Aging is associated with increased cellular senescence, which is hypothesized to drive the eventual development of multiple co-morbidities1. Here, we investigate a role for senescent cells in age-related bone loss by multiple approaches. In particular, we used either genetic (i.e., the INK-ATTAC “suicide” transgene encoding an inducible caspase 8 expressed specifically in senescent cells2–4) or pharmacological (i.e., “senolytic” compounds5,6) means to eliminate senescent cells. We also inhibited the production of the pro-inflammatory secretome of senescent cells using a JAK inhibitor (JAKi)3,7. In old (20–22-months) mice with established bone loss, activation of the INK-ATTAC caspase 8 in senescent cells or treatment with senolytics or the JAKi for 2–4 months resulted in higher bone mass and strength and better bone microarchitecture compared to vehicle-treated mice. The beneficial effects of targeting senescent cells were due to lower bone resorption with either maintained (trabecular bone) or higher (cortical bone) bone formation as compared to vehicle-treated mice. In vitro studies demonstrated that senescent cell-conditioned medium impaired osteoblast mineralization and enhanced osteoclast progenitor survival, leading to increased osteoclastogenesis. Collectively, these data establish a causal role for senescent cells in bone loss with aging and demonstrate that targeting these cells has both anti-resorptive and anabolic effects on bone. As eliminating senescent cells and/or inhibiting their pro-inflammatory secretome also improves cardiovascular function4, enhances insulin sensitivity3, and reduces frailty7, targeting this fundamental mechanism to prevent age-related bone loss suggests a novel treatment strategy not only for osteoporosis but also for multiple age-related co-morbidities.

Estrogen is the major hormonal regulator of bone metabolism in women and men. Therefore, there is considerable interest in unraveling the pathways by which estrogen exerts its protective effects on bone. While the major consequence of the loss of estrogen is an increase in bone resorption, estrogen deficiency is associated with a gap between bone resorption and formation, indicating that estrogen is also important for maintaining bone formation at the cellular level. Direct estrogen effects on osteocytes, osteoclasts, and osteoblasts lead to inhibition of bone remodeling, decreased bone resorption, and maintenance of bone formation, respectively. Estrogen also modulates osteoblast/osteocyte and T-cell regulation of osteoclasts. Unraveling these pleiotropic effects of estrogen may lead to new approaches to prevent and treat osteoporosis.

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.

For more than a decade, Wnt signaling pathways have been the focus of intense research activity in bone biology laboratories because of their importance in skeletal development, bone mass maintenance, and therapeutic potential for regenerative medicine. It is evident that even subtle alterations in the intensity, amplitude, location, and duration of Wnt signaling pathways affects skeletal development, as well as bone remodeling, regeneration, and repair during a lifespan. Here we review recent advances and discrepancies in how Wnt/Lrp5 signaling regulates osteoblasts and osteocytes, introduce new players in Wnt signaling pathways that have important roles in bone development, discuss emerging areas such as the role of Wnt signaling in osteoclastogenesis, and summarize progress made in translating basic studies to clinical therapeutics and diagnostics centered around inhibiting Wnt pathway antagonists, such as sclerostin, Dkk1 and Sfrp1. Emphasis is placed on the plethora of genetic studies in mouse models and genome wide association studies that reveal the requirement for and crucial roles of Wnt pathway components during skeletal development and disease.

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.

