Specificity protein 1-mediated ACSL4 transcription promoted the osteoarthritis progression through suppressing the ferroptosis of chondrocytes

Abstract

Background Chondrocytes are the main cell damage type involved in the occurrence and development of osteoarthritis (OA). Ferroptosis has been confirmed to be related to many degenerative diseases. This research aimed to explore the role of Sp1 and ACSL4 in ferroptosis in the IL-1β-treated human chondrocyte cells line (HCCs). Methods The cell viability was detected with CCK8 assay. The ROS, MDA, GSH, and Fe2+ levels were assessed with corresponding detecting kits. The Col2a1, Acan, Mmp13, Gpx4 and Tfr1 levels were determined by RT-qPCR assay. Western blot was conducted to evaluate the Acsl4 and Sp1 levels. PI staining was carried out to analyze the cell death. The double luciferase report was conducted to verify the interaction between Acsl4 and Sp1. Results The results showed that IL-1β stimulation elevated the LDH release, cell viability, ROS, MDA and Fe2+ levels and declined the GSH levels in the HCCs. Additionally, the mRNA levels of Col2a1, Acan, and Gpx4 were prominently decreased, while Mmp13 and Tfr1 were prominently elevated in the IL-1β stimulated HCCs. Furthermore, Acsl4 protein levels were upregulated in the IL-1β-stimulated HCCs. Both Acsl4 knockdown and ferrostatin-1 treatment neutralized the role of IL-1β in the HCCs. What’s more, Acsl4 was transcriptionally regulated by Specificity protein 1 (Sp1). Sp1 overexpression enhanced the Acsl4 levels and Sp1 knockdown declined it. Conclusion Upregulation of Sp1 activates Ascl4 transcription and thus mediates the occurrence of ferroptosis. Hence, Acsl4 may be a therapeutic target for intervention of OA.

Chondroprotective effects of bone marrow mesenchymal stem cell-derived exosomes in osteoarthritis

“…ACSL4 is a well-established marker of ferroptosis and its expression shows an ascending trend in chondrocytes stimulated by IL-1β; in contrast, this trend is compromised by Fer-1 [32]. In addition, de ciency of ACSL4 in the IL-1β-stimulated chondrocytes can diminish the levels of ROS, MDA and Fe 2+ while elevating GSH levels [19]. Meanwhile, MSC-Exos loaded with miR-194 downregulate the mRNA expression of ACSL4 in an oxygen-glucose deprivation/reoxygenation model [43].…”

“…Levels of Fe 2+ , ROS, MDA and GSH in differently-treated chondrocytes or cartilage tissues of rat knee joints were measured using kits (E-BC-K773-M, E-BC-K138-F, E-BC-K025-M and E-BC-K030-M; Elabscience Biotechnology, Wuhan, Hubei, China) [19,25] following instructions. The results were expressed as percentage (%), with the corresponding control group for each experiment set as 100%.…”

“…The membranes were blocked with 5% skim milk for 1 h at room temperature, and probed with primary antibodies (Abcam) against ACSL4 (ab155282), METTL3 (ab195352), and β-actin (ab8226) at 4°C overnight and with HRP-labelled secondary antibody goat anti-rabbit IgG (H&L) (ab6721, Abcam) at room temperature for 1 h. ECL reagent was used to visualize the immunoblots. Band intensities were quanti ed using Image Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA), normalized to β-actin [19].…”

“…Besides, acyl-CoA synthetase long-chain family member 4 (ACSL4), a member of the long chain family of acyl-CoA synthetase proteins, functions as a critical regulator in ferroptosis [16][17][18]. ACSL4 protein expression is prominently elevated in the IL-1β-stimulated human chondrocytes, while ACSL4 knockdown neutralizes the role of IL-1β in increasing the lactic acid dehydrogenase release, cell viability, levels of reactive oxygen species (ROS), malondialdehyde (MDA) and Fe 2+ and declining the glutathione (GSH) levels in the human chondrocytes [19]. On the basis of these experimental results, we hypothesized that BMSC-Exos might inhibit the ferroptosis of chondrocytes and consequently delay the progression of OA, which was associated with regulation of the METTL3-m6A-ACSL4 axis.…”

SP1‑mediated ADAMTS5 transcription promotes IL‑1β‑induced chondrocyte injury via Wnt/β‑catenin pathway in osteoarthritis

“…These findings offer new evidence for ADAMTS5 as a potential target for OA therapy. The transcription factor SP1 has been shown to mediate OA progression through promoting chondrocyte injury (21,22). Notably, SP1 can regulate the expression of genes that have GC-rich promoters, thereby mediating numerous cellular functions, including cell proliferation, apoptosis and differentiation (31).…”