Marrow Adipocyte Senescence in the Pathogenesis of Bone Loss.

PURPOSE OF REVIEW: Beyond aging, senescent cells accumulate during multiple pathological conditions, including chemotherapy, radiation, glucocorticoids, obesity, and diabetes, even earlier in life. Therefore, cellular senescence represents a unifying pathogenic mechanism driving skeletal and metabolic disorders. However, whether senescent bone marrow adipocytes (BMAds) are causal in mediating skeletal dysfunction has only recently been evaluated. RECENT FINDINGS: Despite evidence of BMAd senescence following glucocorticoid therapy, additional evidence for BMAd senescence in other conditions has thus far been limited. Because the study of BMAds presents unique challenges making these cells difficult to isolate and image, here we review issues and approaches to overcome such challenges, and present advancements in isolation and histological techniques that may help with the future study of senescent BMAds. Further insights into the roles of BMAd senescence in the pathogenesis of skeletal dysfunction may have important basic science and clinical implications for human physiology and disease.

Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair.

Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21+ cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21+ cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21+ cell clearance did not alter bone loss due to aging; conversely, p16+ cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21+ neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21+ OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell-like OCH subset that was p21+ and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells were key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings established contextual roles of p21+ versus p16+ senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

Modest Effects of Osteoclast-Specific ERα Deletion after Skeletal Maturity.

Estrogen regulates bone mass in women and men, but the underlying cellular mechanisms of estrogen action on bone remain unclear. Although both estrogen receptor (ER)α and ERß are expressed in bone cells, ERα is the dominant receptor for skeletal estrogen action. Previous studies using either global or cell-specific ERα deletion provided important insights, but each of these approaches had limitations. Specifically, either high circulating sex steroid levels in global ERα knockout mice or the effects of deletion of ERα during growth and development in constitutive cell-specific knockout mice have made it difficult to clearly define the role of ERα in specific cell types in the adult skeleton. We recently generated and characterized mice with tamoxifen-inducible ERα deletion in osteocytes driven by the 8-kb Dmp1 promoter (ERαΔOcy mice), revealing detrimental effects of osteocyte-specific ERα deletion on trabecular bone volume (-20.1%) and bone formation rate (-18.9%) in female, but not male, mice. Here, we developed and characterized analogous mice with inducible ERα deletion in osteoclasts using the Cathepsin K promoter (ERαΔOcl mice). In a study design identical to that with the previously described ERαΔOcy mice, adult female, but not male, ERαΔOcl mice showed a borderline (-10.2%, p = 0.084) reduction in trabecular bone volume, no change in osteoclast numbers, but a significant increase in serum CTx levels, consistent with increased osteoclast activity. These findings in ERαΔOcl mice differ from previous studies of constitutive osteoclast-specific ERα deletion, which led to clear deficits in trabecular bone and increased osteoclast numbers. Collectively, these data indicate that in adult mice, estrogen action in the osteocyte is likely more important than via the osteoclast and that ERα deletion in osteoclasts from conception onward has more dramatic skeletal effects than inducible osteoclastic ERα deletion in adult mice. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

A new gene set identifies senescent cells and predicts senescence-associated pathways across tissues.

Although cellular senescence drives multiple age-related co-morbidities through the senescence-associated secretory phenotype, in vivo senescent cell identification remains challenging. Here, we generate a gene set (SenMayo) and validate its enrichment in bone biopsies from two aged human cohorts. We further demonstrate reductions in SenMayo in bone following genetic clearance of senescent cells in mice and in adipose tissue from humans following pharmacological senescent cell clearance. We next use SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from human and murine bone marrow/bone scRNA-seq data. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Using this senescence panel, we are able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways. SenMayo also represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.

Skeletal Effects of Inducible ERα Deletion in Osteocytes in Adult Mice.

