Identification of Senescent Cells in the Bone Microenvironment

Abstract

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.

Old age causes de novo intracortical bone remodeling and porosity in mice

“…Importantly, we found that increased osteocyte age was associated with an increase in markers of DNA damage, cellular senescence, and SASP in osteocyte-enriched cortical bone preparations from both male and female B6 mice, consistent with recent findings by others (57). The SASP may well influence the secretory profile of neighboring aged but healthy osteocytes, as well as the relatively young osteocytes formed during endosteal and intracortical remodeling (58).…”

Expression of p16INK4a is a biomarker of chondrocyte aging but does not cause osteoarthritis

“…Furthermore, we used Palbociclib treatment (mimicking high p16 INK4a expression) to demonstrate that chondrocytes are responsive to CDK4/6 inhibition when cultured ex vivo under conditions that support proliferation. In line with reports of senescent features in other postmitotic cells such as neurons (Jurk et al., 2012) and osteocytes (Farr et al., 2016), our results demonstrate that the INK4a/ARF locus is potently activated with aging even in a cell type that is not affected by replicative exhaustion in vivo.…”

“…Of particular relevance is the recent finding that osteocytes exhibit features of cellular senescence with age, including expression of p16 INK4a and SASP markers (Farr et al., 2016). Our in vivo approach focused on the role of p16 INK4a in chondrocytes through the use of the Acan tm1(cre/ERT2)Crm allele that targets articular chondrocytes and meniscal cells (Henry et al., 2009), as well as a fraction of osteoblasts through direct conversion of hypertrophic growth plate chondrocytes (Ono, Ono, Nagasawa & Kronenberg, 2014; Zhou et al., 2014).…”

Osteoprotection Through the Deletion of the Transcription Factor Rorβ in Mice

“…6 A ). Rorβ expression was also significantly increased (fourfold) in osteocyte-enriched bone samples (14) from 12-month-old versus 6-month-old mice (Supporting Fig. 6 B ).…”