Several, but not all, senescent cell types exhibit high levels of the cyclin inhibitor p16. For example, senescent osteocytes have increased levels of p16, while senescent osteoblast progenitors have elevated levels of p21, but not p16 (Kim et al., 2017 ; Piemontese et al., 2017 ). Selective elimination of cells expressing p16 in mouse models increases life‐ and healthspan (Childs et al., 2017 ). Currently, two of such models have been described: the INK‐ATTAC and the p16‐3MR mice (Baker et al., 2011 ; Demaria et al., 2014 ). Using the p16‐3MR model, we have effectively depleted senescent cells in the skin, lungs, muscle, and bone marrow, including senescent hematopoietic and muscle stem cells, and suppressed the SASP in either sub‐lethally irradiated or normally aged mice (Chang et al., 2016 ; Demaria et al., 2014 ). Farr and colleagues have found that elimination of p16‐expressing cells in 20‐month‐old mice for a 4‐month period, using the INK‐ATTAC transgene, increases bone mass (Farr et al., 2017 ). These findings support the notion that senescent cells contribute to age‐related bone loss. However, the identity of the senescent cells that are responsible for skeletal aging remains unknown. Likewise, the extent to which elimination of p16‐expressing cells rescues skeletal aging is unknown. Here, we investigated the skeletal effects of long‐ term ablation of senescent cells using p16‐3MR mice—an alternative to the INK‐ATTAC model of p16‐expressing cell elimination in which the transgene is activated by the administration of GCV. The key objectives of this work were two: first, to eliminate p16 senescent cells from 12 to 24 months of age, the time period during which C57BL/6 mice experience a dramatic age‐related loss of bone mass (Almeida et al., 2007 ), and determine whether the experimental maneuver could prevent the loss of bone. And second, to eliminate p16 senescent cells from 20 to 26 months of age in order to determine whether this intervention could restore bone mass in mice that had already lost it.

Cellular senescence is characterized by a permanent proliferative arrest, and an altered gene expression pattern leading to the production of pro‐inflammatory and matrix‐degrading molecules known as the senescence‐associated secretory phenotype (SASP) (Campisi, 2013 ; Coppe et al., 2008 ; Kuilman et al., 2008 ). Osteocytes, former osteoblasts buried in the bone matrix, are postmitotic and the most abundant cell type in bone (Jilka & O'Brien, 2016 ). Osteocytes modulate bone resorption and formation via the production of RANKL and Sost, respectively (Baron & Kneissel, 2013 ; O'Brien, Nakashima, & Takayanagi, 2013 ). Earlier findings by us and others have elucidated that, like other postmitotic cells, osteocytes in the bone of aged female and male mice show markers of senescence (Farr et al., 2016 ; Piemontese et al., 2017 ).

Soon after the attainment of peak bone mass, the balance between bone resorption and bone formation begins to progressively tilt in favor of the former, in both women and men (Bala, Zebaze, & Seeman, 2015 ; Looker et al., 1993 ; Riggs et al., 2008 ). Both female and male mice exhibit all seminal features of skeletal aging found in humans, including the decline of trabecular and cortical bone mass (Almeida et al., 2007 ; Glatt, Canalis, Stadmeyer, & Bouxsein, 2007 ; Piemontese et al., 2017 ). The age‐related cortical bone loss in mice is associated with an increase in the number of osteoclasts, the cells responsible for degrading the bone matrix (Piemontese et al., 2017 ; Ucer et al., 2017 ). Osteoclasts differentiate from myeloid lineage cells in response to osteoclastogenic signals such as M‐CSF and RANKL. Nonetheless, a decline in bone formation is the seminal culprit of skeletal aging in both humans and rodents (Almeida et al., 2007 ; Parfitt, Villanueva, Foldes, & Rao, 1995 ). A decrease in the number of osteoblasts, the cells that synthesize the bone matrix, underlies the loss of both trabecular and cortical bone in aged mice (Almeida et al., 2007 ). Osteoblasts differentiate from mesenchymal progenitors, and this process is dependent on the activity of the transcription factors Runx2 and Osx1 (Park et al., 2012 ). Importantly, the number of these osteoprogenitors declines with advancing age and this decline is associated with increased markers of cellular senescence (Kim et al., 2017 ).

