of aging-related osteoporosis. This study reveals the underlying mechanism of PQQ's strong antioxidant capacity and provides evidence for PQQ as a potential agent for clinical prevention and treatment of natural aging-induced osteoporosis.

Skeletal Senescence with Aging and Type 2 Diabetes.

Osteoporosis and type 2 diabetes (T2D) are common diseases that often coexist. While both of these diseases are associated with poor bone quality and increased fracture risk, their pathogenesis of increased fracture risk differs and is multifactorial. Mounting evidence now indicates that key fundamental mechanisms that are central to both aging and energy metabolism exist. Importantly, these mechanisms represent potentially modifiable therapeutic targets for interventions that could prevent or alleviate multiple complications of osteoporosis and T2D, including poor bone quality. One such mechanism that has gained increasing momentum is senescence, which is a cell fate that contributes to multiple chronic diseases. Accumulating evidence has established that numerous boneresident cell types become susceptible to cellular senescence with old age. Recent work also demonstrates that T2D causes the premature accumulation of senescent osteocytes during young adulthood, at least in mice, although it remains to be seen which other bone-resident cell types become senescent with T2D. Given that therapeutically removing senescent cells can alleviate age-related bone loss and T2D-induced metabolic dysfunction, it will be important in future studies to rigorously test whether interventions that eliminate senescent cells can also alleviate skeletal dysfunction in context of T2D, as it does with aging.

Deconstructing cellular senescence in bone and beyond.

Osteocytes are specialized bone cells that orchestrate skeletal remodeling. Senescent osteocytes are characterized by an activation of cyclin-dependent kinase inhibitor p16Ink4a and have been implicated in the pathogenesis of several bone loss disorders. In this issue of the JCI, Farr et al. have now shown that systemic removal of senescent cells (termed senolysis) prevented age-related bone loss at the spine and femur and mitigated bone marrow adiposity through a robust effect on osteoblasts and osteoclasts, whereas cell-specific senolysis in osteocytes alone was only partially effective. Surprisingly, transplantation of senescent fibroblasts into the peritoneum of young mice caused host osteocyte senescence associated with bone loss. This refined concept of osteocyte senescence and the effects of remote senolysis may help to develop improved senolytic strategies against multisystem aging in bone and beyond.

Hindlimb unloading in C57BL/6J mice induces bone loss at thermoneutrality without change in osteocyte and lacuno-canalicular network.

Impaired mechanical stimuli during hindlimb unloading (HLU) are believed to exacerbate osteocyte paracrine regulation of osteoclasts. We hypothesized that bone loss and deterioration of the osteocyte lacuno-canalicular network are attenuated in HLU mice housed at thermoneutrality (28°C) compared with those housed at ambient temperature (22°C). Following acclimatization, 20-week-old male C57BL/6J mice were submitted to HLU or kept in pair-fed control cages (CONT), for 5days (5d) or 14d, at 22°C or 28°C. In the femur distal metaphysis, thermoneutral CONT mice had higher bone volume (p=0.0007, BV/TV, in vivo μCT, vs. 14dCONT22) whilst osteoclastic surfaces of CONT and HLU were greater at 22°C (5dCONT22+53%, 5dHLU22+50%, 14dCONT22+186%, 14dHLU22+104%, vs matching 28°C group). In the femur diaphysis and at both temperatures, 14dHLU exhibited thinner cortices distally or proximally compared to controls; the mid-diaphysis being thicker at 28°C than at 22°C in all groups. Expression of cortical genes for proteolytic enzyme (Mmp13), markers for osteoclastogenic differentiation (MCSF, RANKL), and activity (TRAP, Ctsk) were increased following 22°C HLU, whereas only Ctsk expression was increased following 28°C HLU. Expression of cortical genes for apoptosis, senescence, and autophagy were not elevated following HLU at any temperature. Osteocyte density at the posterior mid-diaphysis was similar between groups, as was the proportion of empty lacunae (<0.5%). However, analysis of the lacuno-canalicular network (LCN, fluorescein staining) revealed unstained areas in the 14dHLU22 group only, suggesting disrupted LCN flow in this group alone. In conclusion, 28°C housing influences the HLU bone response but does not prevent bone loss. Furthermore, our results do not show osteocyte senescence or death, and at thermoneutrality, HLU-induced bone resorption is not triggered by osteoclastic activators RANKL and MCSF.

Inflammation produced by senescent osteocytes mediates age-related bone loss.

