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. 2022 Feb 18:10:821014.
doi: 10.3389/fcell.2022.821014. eCollection 2022.

Iron Overload Induces Oxidative Stress, Cell Cycle Arrest and Apoptosis in Chondrocytes

Asima Karim  1 , Khuloud Bajbouj  1   2 , Jasmin Shafarin  2 , Rizwan Qaisar  1 , Andrew C Hall  3 , Mawieh Hamad  2   4
Affiliations

Iron Overload Induces Oxidative Stress, Cell Cycle Arrest and Apoptosis in Chondrocytes

Asima Karim et al. Front Cell Dev Biol. .

Abstract

Clinical and experimental evidence point to the presence of considerable links between arthropathy, osteoarthritis (OA) in particular, and iron overload possibly due to oxidative stress and tissue damage. However, the specific cellular targets of iron overload-related oxidative stress in OA remain ambiguous. We examined the effects of iron overload on chondrocyte health using the C-20/A4 chondrocyte cell line. Cells were treated with increasing concentrations of ferric ammonium citrate (FAC) to mimic iron overload in vitro. Treated cells were assessed for cell viability, cycling, apoptosis, collagen II synthesis, and oxidative stress along with cellular iron content and the expression of key iron regulatory genes. FAC treatment resulted in an increase in ferritin expression and a significant decrease in the expression of hepcidin, ferroportin, transferrin receptors 1 (TfR1) and TfR2. Increased labile iron content was also evident, especially in cells treated with high FAC at 24 h. High doses of FAC treatment also induced higher levels of reactive oxygen species, reduced collagen II production, disrupted cell cycle and higher cell death as compared with untreated controls. In conclusion, findings presented here demonstrate that iron overload disrupts cellular iron homeostasis, which compromises the functional integrity of chondrocytes and leads to oxidative stress and apoptosis.

Keywords: C-20/A4 cells; apoptosis; chondrocytes; iron overload; osteoarthritis; oxidative stress.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
(A) FAC treatment reduces C-20A/4 cell viability. The viability of C-20A/4 chondrocytes was assessed using the MTT assay following treatment with 2, 4, 6, 8, 10, 50, 100, 200 or 300 µM of FAC for 24 or 48 h. Data are the mean ± SEM of three independent experiments. (B) Expression status of key IRGs in FAC-treated C-20A/4 cells. (Bi) Western blotting was used to examine the expression status of some key IRGs, including FTH1, HEP, FPN, TfR1 and TfR2 using lysates obtained from C-20A/4 cells treated with 200 or 300 µM FAC for 24 or 48 h. Untreated C-20A/4 cells cultured under similar conditions served as negative controls; β-actin was used as an internal loading control. (Bii) Fold change ±SD of protein band thickness based on three separate experiments. (C) FAC treatment and size of the labile iron pool (LIP) in C-20A/4 cells. Calcein staining flow cytometry-based analysis was used to determine the LIP content in C-20A/4 cells treated with 200 and 300 µM FAC for 24 and 48 h. Data shown is ΔMFI ±SD based on three independent experiments; ns means not significant. (D) Oxidative stress levels in FAC-treated C-20A/4 cells. To assess the level of oxidative stress under conditions of iron overload, ROS production was measure in C-20A/4 cells following treatment with 200 and 300 µM FAC for (Ci) 24 and (Cii) 48 h. Data shown are the mean ROS level ±SD based on three independent experiments. * (Treated vs. untreated control at 24 h), $ (Treated vs. untreated control at 48 h) and # (Treated at 24 h vs. treated at 48 h) denotes statistical significance at p <0.05; untreated cells cultured under the same conditions served as negative controls.
FIGURE 2
FIGURE 2
Iron overload and cell cycle progression analysis in chondrocytes. Cell cycle progression of C-20A/4 cells was examined following treatment with 200 and 300 µM of FAC and 24 or 48 h using the PI staining flow cytometry-based method. (A) Cell cycle profile in FAC-treated and control C-20A/4 cells; data shown are representative of three independent experiments. (B) Calculated distribution of cell-cycle-related subpopulations based on three independent experiments with ±SD; *,$ denotes statistical significance at p<0.05 as per the indicated group vs. its untreated control at 24 and 48 h respectively. Untreated cells cultured under the same conditions served as negative controls.
FIGURE 3
FIGURE 3
Iron overload induces apoptosis in chondrocytes. (A) Percentage pro-apoptotic and apoptotic C-20A/4 cells was examined following treatment with 200 or 300 µM of FAC and 24 or 48 h using the PI-annexin V staining-based flow cytometry method. (B) Mean ± SD percentage of pro-apoptotic and apoptotic C-20A/4 cells was calculated based on three independent experiments as in (A). *,$ denotes statistical significance at p<0.05 as per the indicated group vs. its untreated control at 24 and 48 h respectively. Untreated cells cultured under the same conditions served as negative controls.
FIGURE 4
FIGURE 4
Iron overload decreases collagen II production in chondrocytes. (A) Expression of collagen II by chondrocytes treated with FAC (200 or 300 µM) and 24 or 48 h were assessed by immunofluorescence microscopy. Collagen II was labelled with Alexafluor®680 (Red), and nuclei were labelled with DAPI (Blue). Images were taken at ×100 magnification. (B) Expression levels of collagen II by FAC treated chondrocytes in comparison to the controls shown as represented in the graph. Data shown are representative of three independent experiments. $ denotes statistical significance at p<0.05 as per the indicated group vs. its untreated control at 48 h.
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