Transforming growth factor ␤-inducible early gene 1 (TIEG1) is a member of the Krüppel-like transcription factor family. To understand the physiological role of TIEG1, we generated TIEG ؊/؊ (null) mice and found that the TIEG ؊/؊ mice had increased osteoblast numbers with no increased bone formation parameters. However, when calvarial osteoblasts (OBs) were isolated from neonatal TIEG ؊/؊ and TIEG ؉/؉ mice and cultured in vitro, the TIEG ؊/؊ cells displayed reduced expression of important OB differentiation markers. When the OBs were differentiated in vitro by treatment with bone morphogenic protein 2, the OBs from TIEG ؉/؉ calvaria displayed several mineralized nodules in culture, whereas those from TIEG ؊/؊ mice showed no nodules. To characterize the OBs' ability to support osteoclast differentiation, the OBs from TIEG ؉/؉ and TIEG ؊/؊ mice were cultured with marrow and spleen cells from TIEG ؉/؉ mice. Significantly fewer osteoclasts developed when TIEG ؊/؊ OBs were used to support osteoclast differentiation than when TIEG ؉/؉ OBs were used. Examination of gene expression in the TIEG ؊/؊ OBs revealed decreased RANKL and increased OPG expression compared to TIEG ؉/؉ OBs. The addition of RANKL to these cocultures only partially restored the ability of TIEG ؊/؊ OBs to support osteoclast differentiation, whereas M-CSF alone or combined with RANKL had no additional effect on osteoclast differentiation. We conclude from these data that TIEG1 expression in OBs is critical for both osteoblast-mediated mineralization and osteoblast support of osteoclast differentiation.Krüppel-like transcription factors (KLFs) are DNA-binding transcriptional regulators which contain C 2 , H 2 -type zinc fingers and play important roles in regulating biological processes such as cell growth, differentiation, and embryogenesis (1, 5, 32). The number of members of the KLF family has been increasing, and it is estimated that 1% of the human genome might contain this family of regulatory factors (5, 11). Our laboratory has cloned a member of this family, the transforming growth factor ␤ (TGF-␤)-inducible early gene 1 (TIEG1), since it represented a primary response gene to TGF-␤ treatment in human osteoblasts (28). Cook et al. (4) identified TIEG2, which shares 91% homology with TIEG1 within the zinc finger region but only 44% homology at the N terminus region. They also showed evidence that overexpression of TIEG2 in Chinese hamster ovary cells inhibits cell proliferation. Recently, Wang et al. (36) identified another member of the TIEG family, TIEG3, which has properties similar to those of TIEG1 and TIEG2.A better understanding of the mechanism of action of TIEG1 is evolving. Using a GAL4-based transcriptional assay, Cook et al. (4) demonstrated that TIEG1 protein has three repression domains. Studies by Zhang et al. (39) identified an alpha-helical repression motif located within the repression domain of TIEG1 and TIEG2. These authors have also shown evidence that these motifs mediate the direct interaction of TIEG1 with mSin3A, whic...

Estrogen (17beta-estradiol, E2) plays pivotal roles in the function and maintenance of the skeleton, including the bone-forming osteoblasts (OBs). The functions of E2 are largely mediated through two distinct estrogen receptor isoforms, ERalpha and ERbeta, both of which are expressed in OBs. The level of each isoform dominates at early or late stages of OB differentiation. To date, only a limited comparison between the transcriptional targets of ERalpha and ERbeta on endogenous gene expression has been reported. We have developed new stable cell lines, which contain doxycycline (Dox)-inducible ERalpha and ERbeta, in the U2OS human osteosarcoma to determine the global transcriptional profile of ERalpha- and ERbeta-regulation of endogenous gene expression. The U2OS-ERalpha and U2OS-ERbeta cell lines were treated with Dox and either vehicle control or E2 for 24 h. Gene expression analysis was performed using a microarray containing approximately 6,800 full-length genes. We detected 63 genes that were regulated solely by ERalpha and 59 genes that were only regulated solely by ERbeta. Of the ERalpha-regulated genes, 81% were upregulated and 19% were inhibited. Similarly 76% of the ERbeta-regulated genes were upregulated whereas 24% were inhibited by E2. Surprisingly, only 17 genes were induced by both ERalpha and ERbeta. Real-time PCR was employed to confirm the expression of a selected number of genes. The regulation of a number of known E2-responsive genes in human OBs, as well as many interesting novel genes, is shown. The distinct patterns of E2-dependent gene regulation in the U2OS cells by ERalpha and ERbeta shown here suggest that during OB differentiation, when either isoform dominates, a unique pattern of gene responses will occur, partially due to the differentiation state and the ER isoform present.

Aging is associated with visceral adiposity, metabolic disorders, and chronic low-grade inflammation. 17α-estradiol (17α-E2), a naturally occurring enantiomer of 17β-estradiol (17β-E2), extends life span in male mice through unresolved mechanisms. We tested whether 17α-E2 could alleviate age-related metabolic dysfunction and inflammation. 17α-E2 reduced body mass, visceral adiposity, and ectopic lipid deposition without decreasing lean mass. These declines were associated with reductions in energy intake due to the activation of hypothalamic anorexigenic pathways and direct effects of 17α-E2 on nutrient-sensing pathways in visceral adipose tissue. 17α-E2 did not alter energy expenditure or excretion. Fasting glucose, insulin, and glycosylated hemoglobin were also reduced by 17α-E2, and hyperinsulinemic-euglycemic clamps revealed improvements in peripheral glucose disposal and hepatic glucose production. Inflammatory mediators in visceral adipose tissue and the circulation were reduced by 17α-E2. 17α-E2 increased AMPKα and reduced mTOR complex 1 activity in visceral adipose tissue but not in liver or quadriceps muscle, which is in contrast to the generalized systemic effects of caloric restriction. These beneficial phenotypic changes occurred in the absence of feminization or cardiac dysfunction, two commonly observed deleterious effects of exogenous estrogen administration. Thus, 17α-E2 holds potential as a novel therapeutic for alleviating age-related metabolic dysfunction through tissue-specific effects.