Development of a DNA damage-induced senescence model in osteoarthritic chondrocytes

Abstract

Senescent cells (SnCs) have been described to accumulate in osteoarthritis (OA) joint tissues in response to injury, thereby participating in OA development and progression. However, clinical therapeutic approaches targeting SnCs using senolysis, although promising in preclinical OA models, have not yet proven their efficacy in patients with knee OA. This pitfall may be due to the lack of understanding of the mechanisms underlying chondrocyte senescence. Therefore, our study aimed to generate models of chondrocyte senescence. This study used etoposide, to induce DNA damage-related senescence or chronic exposure to IL-1β to entail inflammation-related senescence in human OA chondrocytes. Several hallmarks of cellular senescence, such as cell cycle arrest, expression of cyclin-dependent kinase inhibitors, DNA damages, and senescence-associated secretory profile were evaluated. Chronic exposure to IL-1β induces only partial expression of senescence markers and does not allow us to conclude on its ability to induce senescence in chondrocytes. On the other hand, etoposide treatment reliably induces DNA damage-related senescence in human articular chondrocytes evidenced by loss of proliferative capacity, DNA damage accumulation, and expression of some SASP components. Etoposide-induced senescence model may help investigate the initiation of cellular senescence in chondrocytes, and provide a useful model to develop therapeutic approaches to target senescence in OA.

Keywords: IL-1β; chondrocytes; etoposide; osteoarthritis; senescence.

Figure 1

Experimental design. To investigate senescence in chondrocytes, primary human articular chondrocytes (HACs) were stimulated with etoposide for 24 h (blue) or with IL-1β for 8 days (red) with treatment renewal at days 3 and 6. Senescence features were assessed at days 1 and 8 in both conditions by qPCR, WB, and immunofluorescence. Figure created with https://www.biorender.com.

Figure 2

Impact of Etoposide and IL-1β treatments on HACs proliferation. HACs were treated with etoposide (blue) at 5, 10 or 20 μM (A, B) for 24 h and then cultured in normal media or with IL-1β (red) at 1 and 10 ng/mL (C, D) for the length of the experiment. (A, C) The number of viable cells was assessed by trypan blue exclusion dye. (B, D) DNA quantification in Etoposide and IL-1β treated HACs at day 1 and day 8. Data are shown as mean ± SD, (n = 3). P-values were calculated by the two-way ANOVA test, ^*p ≤ 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001. (E) EdU was used to identify proliferative cells and Hoechst staining to visualize the nucleus at day 1 and 8 (scale bar = 50 μm). The images were analyzed by quantification of positive cells for EdU normalized versus the total number of cells obtained with the Hoechst staining at each time. Data are shown as mean ± SD, (n ≥ 3). P-values were calculated by Mann-Whitney test compared to the control on day 1, ^***p < 0.001.

Figure 3

Expression of cyclin-dependent kinase inhibitors in Etoposide, and IL-1β treated HACs. HACs were treated with etoposide (blue) at 20 μM for 24 h or IL-1β (red) at 1 ng/mL for the length of the experiment and the expression of cyclin-dependent kinase inhibitors evaluated by RT-qPCR (A, B) and WB (C, D) at day 1 (A, C) and day 8 (B, D). Data are shown as mean ± SD, (n ≥ 3). P-values were calculated by Mann-Whitney test, *p ≤ 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

Figure 4

DNA damage assessment and senescence-associated nuclear features measure in Etoposide, and IL-1β treated HACs. HACs were treated with Etoposide 20 μM (blue) for 24 h or chronically with 1 ng/mL IL-1β (red) for the length of the experiment. (A) γH2AX immunofluorescence was used to identify DNA damage-associated foci and Hoechst staining to visualize the nucleus at day 1 and day 8. Quantification of the average number of foci per nuclei is shown. (B) Nucleus surface, perimeter and circularity was analyzed using CellProfiler software on the Hoechst channel. Scale bars = 50 μm. Data are shown as mean ± SD, (n = 3). P-values were calculated by Kruskal-Wallis test, ^*p ≤ 0.05; ^**p < 0.01.

Figure 5

SASP markers in Etoposide and IL-1β treated HACs. (A–D) HACs were treated with Etoposide (blue) at 20 μM for 24 h or IL-1β (red) at 1 ng/mL for the length of the experiment. The expressions of SASP markers were evaluated by RT-qPCR at day 1 (A) and day 8 (B), and SASP components were quantified by Luminex assay in the culture medium at day 1 (C) and 8 (D). Data are shown as mean ± SD, (n ≥ 3). P-values were calculated by Mann-Whitney test, ^*p ≤ 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.