It is known that increased inflammation and extracellular matrix (ECM) degradation in chondrocytes can promote the development of osteoarthritis (OA). The FXYD domain containing ion transport regulator 5 (Fxyd5) has been found to promote chronic inflammatory responses. The present study aimed to investigate the role of Fxyd5 in OA. Murine ATDC5 chondrocytes were transfected with short hairpin RNAs specifically targeting Fxyd5 to silence its expression. Subsequently, cells were induced with lipopolysaccharide (LPS). The protein expression levels of Fxyd5, MMPs and proteins related to ECM, apoptosis and NF-κB signaling were detected using western blot analysis. In addition, cell viability was assessed using a Cell Counting Kit-8 assay, while the secretion of the proinflammatory factors and those of the oxidative stress-related markers were measured using the corresponding kits. Finally, cells were treated with the NF-κB activator, betulinic acid (BA) and the above experiments were repeated. The results demonstrated that Fxyd5 was significantly upregulated in ATDC5 cells treated with LPS. Additionally, Fxyd5 knockdown increased cell viability, enhanced the protein expression of Bcl-2, Aggrecan and collagen II, while reduced the expression of Bax, cleaved caspase-3/caspase-3, MMP3 and MMP13 in LPS-induced ATDC5 cells. The production of IL-1β, IL-6 and IL-18 as well as reactive oxygen species and malondialdehyde, and the reduction of superoxide dismutase caused by LPS in ATDC5 cells, were also reversed by Fxyd5 silencing. Fxyd5 silencing inhibited the phosphorylation of p65 and IκBα induced by LPS. Finally, BA reversed the protective effect of Fxyd5 silencing on LPS induced chondrocytes injury. In conclusion, Fxyd5 could enhance chondrocyte inflammation and ECM degradation via activating the NF-κB signaling.
Osteoarthritis (OA), characterized by enhanced cartilage degradation and cartilage cell death, is a degenerative joint disease caused by the progressive erosion of articular cartilage (
FXYD domain containing ion transport regulator 5 (Fxyd5) is a widely expressed single-pass transmembrane protein that increases the apparent affinity for intracellular Na+/K+-ATPase and serves several roles in different cell types (
The significant role of NF-κB signaling in the occurrence and progression of OA has been extensively studied (
The murine ATDC5 chondrocytes were obtained from the American Type Culture Collection and cells were maintained in DMEM/F12 (MilliporeSigma) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin solution (Beyotime Institute of Biotechnology) at 37°C in a humidified CO2 (5%) incubator. To construct the OA cell model
Short hairpin RNAs (shRNAs) against mouse Fxyd5 (accession no. NM_001111073; shFxyd5-1 and shFxyd5-2) were designed using the online design software (
Following cell treatment or transfection, a total of 2×104 ATDC5 cells/well were seeded into a 96-well and were then incubated with CCK-8 regent for 2 h at 37°C. The absorbance at a wavelength of 450 nm was measured in each well using a microplate reader (Multiskan SkyHigh Microplate Spectrophotometer; Thermo Fisher Scientific, Inc.). Each sample was assessed five times.
A total of 1×106 ATDC5 cells were seeded into 6-well plates, washed with PBS and lysed using RIPA Cell lysis buffer (Beyotime Institute of Biotechnology). Following centrifugation at 12,000 × g for 20 min at 4°C, the supernatant was collected to perform western blot analysis. Briefly, total protein concentration was quantified using a BCA kit (Invitrogen; Thermo Fisher Scientific, Inc.), then equal amount of protein (30 µg) was separated through the 10% SDS-PAGE, followed by transfer onto PVDF membranes. After blocking with 5% non-fat milk for 1 h at room temperature, the PVDF membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies were: Rabbit anti-Fxyd5 (1:1,000; cat. no. ABIN2780210), rabbit anti-aggrecan (1:500; cat. no. ABIN6996681; both from antibodies-online), rabbit anti-GAPDH (1:10,000; cat. no. ab181602), rabbit anti-Bcl2 (1:2,000; cat. no. ab182858), rabbit anti-Bax (1:8,000; cat. no. ab32503), rabbit anti-cleaved caspase 3 (1:5,000; cat. no. ab214430), rabbit anti-caspase 3 (1:2,000; cat. no. ab184787), rabbit anti-MMP3 (1:20,000; cat. no. ab52915; all from Abcam), rabbit anti-MMP13 (1:2,000; cat. no. NBP2-66954), rabbit anti-phosphorylated (p)-IκBα (1:800; cat. no. NB100-81987), rabbit anti-IκBα (1:5,000; cat. no. NBP2-67369), rabbit anti-collagen II (1:10,000; cat. no. NBP1-77795; all from Novus Biologicals, Ltd.), rabbit anti-p-p65 (1:1,000; cat. no. 3033S) and rabbit anti-p65 (1:1,000; cat. no. 8242S; Cell Signaling Technology, Inc.). Subsequently, membranes were treated with the goat anti-rabbit IgG (HRP) secondary antibody (1:10,000; cat. no. ab6721; Abcam) at room temperature for 1 h. Then, enhanced chemiluminescence (MilliporeSigma) was used to detect the protein expression levels. Band semi-quantification was performed using Image J software (version 1.54; National Institutes of Health).