Estrogen is known to regulate bone metabolism in both women and men, but substantial gaps remain in our knowledge of estrogen and estrogen receptor alpha (ERα) regulation of adult bone metabolism. Studies using global ERα-knockout mice were confounded by high circulating sex-steroid levels, and osteocyte/osteoblast-specific ERα deletion may be confounded by ERα effects on growth versus the adult skeleton. Thus, we developed mice expressing the tamoxifen-inducible CreERT2 in osteocytes using the 8-kilobase (kb) Dmp1 promoter (Dmp1CreERT2 ). These mice were crossed with ERαfl//fl mice to create ERαΔOcy mice, permitting inducible osteocyte-specific ERα deletion in adulthood. After intermittent tamoxifen treatment of adult 4-month-old mice for 1 month, female, but not male, ERαΔOcy mice exhibited reduced spine bone volume fraction (BV/TV (-20.1%, p = 0.004) accompanied by decreased trabecular bone formation rate (-18.9%, p = 0.0496) and serum P1NP levels (-38.9%, p = 0.014). Periosteal (+65.6%, p = 0.004) and endocortical (+64.1%, p = 0.003) expansion were higher in ERαΔOcy mice compared to control (Dmp1CreERT2 ) mice at the tibial diaphysis, reflecting the known effects of estrogen to inhibit periosteal apposition and promote endocortical formation. Increases in Sost (2.1-fold, p = 0.001) messenger RNA (mRNA) levels were observed in trabecular bone at the spine in ERαΔOcy mice, consistent with previous reports that estrogen deficiency is associated with increased circulating sclerostin as well as bone SOST mRNA levels in humans. Further, the biological consequences of increased Sost expression were reflected in significant overall downregulation in panels of osteoblast and Wnt target genes in osteocyte-enriched bones from ERαΔOcy mice. These findings thus establish that osteocytic ERα is critical for estrogen action in female, but not male, adult bone metabolism. Moreover, the reduction in bone formation accompanied by increased Sost, decreased osteoblast, and decreased Wnt target gene expression in ERαΔOcy mice provides a direct link in vivo between ERα and Wnt signaling. © 2022 American Society for Bone and Mineral Research (ASBMR).

Skeletal Aging.

Aging represents the single greatest risk factor for chronic diseases, including osteoporosis, a skeletal fragility syndrome that increases fracture risk. Optimizing bone strength throughout life reduces fracture risk. Factors critical for bone strength include nutrition, physical activity, and vitamin D status, whereas unhealthy lifestyles, illnesses, and certain medications (eg, glucocorticoids) are detrimental. Hormonal status is another important determinant of skeletal health, with sex steroid concentrations, particularly estrogen, having major effects on bone remodeling. Aging exacerbates bone loss in both sexes and results in imbalanced bone resorption relative to formation; it is associated with increased marrow adiposity, osteoblast/osteocyte apoptosis, and accumulation of senescent cells. The mechanisms underlying skeletal aging are as diverse as the factors that determine the strength (and thus fragility) of bone. This review updates our current understanding of the epidemiology, pathophysiology, and treatment of osteoporosis and provides an overview of the underlying hallmark mechanisms that drive skeletal aging.

Identification of a suitable endogenous control miRNA in bone aging and senescence.

MicroRNAs (miRNAs) are promising tools as biomarkers and therapeutic agents in various chronic diseases such as osteoporosis, cancers, type I and II diabetes, and cardiovascular diseases. Considering the rising interest in the regulatory role of miRNAs in bone metabolism, aging, and cellular senescence, accurate normalization of qPCR-based miRNA expression data using an optimal endogenous control becomes crucial. We used a systematic approach to select candidate endogenous control miRNAs that exhibit high stability with aging from our miRNA sequence data and literature search. Validation of miRNA expression was performed using qPCR and their comprehensive stability was assessed using the RefFinder tool which is based on four statistical algorithms: GeNorm, NormFinder, BestKeeper, and comparative delta CT. The selected endogenous control was then validated for its stability in mice and human bone tissues, and in bone marrow stromal cells (BMSCs) following induction of senescence and senolytic treatment. Finally, the utility of selected endogenous control versus U6 was tested by using each as a normalizer to measure the expression of miR-34a, a miRNA known to increase with age and senescence. Our results show that Let-7f did not change across the groups with aging, senescence or senolytic treatment, and was the most stable miRNA, whereas U6 was the least stable. Moreover, using Let-7f as a normalizer resulted in significantly increased expression of miR-34a with aging and senescence and decreased expression following senolytic treatment. However, the expression pattern for miR-34a reversed for each of these conditions when U6 was used as a normalizer. We show that optimal endogenous control miRNAs, such as Let-7f, are essential for accurate normalization of miRNA expression data to increase the reliability of results and prevent misinterpretation. Moreover, we present a systematic strategy that is transferrable and can easily be used to identify endogenous control miRNAs in other biological systems and conditions.

