Physical function declines in old age, portending disability, increased health expenditures, and mortality. Cellular senescence, leading to tissue dysfunction, may contribute to these consequences of aging, but whether senescence can directly drive age-related pathology and be therapeutically targeted is still unclear. Here we demonstrate that transplanting relatively small numbers of senescent cells into young mice is sufficient to cause persistent physical dysfunction, as well as to spread cellular senescence to host tissues. Transplanting even fewer senescent cells had the same effect in older recipients and was accompanied by reduced survival, indicating the potency of senescent cells in shortening health- and lifespan. The senolytic cocktail, dasatinib plus quercetin, which causes selective elimination of senescent cells, decreased the number of naturally occurring senescent cells and their secretion of frailty-related proinflammatory cytokines in explants of human adipose tissue. Moreover, intermittent oral administration of senolytics to both senescent cell-transplanted young mice and naturally aged mice alleviated physical dysfunction and increased post-treatment survival by 36% while reducing mortality hazard to 65%. Our study provides proof-of-concept evidence that senescent cells can cause physical dysfunction and decreased survival even in young mice, while senolytics can enhance remaining health- and lifespan in old mice.

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.

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.

Osteoblast-lineage cells circulate in physiologically significant numbers, correlate with markers of bone formation, and are markedly higher during pubertal growth; therefore, they may represent a previously unrecognized circulatory component to the process of bone formation.

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...

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