Following cell treatment or transfection, ATDC5 cells were seeded into 6-well plates at a density of 1×106 cells/well and the levels of intracellular ROS, SOD and lipid peroxidation product MDA were determined using ROS Assay kit (cat. no. S0033M; Beyotime Institute of Biotechnology), SOD Assay Kit (cat. no. 7500-100-K; R&D systems, Inc.) and TBARS Parameter Assay Kit (cat. no. KGE013; R&D systems, Inc.), respectively, according to the manufacturer's recommendations.
Control or transfected ATDC5 cells were seeded into 96-well plates at a density of 2×104 cells/well, following indicated treatment, the secretion levels of IL-1β, IL-6 and IL-18 in the culture supernatants were measured using the Mouse IL-1β (cat. no. ab197742), IL-6 (cat. no. ab222503) and IL-18 (cat. no. ab216165) SimpleStep ELISA kits (Abcam) according to the manufacturer's instructions.
All statistical analyses were performed using GraphPad Prism 8.0.1 (GraphPad Software, Inc.). Data were expressed as the mean ± standard deviation from three independent experiments. One-way ANOVA followed by Tukey's multiple comparison test was applied to compare the differences among multiple groups, while those between two groups were compared using an unpaired two-tailed Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
To explore the expression profile of Fxyd5 in the LPS-induced chondrocyte injury model (
To further explore the effect of Fxyd5 on LPS-induced chondrocyte inflammation and oxidative stress, ELISA was carried out to determine the secretion levels of pro-inflammatory cytokines and the content of ROS, MDA and SOD in LPS-induced ATDC5 cells. The results revealed that Fxyd5 was involved in LPS-induced cellular inflammation. Therefore, the secretion levels of the inflammatory factors IL-1β, IL-6 and IL-18 were significantly decreased in Fxyd5-depleted ATDC5 cells treated or not with LPS compared with the shNC group (
To investigate whether Fxyd5 was involved in the regulation of NF-κB pathway in the LPS-induced chondrocyte injury model (
Since Fxyd5 knockdown in ADTC5 cells could inhibit the activation of the NF-κB pathway (
Articular cartilage is a non-self-renewing avascular tissue that lacks inherent repair capability (
LPS is used to establish chondrocyte injury models by inducing cell apoptosis, oxidative stress and inflammation (
The current study aimed to investigate the effect of transport regulator Fxyd5 on LPS-induced ATDC5 cell injury. Unlike other members of the FXYD family, Fxyd5 exhibits several biological functions in different cell types (
The current study also demonstrated that Fxyd5 silencing could relieve LPS-induced oxidative stress in ATDC5 cells as evidenced by reduced ROS and MDA, while increased SOD levels upon Fxyd5 silencing. Excessive production of ROS contributes to the occurrence of oxidative stress; MDA is a production of lipid peroxidation and SOD belongs to a member of the enzyme antioxidant system (
It has been reported that LPS binds to and alters the configuration of its associated receptors, promotes IκBα phosphorylation and activates the classic NF-κB pathway (
The results of the current study revealed that Fxyd5 was significantly upregulated in the LPS-induced chondrocyte injury model. Furthermore, the results demonstrated that the increased expression levels of Fxyd5 could promote inflammation, oxidative stress and ECM degradation in ATDC cells via activating the NF-κB signaling pathway. Overall, these findings could provide novel insights to increase our understanding in the role of NF-κB signaling in the pathogenesis of OA.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
LS guided the project and wrote the manuscript. LS and XL conceived the technical details and designed the experiments. LS, XL and YZ performed the experiments. QS analyzed the data. All authors read and approved the final manuscript. LS and XL confirm the authenticity of all the raw data.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Fxyd5 silencing enhances cell viability and inhibits cell apoptosis and ECM degradation in ATDC5 cells. (A) Western blot analysis showing the upregulated expression levels of Fxyd5 in ATDC5 cells treated with 5 µg/ml LPS for 5 h. ***P<0.001 vs. the control group. (B) shFxyd5-2 exhibited a more potent effect on Fxyd5 silencing compared with shFxyd5-1. Western blot analysis showed that the protein expression levels of Fxyd5 were decreased by ~50% compared with the shNC group. **P<0.01 and ***P<0.001 vs. the shNC group. (C) Cell Counting Kit-8 assay showed that Fxyd5 knockdown reversed the LPS-mediated decreased ATDC5 cell viability. (D) Western blot analysis revealed that Fxyd5 silencing reversed the LPS-induced ATDC5 cell apoptosis, as supported by the decreased expression levels of Bax and cleaved caspase 3 and the increased expression levels of Bcl-2 in Fxyd5-depleted ATDC5 cells compared with the shNC group. The expression levels of caspase 3 remained unchanged. (E) Western blot analysis showed that Fxyd5 knockdown reversed the LPS-induced ECM degradation in ATDC5 cells, as supported by MMP3 and MMP13 downregulation and aggrecan and collagen II upregulation in the shFxyd5 group compared with the shNC group. Untreated and untransfected ATDC5 cells served as the control group. GAPDH served as the loading control. ***P<0.001 vs. the control group; ##P<0.01, ###P<0.001 vs. the LPS + shNC group. Fxyd5, FXYD domain containing ion transport regulator 5; ECM, extracellular matrix; LPS, lipopolysaccharide; sh, short hairpin RNA; NC, negative control.