Targeted clearance of p21- but not p16-positive senescent cells prevents radiation-induced osteoporosis and increased marrow adiposity.

Cellular senescence, which is a major cause of tissue dysfunction with aging and multiple other conditions, is known to be triggered by p16Ink4a or p21Cip1 , but the relative contributions of each pathway toward inducing senescence are unclear. Here, we directly addressed this issue by first developing and validating a p21-ATTAC mouse with the p21Cip1 promoter driving a "suicide" transgene encoding an inducible caspase-8 which, upon induction, selectively kills p21Cip1 -expressing senescent cells. Next, we used the p21-ATTAC mouse and the established p16-INK-ATTAC mouse to directly compare the contributions of p21Cip1 versus p16Ink4a in driving cellular senescence in a condition where a tissue phenotype (bone loss and increased marrow adiposity) is clearly driven by cellular senescence-specifically, radiation-induced osteoporosis. Using RNA in situ hybridization, we confirmed the reduction in radiation-induced p21Cip1 - or p16Ink4a -driven transcripts following senescent cell clearance in both models. However, only clearance of p21Cip1 +, but not p16Ink4a +, senescent cells prevented both radiation-induced osteoporosis and increased marrow adiposity. Reduction in senescent cells with dysfunctional telomeres following clearance of p21Cip1 +, but not p16Ink4a +, senescent cells also reduced several of the radiation-induced pro-inflammatory senescence-associated secretory phenotype factors. Thus, by directly comparing senescent cell clearance using two parallel genetic models, we demonstrate that radiation-induced osteoporosis is driven predominantly by p21Cip1 - rather than p16Ink4a -mediated cellular senescence. Further, this approach can be used to dissect the contributions of these pathways in other senescence-associated conditions, including aging across tissues.

Bone Marrow Adiposity in Models of Radiation- and Aging-Related Bone Loss Is Dependent on Cellular Senescence.

Oxidative stress-induced reactive oxygen species, DNA damage, apoptosis, and cellular senescence have been associated with reduced osteoprogenitors in a reciprocal fashion to bone marrow adipocyte tissue (BMAT); however, a direct (causal) link between cellular senescence and BMAT is still elusive. Accumulation of senescent cells occur in naturally aged and in focally radiated bone tissue, but despite amelioration of age- and radiation-associated bone loss after senescent cell clearance, molecular events that precede BMAT accrual are largely unknown. Here we show by RNA-Sequencing data that BMAT-related genes were the most upregulated gene subset in radiated bones of C57BL/6 mice. Using focal radiation as a model to understand age-associated changes in bone, we performed a longitudinal assessment of cellular senescence and BMAT. Using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), RNA in situ hybridization of p21 transcripts and histological assessment of telomere dysfunction as a marker of senescence, we observed an increase in senescent cell burden of bone cells from day 1 postradiation, without the presence of BMAT. BMAT was significantly elevated in radiated bones at day 7, confirming the qRT-PCR data in which most BMAT-related genes were elevated by day 7, and the trend continued until day 42 postradiation. Similarly, elevation in BMAT-related genes was observed in bones of aged mice. The senolytic cocktail of Dasatinib (D) plus Quercetin (Q) (ie, D + Q), which clears senescent cells, reduced BMAT in aged and radiated bones. MicroRNAs (miRNAs or miRs) linked with senescence marker p21 were downregulated in radiated and aged bones, whereas miR-27a, a miR that is associated with increased BMAT, was elevated both in radiated and aged bones. D + Q downregulated miR-27a in radiated bones at 42 days postradiation. Overall, our study provides evidence that BMAT occurrence in oxidatively stressed bone environments, such as radiation and aging, is induced following a common pathway and is dependent on the presence of senescent cells. © 2022 American Society for Bone and Mineral Research (ASBMR).