Activation of the p16‐3MR transgene does not affect bone mass in aged mice. (a–f) 12‐month‐old female p16‐3MR mice were either vehicle‐administered or Ganciclovir‐administered ( n = 9) for 12 months. (a) Representative micro‐CT images of femur. (b) Femoral cortical thickness, total area, and medullary area by micro‐CT in the midshaft region and cortical porosity in the distal metaphysis. (c) Osteoclast number (Oc. N) and surface (Oc. S) at the endocortical area of the femurs. (d) Representative micro‐CT images of L5 vertebrae. (e) Vertebral trabecular bone parameters (Bone volume per tissue volume, BV/TV; Trabecular Thickness, Tb. Thickness; Trabecular Number, Tb. Number; Trabecular Separation, Tb. Separation) as determined by micro‐CT. (f) Load‐to‐failure, a measure of strength, was determined by compression testing of L1 vertebrae. Maximum load ( left ) and Maximum compressive stress ( Right ). (g and h) 20‐month‐old female p16–3MR mice were either vehicle‐administered ( n = 7) or Ganciclovir‐administered ( n = 7) for 6 months. (g) Representative micro‐CT images of L5 vertebrae. (h) Vertebral trabecular bone parameters (Bone volume per tissue volume, BV/TV; Trabecular Thickness, Tb. Thickness; Trabecular Number, Tb. Number; Trabecular Separation, Tb. Separation) as determined by micro‐CT. Three‐month‐old female p16‐3MR mice ( n = 4) were used as a young control group. * p < 0.05 vs. young mice; unpaired Student's t test. Data represent mean and SD ( error bars )

To examine the skeletal effects of the p16‐3MR transgene, we used micro‐CT. Cortical bone mass, measured in the midshaft of the femur, was not different in 24‐month‐old mice receiving GCV for 12 months, when compared to mice receiving vehicle (Figure 5 a,b). In line with the lack of an effect on bone mass, the number of osteoclasts at the endocortical surface of the femurs was not altered by GCV (Figure 5 c). Similarly, trabecular bone mass and microarchitecture, measured in the vertebrae, were unaffected by the activation of the transgene with GCV (Figure 5 d and e). Because bone strength can be altered independently of bone mass, we measured bone strength using compression testing. Maximum load and maximum compression stress in the vertebrae were indistinguishable between vehicle‐ and GCV‐treated p16‐3MR mice (Figure 5 f). To determine whether GCV could exert effects independently of the activation of the p16‐3MR transgene, we administered GCV to wild‐type C57BL/6 for the same period of time. The administration of GCV to wild‐type mice had no effect on bone mass (Supporting information Figure S2 ).

Activation of the p16‐3MR transgene prevents the increase in adipocyte precursors in the bone marrow. (a and b) Bone marrow stromal cells were isolated from 24‐ (a) and 26‐month‐old (b) female p16‐3MR mice received vehicle or Ganciclovir ( n = 7–9 mice/group) for 12 months (a) or 6 months (b) and cultured with rosiglitazone for 14 days (triplicates). Left , Representative pictures and Right , quantification of Oil Red O Staining in cells. * p < 0.05 vs. young mice, # p < 0.05 vs. vehicle‐treated old mice; unpaired Student's t test. Bars represent mean and SD ( error bars )

Marrow adipocytes increase with aging in humans and rodents (Horowitz et al., 2017 ). Accordingly, the number of adipocytes formed in bone marrow cultures is higher in cells obtained from old than young mice (Moerman, Teng, Lipschitz, & Lecka‐Czernik, 2004 ). GCV administration decreased the adipogenic potential of bone marrow cells (Figure 4 a–b). Specifically, the number of Oil Red O‐positive cells, formed in cultures of stromal cells in the presence of the PPARγ stimulator rosiglitazone, was higher in cells obtained from p16‐3MR mice receiving vehicle when compared to cultures from young mice. The number of adipocytes was reduced in cultures derived from mice treated for 12 (Figure 4 a) or 6 months (Figure 4 b) with GCV, when compared to cells obtained from the age‐matched control mice.