The molecular mechanisms of age-related bone loss are unclear and without valid drugs yet. The aims of this study were to explore the molecular changes that occur in bone tissue during age-related bone loss, to further clarify the changes in function, and to predict potential therapeutic drugs. We collected bone tissues from children, middle-aged individuals, and elderly people for protein sequencing and compared the three groups of proteins pairwise, and the differentially expressed proteins (DEPs) in each group were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). K-means cluster analysis was then used to screen out proteins that continuously increased/decreased with age. Canonical signaling pathways that were activated or inhibited in bone tissue along with increasing age were identified by Ingenuity Pathway Analysis (IPA). Prediction of potential drugs was performed using the Connectivity Map (CMap). Finally, DEPs from sequencing were verified by Western blot, and the drug treatment effect was verified by quantitative real-time PCR. The GO and KEGG analyses show that the DEPs were associated with inflammation and bone formation with aging, and the IPA analysis shows that pathways such as IL-8 signaling and acute-phase response signaling were activated, while glycolysis I and EIF2 signaling were inhibited. A total of nine potential drugs were predicted, with rapamycin ranking the highest. In cellular experiments, rapamycin reduced the senescence phenotype produced by the H2O2-stimulated osteocyte-like cell MLO-Y4. With age, inflammatory pathways are activated in bone tissue, and signals that promote bone formation are inhibited. This study contributes to the understanding of the molecular changes that occur in bone tissue during age-related bone loss and provides evidence that rapamycin is a drug of potential clinical value for this disease. The therapeutic effects of the drug are to be further studied in animals.

Abstract 5675: Pathological crosstalk between osteocytes and breast cancer cells in bone metastasis

Abstract Bone provides an ideal niche for breast cancer cells (BCa) to reside and proliferate and is a frequent site of BCa metastasis. Osteocytes are the most abundant skeletal cells and permanent residents of bone. However, the role of osteocytes in BCa bone metastasis remains understudied. Here, we investigated the crosstalk between metastatic BCa cells and osteocytes. In vitro cultures revealed that conditioned media (CM) derived from metastatic E0771 BCa cells decreased by 50% the number of alive MLO-A5 and MLO-Y4 osteocyte-like cell lines. BCa cells modestly increased osteocyte apoptosis (2% vs. 6%, alone vs. exposed to BCa, respectively). Treatment with DEVD, a caspase 3 inhibitor, blocked osteocyte apoptosis but did not prevent the decrease in osteocytes induced by BCa. Thus, we studied osteocyte senescence, a process that causes irreversible cell cycle arrest and profound changes in gene expression. We found that BCa-CM upregulated the expression of markers of senescence markers (p16, p21) and senescence-associated secretory phenotype (Mmp13, Mmp9, Vcam-1) in osteocyte-like cell lines. Similarly, BCa-CM increased the expression of senescent markers in ex vivo bone organ cultures containing primary osteocytes. Osteocytes increased BCa cell proliferation by increasing cyclin D1 expression Through both cell-to-cell interactions and secretion of soluble factors., osteocytes increased BCa cell proliferation by increasing cyclin D1 expression. Moreover, osteocyte-derived CM stimulated BCa cells migration and invasion capabilities and upregulated the expression of genes involved in migration (Vcam-1 and N-cadherin). Co-culture with stromal cells also stimulated BCa cell proliferation, invasion, and migration. However, the increases induced by stromal cells were four-fold lower than those seen with osteocytes. Overall, our results suggest that BCa cells reprogram osteocytes, which acquire a senescent phenotype, whereas osteocytes produce factors that stimulate tumor growth, migration, and invasion. Together, these findings support the existence of a crosstalk between osteocytes and BCa cells in the metastatic niche that favors the progression of breast cancer in bone. Future studies are warranted to examine the contribution of osteocyte senescence and osteocytes-derived factors in animal models of breast cancer metastasis. Citation Format: Manish Adhikari, Hayley Sabol, Aric anloague, Sharmin Khan, Jesus Delgado-Calle. Pathological crosstalk between osteocytes and breast cancer cells in bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5675.

The mechanotransduction of MLO-Y4 cells is disrupted by the senescence-associated secretory phenotype of neighboring cells.