The role of senolytics in osteoporosis and other skeletal pathologies.

The skeletal system undergoes irreversible structural deterioration with aging, leading to increased fracture risk and detrimental changes in mobility, posture, and gait. This state of low bone mass and microarchitectural changes, diagnosed as osteoporosis, affects millions of individuals worldwide and has high clinical and economic burdens. Recently, pre-clinical studies have linked the onset of age-related bone loss with an accumulation of senescent cells in the bone microenvironment. These senescent cells appear to be causal to age-related bone loss, as targeted clearance of these cells leads to improved bone mass and microarchitecture in old mice. Additionally, other pathologies leading to bone loss that result from DNA damage, such as cancer treatments, have shown improvements after clearance of senescent cells. The development of new therapies that clear senescent cells, termed "senolytics", is currently underway and may allow for the modulation of bone loss that results from states of high senescent cell burden, such as aging.

Osteocyte Cellular Senescence.

PURPOSE OF REVIEW: Senescent cells are now known to accumulate in multiple tissues with aging and through their inflammation (the senescence-associated secretory phenotype, SASP) contribute to aging and chronic diseases. Here, we review the roles of senescent osteocytes in the context of bone loss. RECENT FINDINGS: Numerous studies have established that senescent osteocytes accumulate in the bone microenvironment with aging in mice and in humans. Moreover, at least in mice, elimination of senescent cells results in attenuation of age-related bone loss. Osteocyte senescence also occurs in response to other cellular stressors, including radiotherapy, chemotherapy, and metabolic dysfunction, where it appears to mediate skeletal deterioration. Osteocyte senescence is linked to bone loss associated with aging and other conditions. Senescent osteocytes are potential therapeutic targets to alleviate skeletal dysfunction. Additional studies better defining the underlying mechanisms as well as translating these exciting findings from mouse models to humans are needed.

Cellular senescence in age-related disorders.

Much of the population is now faced with an enormous burden of age-associated chronic diseases. Recent discoveries in geroscience indicate that healthspan in model organisms such as mice can be manipulated by targeting cellular senescence, a hallmark mechanism of aging, defined as an irreversible proliferative arrest that occurs when cells experience oncogenic or other diverse forms of damage. Senescent cells and their proinflammatory secretome have emerged as contributors to age-related tissue dysfunction and morbidity. Cellular senescence has causal roles in mediating osteoporosis, frailty, cardiovascular diseases, osteoarthritis, pulmonary fibrosis, renal diseases, neurodegenerative diseases, hepatic steatosis, and metabolic dysfunction. Therapeutically targeting senescent cells in mice can prevent, delay, or alleviate each of these conditions. Therefore, senotherapeutic approaches, including senolytics and senomorphics, that either selectively eliminate senescent cells or interfere with their ability to promote tissue dysfunction, are gaining momentum as potential realistic strategies to abrogate human senescence to thereby compress morbidity and extend healthspan.

Targeted Reduction of Senescent Cell Burden Alleviates Focal Radiotherapy-Related Bone Loss.

Clinical radiotherapy treats life-threatening cancers, but the radiation often affects neighboring normal tissues including bone. Acute effects of ionizing radiation include oxidative stress, DNA damage, and cellular apoptosis. We show in this study that a large proportion of bone marrow cells, osteoblasts, and matrix-embedded osteocytes recover from these insults only to attain a senescent profile. Bone analyses of senescence-associated genes, senescence-associated beta-galactosidase (SA-ß-gal) activity, and presence of telomere dysfunction-induced foci (TIF) at 1, 7, 14, 21, and 42 days post-focal radiation treatment (FRT) in C57BL/6 male mice confirmed the development of senescent cells and the senescence-associated secretory phenotype (SASP). Accumulation of senescent cells and SASP markers were correlated with a significant reduction in bone architecture at 42 days post-FRT. To test if senolytic drugs, which clear senescent cells, alleviate FRT-related bone damage, we administered the senolytic agents, dasatinib (D), quercetin (Q), fisetin (F), and a cocktail of D and Q (D+Q). We found moderate alleviation of radiation-induced bone damage with D and Q as stand-alone compounds, but no such improvement was seen with F. However, the senolytic cocktail of D+Q reduced senescent cell burden as assessed by TIF+ osteoblasts and osteocytes, markers of senescence (p16 Ink4a and p21), and key SASP factors, resulting in significant recovery in the bone architecture of radiated femurs. In summary, this study provides proof of concept that senescent cells play a role in radiotherapy-associated bone damage, and that reduction in senescent cell burden by senolytic agents is a potential therapeutic option for alleviating radiotherapy-related bone deterioration. © 2020 American Society for Bone and Mineral Research.