Activation of the p16‐3MR transgene does not alter osteoblastogenesis. (a‐d) Bone marrow stromal cells were isolated from 24‐ (a and b) and 26‐month‐old (c and d) female p16‐3MR mice received vehicle or Ganciclovir ( n = 7–9 mice/group) for 12 months (a and b) or 6 months (c and d) and cultured with ascorbate and β‐glycerophosphate (triplicates). (a and c) Left , Representative pictures and Right , quantification of Alizarin Red Staining in cells cultured for 21 days and (b) protein levels by Western blot and (d) mRNA levels by qRT‐PCR in cells cultured for 7 days. * p < 0.05 vs. young mice; unpaired Student's t test. Bars represent mean and SD ( error bars )

We next examined whether ablation of p16‐expressing cells in the bone marrow of p16‐3MR mice altered osteoblast or adipocyte formation in culture. Bone marrow stromal cells from 24‐month‐old p16‐3MR mice treated with GCV showed the same degree of mineralization as cells from mice treated with vehicle when cultured under osteogenic conditions (Figure 3 a). As described before (Kim et al., 2017 ), p16 protein was undetectable in stromal cells from old mice (Figure 3 b). Nevertheless, the levels of p21, GATA4, and SASP are elevated in osteoblast progenitors from old mice (Kim et al., 2017 ). GCV administration to p16‐3MR mice did not alter the levels of p21 or GATA4 (Figure 3 b). Mineralization was also unaffected in bone marrow cultures from 26‐month‐old mice in which the p16‐3MR was activated for 6 months (Figure 3 c). Cultures from 3‐month‐old mice were used as young control. Similar to earlier findings (Kim et al., 2017 ), the mRNA levels of p21, IL‐1α, Mmp‐13, and RANKL were increased with age in the stromal cell cultures (Figure 3 d). GCV administration to mice did not alter the expression of any of these genes (Figure 3 d).

Activation of the p16‐3MR transgene eliminates osteoclast progenitors expressing p16. (a–c) Cultures of BMMs isolated from 24‐month‐old female p16‐3MR mice administered vehicle or Ganciclovir ( n = 9 mice/group) for 12 months. (d) Cultures of BMMs isolated from 26‐month‐old female p16‐3MR mice were either vehicle‐ or Ganciclovir‐administered ( n = 7 mice/group) for 6 months. Three‐month‐old female p16–3MR mice were used as a control. (a) p16 levels detected by Western blot and (b) p16 and SASP levels detected by qRT‐PCR after 48 hr in the presence of M‐CSF and (c and d) Top , Representative pictures and Bottom , number of TRAP‐positive multinucleated osteoclasts derived from BMMs of the mice cultured with M‐CSF (30 ng/ml) and RANKL (30 ng/ml) for 5 days (triplicates). * p < 0.05 vs. young mice, # p < 0.05 vs. vehicle‐treated old mice; unpaired Student's t test. Bars represent mean and S.D. ( error bars )

Because the osteoclastogenic potential of myeloid cells and the number of osteoclast in endocortical bone increases with age, we next examined the levels of p16 in osteoclast progenitors. To do this, we obtained bone marrow‐derived macrophages (BMMs) from mice in which the p16‐3MR transgene was activated from 12 to 24 month of age and cultured them in the presence of M‐CSF (Figure 2 a). Cultures from 3‐month‐old mice were used as young controls. P16 protein levels were greatly increased in 24‐month‐old p16‐3MR mice receiving vehicle. In contrast, cells from 24‐month‐old p16‐3MR mice receiving GCV had similar p16 levels to those of young mice (Figure 2 a). The mRNA levels of Cdkn2a (the gene that encodes both p16Ink4a and p19Arf) and common elements of the SASP such as IL‐1α, IL‐6, and TNFα were also increased with age in the osteoclast progenitor cultures; and all, except TNFα, were greatly decreased by GCV administration (Figure 2 b). Likewise, the number of osteoclasts formed in cultures of BMMs from the aged control mice was much greater than that the young, and this increase was also prevented by elimination of senescent cells with GCV (Figure 2 c). Administration of GCV to wild‐type mice had no impact on osteoclast formation in vitro (data not shown).

In a second study, we administered GCV or PBS to p16–3MR female mice from 20 to 26 months of age (Supporting information Figure S1 c). At 20 months of age, mice have already experienced significant loss of bone mass (Almeida et al., 2007 ; Piemontese et al., 2017 ). Like the findings in our first study, the levels of p16 in the cortical bone of GCV‐treated mice were undistinguishable from the vehicle‐treated mice at 26 months (Figure 1 d).

