Telomere shortening impacts bone cell differentiation
An important mechanism that contributes to cell senescence, characterized by irreversible cycle arrest, is telomere shortening. Telomere length is maintained by telomerase and protein complexes composed of shelterin and other proteins that regulate telomere maintenance by telomerase,[10, 11] which allows cells to extend telomere length and to maintain self-renewal and stem cell population. Telomere shortening has been involved in several age-related diseases and premature aging syndromes. Age-related shortening of telomeres leads to reduced cell replication, which contributes to impaired DNA repair and to accumulation of damaged cells. In osteoblast progenitor cells, this leads to limited stem cell pools and impaired differentiation into bone-forming cells. The role of telomeres in skeletal aging is supported by the finding that mice with invalidated telomerase reverse transcriptase (Terc) show decreased bone mass associated with reduced osteoblast differentiation and function and enhanced osteoclastogenesis resulting from a proinflammatory osteoclast-activating microenvironment. Mechanistically, the reduced osteoblast differentiation in Terc−/− mice involves increased p53 expression, whose activation induces cell cycle arrest, apoptosis, and reduced Runx2 expression. Thus, telomere dysfunction limits osteoblast differentiation of skeletal progenitor cells and contributes to age-related bone loss (Fig. 2). Consistent with this concept, telomerase overexpression in human mesenchymal stem cells (MSCs) was found to maintain differentiation in vitro[16-18] and to enhance bone formation in vivo.[19, 20] This suggests that increasing telomerase expression could prevent telomere shortening and replicative senescence in osteoblast precursor cells. Recent data indicate that telomerase gene therapy in old mice delays aging without increasing cancer, which may open a new area focused on telomerase reactivation to limit bone cell senescence and attenuate age-related bone loss.
Figure 2. Integrated view of the current mechanisms involved in bone cell senescence. Current data indicate that skeletal aging results from a complex network of interacting effector programs including intrinsic cellular processes, alterations in endogenous factors and signals, and possibly inflammatory processes (dashed lines). This hierarchical basis of senescence mechanisms results in altered bone cell differentiation, function, and fate, which contribute to altering skeletal integrity.
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Increased oxidative stress causes bone cell damage
Damage to cellular components induced by reactive oxygen species is an important mechanism contributing to aging. As is the case in other tissues, the accumulation of cellular and tissue damage caused by oxidative stress contributes to the adverse effects of aging on bone cells. Reactive oxygen species (ROS) increase with age and cause cell damage and cell death in bone cells, supporting the concept that the increased oxidative stress plays a pathological role in the development of bone fragility with aging. Mechanistically, the age-related increase in ROS and decrease in glutathione reductase activity are associated with increased phosphorylation of p53 and p66shc that regulate cell senescence and aging, leading to activation of osteoblast/osteocyte apoptosis and decreased bone mass in mice. This mechanism can be reproduced in mice deficient in the cytoplasmic copper/zinc superoxide dismutase (Sod1) gene, which exhibit increased intracellular ROS, skeletal fragility, and low-turnover osteopenia related to reduced osteoblastic cell proliferation and impaired osteoblast viability, a mechanism that is exacerbated by mechanical unloading. The age-related ROS accumulation may also contribute to the age-related shift in bone marrow stromal cell differentiation toward adipocytes instead of osteoblasts, resulting in increased bone marrow adipogenesis associated with decreased osteoblastogenesis. This effect results from the activation of PPARγ signaling in osteoblastic cells by oxidized lipids. Additionally, the increased endogenous glucocorticoid expression with age contributes to increased ROS production and FOXO activity, which in turn antagonizes Wnt signaling.[29, 31] Both the ROS-mediated activation of FOXOs and the sequestration of β-catenin by PPARγ can account for the age-related decrease in Wnt signaling, which is an important pathway that controls osteoblast and adipogenic differentiation. The role of FOXOs as inhibitors of Wnt/β-catenin signaling during aging is supported by the finding that mice lacking FOXOs in skeletal progenitor cells show increased bone formation and bone mass during aging. ROS are also known to stimulate osteoclastic bone resorption, suggesting that the age-related increase in ROS may contribute to the promotion of bone resorption in addition to reduced osteoblastogenesis (Fig. 2). Consistently, oxidative stress was found to be associated with increased bone resorption and low bone mass in women.
Although the accumulation of oxidative DNA damage contributes to age-related osteoblast dysfunction, the mechanism by which DNA damage leads to accelerated bone aging is not fully understood. Recent data have pointed out a role of the NF-κB pathway in age-related bone loss. This pathway is an important modulator of both osteoclasts and osteoblasts.[38, 39] Consistent with a role of NF-κB in DNA damage, Chen and colleagues showed that mice that are deficient in ERCC1-XPF, an endonuclease that is required for DNA repair, exhibit severe bone loss caused by increased NF-κB transcription in osteoblastic and osteoclastic cells. This resulted in senescence of osteoblastic cells, reduced bone formation, and increased osteoclastogenesis. These studies indicate that DNA damage induces bone cell dysfunctions in part through an NF-κB-dependent mechanism (Fig. 2).