LPS-induced premature osteocyte senescence: Implications in inflammatory alveolar bone loss and periodontal disease pathogenesis.

Cellular senescence is associated with inflammation and extracellular matrix tissue remodeling through the secretion of proteins termed the senescence-associated secretory phenotype (SASP). Although osteocyte senescence in older individuals in the skeleton is well recognized, whether young alveolar osteocytes can also become senescent is unknown. This is potentially important in the context of periodontal disease, which is an inflammatory condition caused by a gradual change from symbiotic to pathogenic oral microflora that can lead to tooth loss. Our aim was to identify whether senescent osteocytes accumulate in young alveolar bone and whether bacterial-derived lipopolysaccharide (LPS) can influence cellular senescence in alveolar bone. An osteocyte-enriched cell population isolated from alveolar bone expressed increased levels of the known senescence marker p16Ink4a, as well as select SASP markers known to be implicated alveolar bone resorption (Icam1, Il6, Il17, Mmp13 and Tnfα), compared to ramus control cells. Increased senescence of alveolar bone osteocytes was also observed in vivo using the senescence-associated distension of satellites (SADS) assay and increased γH2AX, a marker of DNA damage associated with senescent cells. To approximate a bacterial infection in vitro, alveolar osteocytes were treated with LPS. We found increased expression of various senescence and SASP markers, increased γH2AX staining, increased SA-ß-Gal activity and the redistribution of F-actin leading to a larger and flattened cell morphology, all hallmarks of cellular senescence. In conclusion, our data suggests a model whereby bacterial-derived LPS stimulates premature alveolar osteocyte senescence, which in combination with the resultant SASP, could potentially contribute to the onset of alveolar bone loss.

Identification of osteoclast-osteoblast coupling factors in humans reveals links between bone and energy metabolism.

Bone remodeling consists of resorption by osteoclasts followed by formation by osteoblasts, and osteoclasts are a source of bone formation-stimulating factors. Here we utilize osteoclast ablation by denosumab (DMAb) and RNA-sequencing of bone biopsies from postmenopausal women to identify osteoclast-secreted factors suppressed by DMAb. Based on these analyses, LIF, CREG2, CST3, CCBE1, and DPP4 are likely osteoclast-derived coupling factors in humans. Given the role of Dipeptidyl Peptidase-4 (DPP4) in glucose homeostasis, we further demonstrate that DMAb-treated participants have a significant reduction in circulating DPP4 and increase in Glucagon-like peptide (GLP)-1 levels as compared to the placebo-treated group, and also that type 2 diabetic patients treated with DMAb show significant reductions in HbA1c as compared to patients treated either with bisphosphonates or calcium and vitamin D. Thus, our results identify several coupling factors in humans and uncover osteoclast-derived DPP4 as a potential link between bone remodeling and energy metabolism.

Independent Roles of Estrogen Deficiency and Cellular Senescence in the Pathogenesis of Osteoporosis: Evidence in Young Adult Mice and Older Humans.