Levels of p16 in several tissues of old p16‐3MR mice. (a–d) Protein was isolated from the indicated tissues of (a–c) 24‐month‐old mice which received vehicle or Ganciclovir for 12 months, and (d) 26‐month‐old mice which received vehicle or Ganciclovir for 6 months. Each lane on the immunoblots represents one mouse (brain and liver, n = 4; bone shaft, n = 9). On the right, the expression of p16 was calculated as a ratio to β‐actin levels within in each lane. * p < 0.05 by Student's t test. Bars represent mean and SD ( error bars )

We administered GCV or PBS (vehicle) to p16‐3MR female mice, in the C57BL/6 genetic background, from 12 to 24 months of age (Supporting information Figure S1 b). Mice receiving GCV exhibited the same weight at 24 months as the control mice (data not shown). As expected, p16 protein levels in the brain and liver of mice treated for one year with GCV were greatly diminished (Figure 1 a and b). However, p16 protein levels were not affected in osteocyte‐enriched bone shafts (Figure 1 c), indicating that the 3MR transgene does not ablate p16‐expressing osteocytes. We have previously shown that the increase in p16 levels in cortical bone osteocytes is associated with increased levels of γH2AX, a marker of DNA damage (Piemontese et al., 2017 ). Consistent with the lack of an effect on p16 levels, GCV administration did not alter γH2AX in osteocytes (Figure 1 c).

3 DISCUSSION

Aging is a critical risk factor for the development of osteoporosis (Almeida et al., 2017; Manolagas, 2018), and cellular senescence has emerged as one of the hallmarks of aging and major contributor to age‐associated diseases, including osteoporosis (Childs et al., 2017; Lopez‐Otin, Blasco, Partridge, Serrano, & Kroemer, 2013). In aged mice, several cell types within bone exhibit markers of senescence (Farr et al., 2016; Kim et al., 2017; Piemontese et al., 2017). The p16‐3MR transgene ablates p16‐expressing cells from a diverse array of cells and tissues including skin, kidney, articular cartilage, hematopoietic system, and arteries (Baar et al., 2017; Chang et al., 2016; Childs et al., 2016; Demaria et al., 2017; Jeon et al., 2017). This maneuver rejuvenates hematopoietic stem cells and counters the development of kidney failure, osteoarthritis, atherosclerosis, and cancer relapse. On the other hand, the elimination of p16‐expressing cells in the skin delays wound healing (Demaria et al., 2014). In the present report, we show that activation of the p16‐3MR transgene efficiently eliminates p16‐expressing cells in the brain and liver of old mice. Unexpectedly, however, activation of the p16‐3MR transgene did not ablate senescent osteocytes in cortical bone. In contrast to our findings, Farr et al. have shown that the INK‐ATTAC transgene effectively decreases the number of senescent osteocytes in old mice (Farr et al., 2017). In that earlier work, the changes in Cdkn2 mRNA levels in cortical bone were very similar to the changes seen by enumeration of osteocytes exhibiting senescence‐associated distension of satellites (SADS). Be that as it may, it remains unknown whether elimination of senescent osteocytes, in and of itself, is responsible for the increase in bone mass seen in the INK‐ATTAC model. The reason(s) for the lack of efficacy of the p16‐3MR transgene in osteocytes remains unclear. Nevertheless, our findings demonstrate that elimination of p16‐expressing cells by the p16‐3MR transgene is tissue selective. Notably, the INK‐ATTAC mediated clearance of senescent cells is also partial and tissue selective (Baker et al., 2016).

