Senescence of osteoclast precursors in the bone microenvironment

Objectives: Recent studies suggest that stem cells may undergo senescence, what plays a crucial role in organismal aging. It has become clear that aging cells do not simply stop cell growth but secrete various proteins such as inflammatory cytokines and chemokines. This secretory phenomenon is called SASP (senescence-associated secretory phenotype). Osteoclasts (OC) are differentiated from monocyte/macrophage-lineage hematopoietic precursor cells which were termed OC precursors (OCPs) in the bone microenvironment. The purpose of this study is to elucidate the effects of senescence on the differentiation and SASP of OCPs in the bone microenvironment.
Methods: RAW264.7 cells were used as osteoclast precursors, and cultured for 5, 10, and 20 passages (P5, P10, and P20). We evaluated cell proliferation assay and senescence-associated β-galactosidase (SA-β-gal) staining to confirm replicative senescence of RAW264.7 cells. TRAP activity was determined by TRAP solution assay. RANK, NFATc1, mTOR, p53, HIF, iNOS and TGF-β1 were detected by Western blot analysis. We evaluated nitric oxide (NO) and IL-6 production in culture supernatant.
Results: The results of cell proliferation assay and SA-β-gal staining indicated P20 cells were induced replicative senescence. The ability to differentiate into osteoclasts of P20 were reduced, and produced high amounts of SASP factors (TGF-β1, iNOS, HIF) compared with P5. NO and IL-6 production were much more in P20 than P5.
Conclusions: The results of this study indicate that an accumulation of senescence osteoclast precursors may induce further senescence in the bone microenvironment.