The finding that oxidative stress in bone cells is an important mechanism underlying the age-related skeletal fragility raises the hypothesis that this mechanism may be antagonized by antioxidants. Consistently, treatment with antioxidants was found to prevent bone loss in animal models[41-43] and to abrogate the increased apoptosis in aged mice, although this effect was limited by concomitant inhibition of Wnt signaling. Similarly, treatment of SOD1-deficient mice with the antioxidant vitamin C decreased bone fragility and osteoblastic survival and attenuated bone loss during unloading.[26, 27] Intermittent parathyroid hormone (iPTH) administration was also shown to attenuate the rise in intracellular ROS, p66shc phosphorylation, and FOXO transcriptional activity induced by oxidative stress in osteoblastic cells, as well as their adverse effects on Wnt signaling and osteoblast apoptosis, indicating that this agent can antagonize the age-related increase in oxidative stress in aging mice. In addtition to iPTH, estrogens and androgens exert direct antioxidant actions on bone cells, which may contribute to their bone-protective effects in mice. Oxidative stress can also be targeted in osteoclasts to reduce bone loss. Osteoclasts express NADPH oxidase 4 (NOX4), an enzyme involved in ROS production, and mice deficient in NOX4 display high bone density and reduced osteoclast number. Inhibition of NOX4 leads to reduced bone resorption and bone loss induced by ovariectomy in mice. Thus, combating the age-associated oxidative stress in bone cells can lead to an attenuation of the adverse effects of aging on bone (Fig. 3).
Figure 3. Potential targets to overcome bone forming cell senescence. Bone cell senescence may possibly be attenuated by agents (in italics) that target some of the major mechanisms that are involved in cell senescence. Such therapeutic approaches aimed at reducing bone cell senescence may have a positive impact on bone formation and skeletal integrity.
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Epigenetics: implications on bone cell aging
Changes in epigenetic pathways such as DNA methylation, histone methylation, and histone acetylation are clearly linked to cellular senescence and aging through their control of chromatin structure and telomere length.[48, 49] In bone, several epigenetic mechanisms were found to influence the transcription of genes that control osteoclastogenesis and osteoblastogenesis. Notably, DNA methylation controls RANKL and osteoprotegerin (OPG), osteocalcin, BMP2, sclerostin, and C/EBPα.[54, 55] In addition, histone demethylases regulate the expression of H3 histones that control RANKL-induced NFATc1 expression and osteoclast differentiation. Histone demethylases also regulate osteogenic differentiation[57, 58] and modulate the expression of Runx2 and Osterix,[59, 60] whereas histone methylation controls PPARγ target gene promoters. Histone acetylases and deactelyases (HDACs) are highly involved in the regulation of osteoblast differentiation by controlling histone H4, C/EBPα, and Runx2.[62, 64] Given these important effects, it is expected that disturbances in epigenetic regulators in bone cells may contribute to the pathophysiology of age-related bone loss (Fig. 2). In support of this concept, mice deficient in DNA methyltransferase 1 (Dnmt1), the most prevalent DNA methyltransferase, exhibit decreased bone mineral density without altered survival or mortality. One particular group of HDACs called sirtuins play an essential role in the regulation of chromatin structure, telomere stability, senescence, DNA repair, cell differentiation, and apoptosis. A member of the sirtuin family, the NAD-dependent histone deacetylase silent information regulator T1 (SIRT-1), regulates transcription factors involved in aging. In bone, SIRT-1 was shown to promote osteoblast differentiation and to attenuate adipogenesis through the regulation of SOST, PPARγ, Runx2, and β-catenin. SIRT-1 also negatively controls NF-κB signaling. In mice, repression of SIRT-1 in osteoblasts or osteoclasts activates NF-κB signaling, resulting in reduced bone formation and enhanced bone resorption, a phenotype rescued by inhibition of NF-κB. Interestingly, SIRT-1 overexpression protects against age-related bone loss, whereas reducing its expression causes decreased bone formation in mice,[68, 73] further indicating that SIRT-1 is an important epigenetic regulator in aging bone cells.
MicroRNA (miRNA) are other epigenetic regulators that control gene expression directly or through crosstalks with DNA methylation and may thereby play an important role during aging.[74, 75] In osteoclasts, several miRNAs were shown to regulate key signaling molecules such as Notch, RANKL, RANK, calcitonin receptor, and RhoA. In bone-forming cells, some miRNAs were found to enhance osteoblast differentiation in MSCs by enhancing promoting signals (such as Runx2, Osx, ATF4, Smad5, ERK, Cx43, and LRP6) or by suppressing inhibitory signals (such as Foxo1, DKK1, and PPARγ).[77, 78] Moreover, miRNA were found to regulate HDACs that control osteoblast differentiation and bone formation.[79, 80] In support of a role of miRNA in bone cell functions in the aging skeleton, dysfunctions in miRNA-dependent mechanisms have been reported in osteoporosis.