In the present work, we focused on macrophages because these cells include the osteoclast progenitors and an increase in osteoclast number is associated with the thinning of cortical bone and the development of cortical porosity in old mice (Piemontese et al., 2017; Ucer et al., 2017). We found that the levels of p16 in macrophage cultures from the bone marrow of old mice were significantly higher as compared to cells from young mice. This finding is in line with evidence that CD14+ myeloid‐enriched cell populations isolated from the bone marrow of old mice exhibit elevated levels of p16 and SASP (Farr et al., 2016). F4/80‐positive macrophages in visceral adipose tissue and spleen of old mice also exhibit increased levels of p16 and β‐galactosidase activity (Hall et al., 2016). The activation of the p16‐3MR transgene by GCV abrogated the age‐associated increase in p16 and osteoclastogenic potential of bone marrow‐derived myeloid cells. Albeit, the assays used for this work cannot distinguish whether the changes in cell number are due to altered progenitor number or differentiation capacity. Be that as it may, our findings indicate that cell senescence is a major contributor to the age‐associated increase in osteoclastogenic potential of myeloid cells. Nonetheless, the elimination of the senescent osteoclast progenitors, in and of itself, does not alter endocortical osteoclast number and the loss of bone mass with age.

Cells of the mesenchymal lineage are a critical source of pro‐osteoclastogenic cytokines. We and others have shown earlier that stromal cell cultures obtained from the bone marrow of old mice provide greater support for osteoclastogenesis than cells from young mice (Cao et al., 2005; Kim et al., 2017). Elimination of senescent cells in cultures from old mice, using the senolytic ABT263, decreases the SASP and the pro‐osteoclastogenic effect (Kim et al., 2017). These lines of evidence, along with our present findings, support the notion that senescent cells of the mesenchymal lineage are major contributors to the increase in osteoclast number in the aged skeleton.

We have also shown earlier that a decrease in bone formation contributes to skeletal involution and is associated with a decline in the number of osteoprogenitor and their osteoblast descendants (Almeida et al., 2007; Kim et al., 2017). Specifically, we found that osteoblast progenitor cells, freshly isolated from the bone marrow of old mice, exhibit several markers of senescence (Kim et al., 2017). Nevertheless, osteoblast progenitor senescence is associated with increased p21, but not p16 levels. In view of this earlier finding, we had predicted that activation of the p16‐3MR would not eliminate senescent osteoblast progenitors. In line with our prediction, activation of the p16–3MR transgene by GCV did not alter the markers of senescence or the osteoblastogenic potential of bone marrow‐derived stromal cells. A decrease in osteoblast number is the seminal cellular mechanism for the age‐related loss of trabecular bone in the spine, as indicated by the fact that the number of osteoclasts does not increase with age in this compartment (Almeida et al., 2007). In agreement with the contention that osteoprogenitor senescence is independent of p16, elimination of p16‐expressing cells in old INK‐ATTAC mice does not affect osteoblast number in the trabecular bone of the spine (Farr et al., 2017).

Bone marrow adipocytes increase with age in bone, but the mechanism(s) responsible for this effect and its contribution to age‐related bone loss remains unclear. It is generally accepted that marrow adipocytes arise from mesenchymal progenitor cells (Horowitz et al., 2017). We previously suggested that an increase in oxidized lipids contributes to the age‐related marrow fat accumulation (Almeida, Ambrogini, Han, Manolagas, & Jilka, 2009). In the present study, we found that ablation of p16‐expressing cells in aged p16‐3MR mice decreased the adipogenic potential of bone marrow cells. Similarly, ablation of p16‐expressing cells in aged INK‐ATTAC mice reduces the number of bone marrow adipocytes (Farr et al., 2017). In line with these findings, hepatocyte‐specific senescence leads to fat accumulation in the liver (Ogrodnik et al., 2017). Taken together with the evidence that senescent cells of the mesenchymal lineage were not eliminated by the p16‐3MR transgene, these observations suggest that senescence of other cell types, perhaps within the hematopoietic lineage, contribute via SASP to the increase in adipocyte precursors.

In closing, even though the identity of the senescent cells responsible for the age‐related loss of bone mass remains unclear, the results of the present work strongly suggest that senescent hematopoietic lineage cells are not major culprits. Earlier observations by Farr et al., using the INK‐ATTAC model, indicate that p16‐expressing cells in old mice increase osteoclast number (Farr et al., 2017). Together with our results with the p16‐3MR model, the findings of Farr and colleagues suggest that pro‐osteoclastogenic cytokines of the SASP originating from cells of the osteoblastic lineage contribute to the increase in osteoclast number in the aging skeleton. Because the long‐lived osteocytes are the primary cellular source of the RANKL required for adult bone remodeling (Xiong et al., 2011), accumulation of senescent osteocytes is, likely, a critical mechanism of skeletal aging.