Fxyd5 knockdown attenuates inflammation and oxidative stress in ATDC5 cells. (A) ELISA showed that Fxyd5 knockdown in ATDC5 cells reduced the LPS-mediated release of the pro-inflammatory cytokines IL-1β, IL-6 and IL-18 compared with the shNC group. (B) Fxyd5 silencing alleviated LPS-induced oxidative stress in ATDC5 cells compared with the shNC group, as supported by the decreased levels of ROS and MDA and the increased ones of SOD. Untreated and untransfected ATDC5 cells served as the control group. ***P<0.001 vs. the control group; ###P<0.001 vs. the LPS + shNC group. Fxyd5, FXYD domain containing ion transport regulator 5; LPS, lipopolysaccharide; NC, negative control; shRNA, short hairpin RNA; ROS, reactive oxygen species; MDA, malondialdehyde; SOD, superoxide dismutase.
Fxyd5 regulates NF-κB signaling in ATDC5 cells. Western blot analysis revealed that Fxyd5 silencing in ATDC5 cells reduced the LPS-induced phosphorylation levels of p65 and IκBα compared with the shNC group. Untreated and untransfected ATDC5 cells served as the control group. GAPDH served as the loading control. ***P<0.001 vs. the control group; ###P<0.001 vs the. LPS + shNC group. Fxyd5, FXYD domain containing ion transport regulator 5; LPS, lipopolysaccharide; shRNA, short hairpin RNA; NC, negative control short hairpin RNA; p-, phosphorylated; t-, total.
Fxyd5 regulates cell viability, apoptosis and ECM degradation via activating the NF-κB signaling pathway in ATDC5 cells. Following cell treatment with 10 nM BA, a NF-κB activator, for an additional 48 h (A) the phosphorylation levels of p65 and IκBα were increased, as demonstrated by western blot analysis. (B) Cell Counting Kit 8 assay showed that cell viability was increased in Fxyd5-depleted ATDC5 cells co-treated with LPS and BA compared with cells treated only with LPS. (C and D) Western blotting was used to evaluate the expression of proteins involved in cell apoptosis and ECM degradation. Untreated and untransfected ATDC5 cells served as the control group. GAPDH served as the loading control. ***P<0.001 vs. the control group; ##P<0.01 and ###P<0.001 vs. the LPS + shNC group; $P<0.05, $$P<0.01 and $$$P<0.001 vs. the LPS + shFxyd5-2 + BA group. Fxyd5, FXYD domain containing ion transport regulator 5; ECM, extracellular matrix; BA, betulinic acid; LPS, lipopolysaccharide; shRNA, short hairpin RNA; NC, negative control.
Fxyd5 regulates cellular inflammation and oxidative stress in ATDC5 cells via activating NF-κB signaling. Following cell treatment with 10 nM BA for additional 48 h, the secretion levels of the (A) pro-inflammatory factors IL-1β, IL-6 and IL-18 were increased in ELISA. (B) The content of ROS and MDA was increased and that of SOD was decreased in Fxyd5-depleted ATDC5 cells co-treated with LPS and BA compared with cells treated only with LPS. Untreated and untransfected ATDC5 cells served as the control group. GAPDH served as the loading control. ***P<0.001 vs. the control group; ###P<0.001 vs. the LPS + shNC group; $$P<0.01 and $$$P<0.001 vs. the LPS + shFxyd5-2 + BA group. Fxyd5, FXYD domain containing ion transport regulator 5; BA, betulinic acid; LPS, lipopolysaccharide; shRNA, short hairpin RNA; NC, negative control; ROS, reactive oxygen species MDA, malondialdehyde; SOD, superoxide dismutase.