Given the importance of epigenetic mechanisms in the regulation of bone cells, it may be of interest to target these processes to optimize bone cell differentiation or function during aging. For example, recent data suggest that miRNAs can be targeted to promote osteoblast differentiation and bone formation and repair in vivo.[78, 81, 82] In contrast, it is still uncertain whether targeting HDACs may have beneficial effects on bone cells. Indeed, HDAC inhibition was found to inhibit or increase osteoclast differentiation in vitro or bone resorption in vivo, presumably because of the distinct role of specific HDACs in bone. Similarly, HDACs inhibitors were shown to stimulate osteoblast differentiation in vitro but this has not been confirmed in animals, possibly because these inhibitors promote apoptosis in immature osteoblast progenitor cells. Pharmacological enhancement of SIRT-1 could be one interesting option to promote osteogenic differentiation. Indeed, increasing the activity of the SIRT-1 protein by the phytoestrogen resveratrol in MSCs resulted in increased osteoblast differentiation and decreased adipocyte differentiation in vitro.[70, 84, 85] Resveratrol also inhibits NF-kB activity and osteoclast differentiation in vitro.[84, 86] In vivo, resveratrol administration was found to preserve bone mineral density in elderly mice, in part through the attenuation of the age-associated increase in oxidative stress. In a mouse model of progeria, resveratrol treatment improved trabecular bone structure and mineral density through enhanced binding between SIRT-1 and lamin A. Overall, these studies suggest that SIRT-1 could be targeted to alleviate some of the senescence mechanisms in bone cells that contribute to skeletal aging. Further studies in humans are needed to identify specific epigenetic changes associated with aging that could be targeted to counteract the age-related dysfunctions in bone cells.
Decreased autophagy and proteasome degradation: impact on bone cell differentiation and function
The maintenance of cellular homeostasis requires efficient proteolysis of damaged proteins. Autophagy is a lysosome-dependent degradation pathway whose primary role is to break down damaged cellular components. Under stressful conditions, autophagy is increased, which helps cells to survive stress through increased recycling of cellular components. Thereby, autophagy plays a critical role in the maintenance, differentiation, and survival of stem cells as well as more differentiated cells.[89, 90] Autophagy was recently identified as a new effector of senescence. Autophagic efficiency declines with age, which may contribute to the age-related accumulation of damaged molecules and organelles induced by starvation and ROS in aging cells.[93, 94] In the skeleton, all bone cells exhibit autophagy and current evidence indicates that this process plays a role in bone physiology and pathology (Fig. 2). In bone-resorbing cells, autophagy in response to oxidative stress induces osteoclast precursor cell differentiation. In addition, autophagy proteins participate in the secretion of lysosomal contents into the extracellular space, an important mechanism involved in bone resorption. However, the actual importance of autophagy in regulating osteoclastogenesis and function during aging remains unclear. In contrast, a role of autophagy in osteoblastogenesis is supported by the finding that specific deletion of FIP200, an essential component of mammalian autophagy, leads to defective osteoblast differentiation, reduced bone formation, and osteopenia in mice. Although the role of autophagy in osteocytes during aging is not fully established, it was recently found that conditional deletion in osteocytes of Atg7, a gene essential for autophagy, leads to decreased bone formation and bone mass associated with increased oxidative stress, suggesting that suppression of autophagy in osteocytes induces a phenotype resembling aging. Thus, current data suggest that a decline in autophagy with age may contribute to bone aging. Whether autophagy may be targeted to reduce bone cell senescence and bone loss associated with aging remains to be determined.
Another important mechanism regulating intracellular protein degradation is the ubiquitin-dependent proteolysis system (UPS). This system mediates the ubiquitination of proteins through the actions of ubiquitin ligases, which direct their degradation through the proteasome. The UPS-mediated clearance of damaged and short-lived proteins ensures a protein quality control, a mechanism that impacts cell proliferation, differentiation, and apoptosis. Because of its important function, a decrease in proteasome activity with age may lead to the accumulation of oxidized and damaged proteins,[103-105] which are directly linked to aging. In bone cells, significant advances have been made in our understanding of the role of UPS and E3 ubiquitin ligases in the proteasomal degradation of key regulatory proteins such as receptor tyrosine kinases (RTK), signaling molecules, and transcription factors that control osteoclast and osteoblast differentiation and function. Because the ubiquitination/degradation system controls many processes in bone cells, this mechanism could be targeted to improve bone cell function (Fig. 3). For example, proteasome inhibitors were shown to increase MSC osteogenic differentiation and bone formation in mice.[108, 109] A more specific approach for promoting bone formation may be to target E3 ubiquitin ligases that control proteasome degradation. In support of this concept, it was shown that inhibiting the activity of E3 ligase c-Cbl in murine and human MSCs promoted osteoblast differentiation and survival through activation of RTK signaling and STAT5-Runx2 transcriptional activity. Whether targeting E3 ubiquitin ligases or other proteins controlling proteasome degradation in bone cells may attenuate age-related bone cell senescence warrants further investigation.