Age-related bone loss is attributed to the accumulation of senescent cells and their increasing production of inflammatory cytokines as part of the senescence-associated secretory phenotype (SASP). In otherwise healthy individuals, osteocytes play a key role in maintaining bone mass through their primary function of responding to skeletal loading. Given that osteocytes' response to loading is known to steadily decline with age, we hypothesized that the increasing presence of senescent cells and their SASP inhibit osteocytes' response to loading. To test this hypothesis, we developed two in vitro models of senescent osteocytes and osteoblasts derived from MLO-Y4 and MC3T3 cell lines, respectively. The senescent phenotype was unique to each cell type based on distinct changes in cell cycle inhibitors and SASP profile. The SASP profile of senescent osteocytes was in part dependent on nuclear factor-κB signaling and presents a new potential mechanism to targetthe SASP in bone. Nonsenescent MLO-Y4 cells cultured with the SASP of each senescent cell type failed to exhibit changes in gene expression as well as ERK phosphorylation and prostaglandin E2 release. The SASP of senescent osteocytes had the largest effect and neutralizing interleukin-6 (IL-6) as part of the SASP restored osteocytes' response to loading. The loss in mechanotransduction due to IL-6 was attributed to a decrease in P2X7 expression and overall sensitivity to purinergic signaling. Altogether, these findings demonstrate that the SASP of senescent cells have a negative effect on the mechanotransduction of osteocytes and that IL-6 is a key SASP component that contributes to the loss in mechanotransduction.

Astaxanthin attenuates irradiation-induced osteoporosis in mice by inhibiting oxidative stress, osteocyte senescence, and SASP.

Radiation therapy (RT) is a crucial part of many treatment plans for cancer patients. However, major undesired side effects are associated with this treatment, including impaired bone remodeling and bone loss. Irradiation induces bone loss due to promoted osteoclastic bone resorption and reduced osteoblastic bone formation. Astaxanthin (AST) is a natural antioxidant with anti-oxidative and anti-aging properties. However, it is unclear whether AST is also protective against osteoporosis induced by ionizing radiation (IR). Here, we evaluate the efficacy of AST in mitigating IR-induced bone loss in a mouse model where both hindlimbs received radiation. Reduced BMD, bone biomechanical strength, bone formation, elevated oxidative stress, and osteoclast activity with microarchitectural deterioration of trabecular and cortical bones were observed in IR mice. Supplementation with AST corrected these osteoporotic phenotypes, caused by IR, by inhibiting oxidative stress, DNA damage, osteocyte senescence, and senescence-associated secretory phenotype (SASP), subsequently promoting osteoblastic bone formation and inhibiting osteoclastic bone resorption. The results from our study provide experimental evidence for the clinical use of AST to prevent IR-induced osteoporosis in cancer patients.

Induction of somatopause in adult mice compromises bone morphology and exacerbates bone loss during aging.

Somatopause refers to the gradual declines in growth hormone (GH) and insulin‐like growth factor‐1 throughout aging. To define how induced somatopause affects skeletal integrity, we used an inducible GH receptor knockout (iGHRKO) mouse model. Somatopause, induced globally at 6 months of age, resulted in significantly more slender bones in both male and female iGHRKO mice. In males, induced somatopause was associated with progressive expansion of the marrow cavity leading to significant thinning of the cortices, which compromised bone strength. We report progressive declines in osteocyte lacunar number, and increases in lacunar volume, in iGHRKO males, and reductions in lacunar number accompanied by ~20% loss of overall canalicular connectivity in iGHRKO females by 30 months of age. Induced somatopause did not affect mineral/matrix ratio assessed by Raman microspectroscopy. We found significant increases in bone marrow adiposity and high levels of sclerostin, a negative regulator of bone formation in iGHRKO mice. Surprisingly, however, despite compromised bone morphology, osteocyte senescence was reduced in the iGHRKO mice. In this study, we avoided the confounded effects of constitutive deficiency in the GH/IGF‐1 axis on the skeleton during growth, and specifically dissected its effects on the aging skeleton. We show here, for the first time, that induced somatopause compromises bone morphology and the bone marrow environment.

Radiation-Induced Osteocyte Senescence Alters Bone Marrow Mesenchymal Stem Cell Differentiation Potential via Paracrine Signaling.

