Hypoxia negatively affects senescence in osteoclasts and delays osteoclastogenesis

Abstract

Cellular senescence, that is, the withdrawal from the cell cycle, combined with the acquirement of the senescence associated secretory phenotype has important roles during health and disease and is essential for tissue remodeling during embryonic development. Osteoclasts are multinucleated cells, responsible for bone resorption, and cell cycle arrest during osteoclastogenesis is well recognized. Therefore, the aim of this study was to investigate whether these cells should be considered senescent and to assess the influence of hypoxia on their potential senescence status. Osteoclastogenesis and bone resorption capacity of osteoclasts, cultured from CD14+ monocytes, were evaluated in two oxygen concentrations, normoxia (21% O₂ ) and hypoxia (5% O₂ ). Osteoclasts were profiled by using specific staining for proliferation and senescence markers, qPCR of a number of osteoclast and senescence-related genes and a bone resorption assay. Results show that during in vitro osteoclastogenesis, osteoclasts heterogeneously obtain a senescent phenotype. Furthermore, osteoclastogenesis was delayed at hypoxic compared to normoxic conditions, without negatively affecting the bone resorption capacity. It is concluded that osteoclasts can be considered senescent, although senescence is not uniformly present in the osteoclast population. Hypoxia negatively affects the expression of some senescence markers. Based on the direct relationship between senescence and osteoclastogenesis, it is tempting to hypothesize that contents of the so-called senescence associated secretory phenotype (SASP) not only play a functional role in matrix resorption, but also may regulate osteoclastogenesis.

Keywords: cellular senescence; hypoxia; osteoclastogenesis; osteoclasts.

Figure 1

Osteoclasts seem to experience hypoxia as there is a tendency toward increased average quantity (+/− SD) of HIF‐1α present at week 2 in 5% versus 21% O₂ (a, n = 3 donors per time point) and corresponding blots of HIF‐1a (upper) and tubulin (lower) (c) qRT‐PCR results of the HIF target gene NIX (BCL2 Interacting Protein 3 Like) over time (c, n = 3 donors per time point). There were no significant differences between culture conditions

Figure 2

Hypoxia delays cellular fusion and with that osteoclastogenesis as indicated by the number of multinucleated, TRAP positive osteoclasts (OCL) over time (a) with representative examples (b), and corresponding results of the RT‐qPCR analysis of genes associated with osteoclast differentiation and function (c). TRAP, Tartrate‐resistant acid phosphatase, CA II, Carbonic Anhydrase II, CATK, Cathepsin K, DCSTAMP, Dendritic Cells (DC)‐Specific Transmembrane Protein, Integrin β3, Integrin subunit β3. Each symbol represents a single donor. *0.01 < p < 0.05; **p < 0.01 hypoxia vs normoxia at the same time point

Figure 3

Number of single (left) and double nucleated cells after over time in normoxic (21% O₂) and hypoxic (5% O₂) culture conditions 21% (*0.01 < p < 0.05; **p < 0.01)

Figure 4

Oxygen concentration during culture does not seem to affect the bone resorption capacity of osteoclasts as indicated by the integrated intensity of bisphosphonate staining after 3 weeks of culturing osteoclasts on bovine bone chips in 21% and 5% of O₂ (a). Representative examples with resorption lacuna stained blue and nuclei stained red (b). There were no significant differences between culture conditions

Figure 5

The proliferative marker Ki67 is expressed heterogeneously in the nuclei during osteoclastogenesis and seems not to be affected by oxygen tension. Percentage of Ki67 positive, multinucleated, TRAP positive osteoclasts (OCL) over time (left) with representative examples (right) in normoxic (21% O₂) and hypoxic (5% O₂) culture conditions. The arrows indicate Ki67 positive nuclei in multinucleated cells. There were no significant differences between culture conditions

Figure 6

The senescence marker p16 is limited expressed during osteoclastogenesis. Percentage of p16 positive nuclei in multinucleated, TRAP positive osteoclasts (OCL) over time (left) and representative examples (right) in normoxic (21% O₂) and hypoxic (5% O₂) culture conditions. The arrows indicate p16 positive nuclei in multinucleated cells. Note that while mononuclear cells were p16 positive, no osteoclasts were present after 1 week of culture. There were no significant differences between culture conditions

Figure 7

The senescence marker p21 is abundantly expressed during osteoclastogenesis and negatively affected by hypoxia. Percentage of p21 positive nuclei in multinucleated, TRAP positive osteoclasts (OCL) over time and representative examples (right) in normoxic (21% O₂) and hypoxic (5% O₂) culture conditions. The arrows indicate p21 positive nuclei in multinucleated cells. *0.01 < p < 0.05; **p < 0.01 hypoxia vs normoxia at the same time point

Figure 8

Senescence associated beta galactosidase staining is present at week 1 and 2 (a) supporting the senescent phenotype of osteoclasts. RT‐qPCR analysis of the senescence associated genes CCL 2, CCL5, p21, and MMP9 (b; n = 3 donors) shows differential gene expression profiles in normoxia vs hypoxia. CCL2, C‐C Motif Chemokine Ligand 2; CCL 5, C‐C Motif Chemokine Ligand 5; p21, Cyclin Dependent Kinase Inhibitor 1A; MMP9, Matrix Metalloproteinase 9 after 1 day of culturing (indicated with “0”) and 1–3 weeks of culturing. *0.01 < p < 0.05; **p < 0.01 hypoxia vs normoxia at the same time point