Estrogen deficiency is a seminal mechanism in the pathogenesis of osteoporosis. Mounting evidence, however, establishes that cellular senescence, a fundamental mechanism that drives multiple age-related diseases, also causes osteoporosis. Recently, we systematically identified an accumulation of senescent cells, characterized by increased p16Ink4a and p21Cip1 levels and development of a senescence-associated secretory phenotype (SASP), in mouse bone/marrow and human bone with aging. We then demonstrated that elimination of senescent cells prevented age-related bone loss using multiple approaches, eg, treating old mice expressing a "suicide" transgene, INK-ATTAC, with AP20187 to induce apoptosis of p16Ink4a -senescent cells or periodically treating old wild-type mice with "senolytics," ie, drugs that eliminate senescent cells. Here, we investigate a possible role for estrogen in the regulation of cellular senescence using multiple approaches. First, sex steroid deficiency 2 months after ovariectomy (OVX, n = 15) or orchidectomy (ORCH, n = 15) versus sham surgery (SHAM, n = 15/sex) in young adult (4-month-old) wild-type mice did not alter senescence biomarkers or induce a SASP in bone. Next, in elderly postmenopausal women, 3 weeks of estrogen therapy (n = 10; 74 ± 5 years) compared with no treatment (n = 10; 78 ± 5 years) did not alter senescence biomarkers or the SASP in human bone biopsies. Finally, young adult (4-month-old) female INK-ATTAC mice were randomized (n = 17/group) to SHAM+Vehicle, OVX+Vehicle, or OVX+AP20187 for 2 months. As anticipated, OVX+Vehicle caused significant trabecular/cortical bone loss compared with SHAM+Vehicle. However, treatment with AP20187, which eliminates senescent cells in INK-ATTAC mice, did not rescue the OVX-induced bone loss or alter senescence biomarkers. Collectively, our data establish independent roles of estrogen deficiency and cellular senescence in the pathogenesis of osteoporosis, which has important implications for testing novel senolytics for skeletal efficacy, as these drugs will need to be evaluated in preclinical models of aging as opposed to the current FDA model of prevention of OVX-induced bone loss. © 2019 American Society for Bone and Mineral Research.

Sympathetic ß1-adrenergic signaling contributes to regulation of human bone metabolism.

BACKGROUND: Evidence from rodent studies indicates that the sympathetic nervous system (SNS) regulates bone metabolism, principally via ß2-adrenergic receptors (ß2-ARs). Given the conflicting human data, we used multiple approaches to evaluate the role of the SNS in regulating human bone metabolism. METHODS: Bone biopsies were obtained from 19 young and 19 elderly women for assessment of ADRB1, ADRB2, and ADRB3 mRNA expression. We examined the relationship of ß-blocker use to bone microarchitecture by high-resolution peripheral quantitative CT in a population sample of 248 subjects. A total of 155 postmenopausal women were randomized to 1 of 5 treatment groups for 20 weeks: placebo; propranolol, 20 mg b.i.d.; propranolol, 40 mg b.i.d.; atenolol, 50 mg/day; or nebivolol, 5 mg/day. We took advantage of the ß1-AR selectivity gradient of these drugs (propranolol [nonselective] << atenolol [relatively ß1-AR selective] < nebivolol [highly ß1-AR selective]) to define the ß-AR selectivity for SNS effects on bone. RESULTS: ADRB1 and ADRB2, but not ADRB3, were expressed in human bone; patients treated clinically with ß1-AR-selective blockers had better bone microarchitecture than did nonusers, and relative to placebo, atenolol and nebivolol, but not propranolol, reduced the bone resorption marker serum C-telopeptide of type I collagen (by 19.5% and 20.6%, respectively; P < 0.01) and increased bone mineral density of the ultradistal radius (by 3.6% and 2.9%; P < 0.01 and P < 0.05, respectively). CONCLUSIONS: These 3 independent lines of evidence strongly support a role for adrenergic signaling in the regulation of bone metabolism in humans, principally via ß1-ARs. TRIAL REGISTRATION: ClinicalTrials.gov NCT02467400. FUNDING: This research was supported by the NIH (AG004875 and AR027065) and a Mayo Clinic Clinical and Translational Science Award (CTSA) (UL1 TR002377).

Identification of Senescent Cells in the Bone Microenvironment.

Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16Ink4a , profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence-associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age-related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16Ink4a expression by real-time quantitative polymerase chain reaction (rt-qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence-associated distension of satellites (SADS), ie, large-scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction-induced foci (TIFs) than osteocytes from young mice. Corresponding with the age-associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age-associated upregulation in myeloid cells. These data show that with aging, a subset of cells of various lineages within the bone microenvironment become senescent, although senescent myeloid cells and senescent osteocytes predominantly develop the SASP. Given the critical roles of osteocytes in orchestrating bone remodeling, our findings suggest that senescent osteocytes and their SASP may contribute to age-related bone loss. © 2016 American Society for Bone and Mineral Research.

Hdac3 Deficiency Increases Marrow Adiposity and Induces Lipid Storage and Glucocorticoid Metabolism in Osteochondroprogenitor Cells.

Bone loss and increased marrow adiposity are hallmarks of aging skeletons. Conditional deletion of histone deacetylase 3 (Hdac3) in murine osteochondroprogenitor cells causes osteopenia and increases marrow adiposity, even in young animals, but the origins of the increased adiposity are unclear. To explore this, bone marrow stromal cells (BMSCs) from Hdac3-depleted and control mice were cultured in osteogenic medium. Hdac3-deficient cultures accumulated lipid droplets in greater abundance than control cultures and expressed high levels of genes related to lipid storage (Fsp27/Cidec, Plin1) and glucocorticoid metabolism (Hsd11b1) despite normal levels of Pparγ2. Approximately 5% of the lipid containing cells in the wild-type cultures expressed the master osteoblast transcription factor Runx2, but this population was threefold greater in the Hdac3-depleted cultures. Adenoviral expression of Hdac3 restored normal gene expression, indicating that Hdac3 controls glucocorticoid activation and lipid storage within osteoblast lineage cells. HDAC3 expression was reduced in bone cells from postmenopausal as compared to young women, and in osteoblasts from aged as compared to younger mice. Moreover, phosphorylation of S424 in Hdac3, a posttranslational mark necessary for deacetylase activity, was suppressed in osseous cells from old mice. Thus, concurrent declines in transcription and phosphorylation combine to suppress Hdac3 activity in aging bone, and reduced Hdac3 activity in osteochondroprogenitor cells contributes to increased marrow adiposity associated with aging. © 2015 American Society for Bone and Mineral Research.

Effects of Age and Estrogen on Skeletal Gene Expression in Humans as Assessed by RNA Sequencing.

UNLABELLED: Precise delineation of the specific genes and pathways altered with aging and estrogen (E) therapy may lead to new skeletal biomarkers and the development of novel bone therapeutics. Previous human bone studies, however, have been limited by only examining pre-specified genes and pathways. High-throughput RNA sequencing (RNAseq), on the other hand, offers an unbiased approach to examine the entire transcriptome. Here we present an RNAseq analysis of human bone samples, obtained from iliac crest needle biopsies, to yield the first in vivo interrogation of all genes and pathways that may be altered in bone with aging and E therapy in humans. 58 healthy women were studied, including 19 young women (mean age ± SD, 30.3 ± 5.4 years), 19 old women (73.1 ± 6.6 years), and 20 old women treated with 3 weeks of E therapy (70.5 ± 5.2 years). Using generally accepted criteria (false discovery rate [q] < 0.10), aging altered a total of 678 genes and 12 pathways, including a subset known to regulate bone metabolism (e.g., Notch). Interestingly, the LEF1 transcription factor, which is a classical downstream target of the Wnt/ß-catenin signaling pathway, was significantly downregulated in the bones from the old versus young women; consistent with this, LEF1 binding sites were significantly enriched in the promoter regions of the differentially expressed genes in the old versus young women, suggesting that aging was associated with alterations in Wnt signaling in bone. Further, of the 21 unique genes altered in bone by E therapy, the expression of INHBB (encoding for the inhibin, beta B polypeptide), which decreased with aging (by 0.6-fold), was restored to young adult levels in response to E therapy. In conclusion, our data demonstrate that aging alters a substantial portion of the skeletal transcriptome, whereas E therapy appears to have significant, albeit less wide-ranging effects. These data provide a valuable resource for the potential identification of novel biomarkers associated with age-related bone loss and also highlight potential pathways that could be targeted to treat osteoporosis. TRIAL REGISTRATION: ClinicalTrials.gov NCT02349113.