Cellular senescence and its senescence-associated secretory phenotype (SASP) are widely regarded as promising therapeutic targets for aging-related diseases, such as osteoporosis. However, the expression pattern of cellular senescence and multiple SASP secretion remains unclear, thus leaving a large gap in the knowledge for a desirable intervention targeting cellular senescence. Therefore, there is a critical need to understand the molecular mechanism of SASP secretion in the bone microenvironment that can ameliorate aging-related degenerative pathologies including osteoporosis. In this study, osteocyte-like cells (MLO-Y4) were induced to cellular senescence by 2 Gy γ-rays; then, senescence phenotype changes and adverse effects of SASP on bone marrow mesenchymal stem cell (BMSC) differentiation potential were investigated. The results revealed that 2 Gy irradiation could hinder cell viability, shorten cell dendrites, and induce cellular senescence, as evidenced by the higher expression of senescence markers p16 and p21 and the elevated formation of senescence-associated heterochromatin foci (SAHF), which was accompanied by the enhanced secretion of SASP markers such as IL-1α, IL-6, MMP-3, IGFBP-6, resistin, and adiponectin. When 0.8 μM JAK1 inhibitors were added to block SASP secretion, the higher expression of SASP was blunted, but the inhibition in osteogenic and adipogenic differentiation potential of BMSCs co-cultured with irradiated MLO-Y4 cell conditioned medium (CM- 2 Gy) was alleviated. These results suggest that senescent osteocytes can perturb BMSCs’ differential potential via the paracrine signaling of SASP, which was also demonstrated by in vivo experiments. In conclusion, we identified the SASP factor partially responsible for the degenerative differentiation of BMSCs, which allowed us to hypothesize that senescent osteocytes and their SASPs may contribute to radiation-induced bone loss.

Radiation induces primary osteocyte senescencephenotype and affects osteoclastogenesis invitro.

Irradiation-induced bone remodeling imbalances arise as a consequence of the dysregulation of bone formation and resorption. Due to the abundance of osteocytes, their long life and their dual-regulatory effects on both osteoblast and osteoclast function, they serve as critical coordinators of bone remolding. In the present study, femur and tibia-derived primary osteocytes were cultured and irradiated to observe the functional changes and the cellular senescence phenotype in vitro. Irradiation directly reduced cell viability, affected the crucial dendritic morphology and altered the expression of functional proteins, including upregulation of receptor activator of nuclear factor-κB ligand and sclerostin, and downregulation of osteoprotegerin. Irradiated osteocytes were shown to exhibit notable DNA damage, which resulted in the initiation of a typical cellular senescence phenotype. Furthermore, it was found that irradiation-induced prematurely senescent osteocytes stimulate molecular secretion, referred to as senescence-associated secretory phenotype (SASP), which may be involved in modulation of the bone microenvironment, including the promotion of osteoclastogenesis. Taken together, the results showed that irradiation triggered osteocyte senescence and the acquisition of an associated secretory phenotype. This further resulted in an imbalance of bone remodeling through senescent influence on proliferation, morphology and marker protein production, but also indirectly via a paracrine pathway through SASP secretion. The results of the present study may highlight the potential of SASP-targeted interventions for the management of radiation-induced bone loss.

Senescent cells exacerbate chronic inflammation and contribute to periodontal disease progression in old mice.

Coinciding with other chronic comorbidities, the prevalence of periodontal disease increases with aging. Mounting evidence has established that senescent cells accumulate at sites of age-related pathologies, where they promote "non-microbial" inflammation. We hypothesized that alveolar bone osteocytes develop senescence characteristics in old age. Alveolar bone samples were obtained from young (6 months) and old (20 to 22 months) mice to evaluate the expression of senescence biomarkers by immunofluorescent staining. Osteocyte-enriched fractions were used to characterize the age-related senescence-associated secretory phenotype (SASP) gene expression profile. Primary alveolar bone cells were exposed to the SASP via in vitro senescent conditioned media (SCM) administration. A multiplex assay confirmed protein levels of specific cytokines. Interactions with bacterial components were evaluated by stimulating cells with lipopolysaccharide (LPS). Increased senescence-associated distension of satellites (SADS) and p16Ink4a mRNA expression were identified in alveolar bone osteocytes with aging. These findings were associated with increased levels of DNA damage, and activated p38 MAPK, both inducers of senescence. Furthermore, interleukin-6 (IL6), IL17, IGFBP4, and MMP13 were significantly upregulated with aging in osteocyte-enriched samples. Interestingly, SCM potentiated the LPS-induced expression of IL1α,IL1β,and IL6. Cell migration and differentiation were also impeded by SCM. These in vitro effects were ameliorated by the p38 MAPK inhibitor SB202190. Accumulation of senescent osteocytes contributes to deterioration of the periodontal environment by exacerbating chronic inflammation and reducing regeneration in old age. Cellular senescence is a cell-intrinsic response to DNA damage, and a host-related mechanism associated with aging that could potentiate inflammation induced by bacterial components.