IRF1 governs the expression of SMARCC1 via the GCN5-SETD2 axis and actively engages in the advancement of osteoarthritis

Abstract

Background: Osteoarthritis (OA) is a degenerative joint disease characterized by the breakdown of joint cartilage and underlying bone. Macrophages are a type of white blood cell that plays a critical role in the immune system and can be found in various tissues, including joints. Research on the relationship between OA and macrophages is essential to understand the mechanisms underlying the development and progression of OA.

Objective: This study was performed to analyze the functions of the IRF1-GCN5-SETD2-SMARCC1 axis in osteoarthritis (OA) development.

Methods: A single-cell RNA sequencing (scRNA-seq) dataset, was subjected to a comprehensive analysis aiming to identify potential regulators implicated in the progression of osteoarthritis (OA). In order to investigate the role of IRF1 and SMARCC1, knockdown experiments were conducted in both OA-induced rats and interleukin (IL)-1β-stimulated chondrocytes, followed by the assessment of OA-like symptoms, secretion of inflammatory cytokines, and polarization of macrophages. Furthermore, the study delved into the identification of aberrant epigenetic modifications and functional enzymes responsible for the regulation of SMARCC1 by IRF1. To evaluate the clinical significance of the factors under scrutiny, a cohort comprising 13 patients diagnosed with OA and 7 fracture patients without OA was included in the analysis.

Results: IRF1 was found to exert regulatory control over the expression of SMARCC1, thus playing a significant role in the development of osteoarthritis (OA). The knockdown of either IRF1 or SMARCC1 disrupted the pro-inflammatory effects induced by IL-1β in chondrocytes, leading to a mitigation of OA-like symptoms, including inflammatory infiltration, cartilage degradation, and tissue injury, in rat models. Additionally, this intervention resulted in a reduction in the predominance of M1 macrophages both in vitro and in vivo. Significant epigenetic modifications, such as abundant H3K27ac and H3K4me3 marks, were observed near the SMARCC1 promoter and 10 kb upstream region. These modifications were attributed to the recruitment of GCN5 and SETD2, which are functional enzymes responsible for these modifications. Remarkably, the overexpression of either GCN5 or SETD2 restored SMARCC1 expression in rat cartilages or chondrocytes, consequently exacerbating the OA-like symptoms.

Conclusion: This research postulates that the transcriptional activity of SMARCC1 can be influenced by IRF1 through the recruitment of GCN5 and SETD2, consequently regulating the H3K27ac and H3K4me3 modifications in close proximity to the SMARCC1 promoter and 10 kb upstream region. These modifications, in turn, facilitate the M1 skewing of macrophages and contribute to the progression of osteoarthritis (OA).

The translational potential of this article: The study demonstrated that the regulation of SMARCC1 by IRF1 plays a crucial role in the development of OA. Knocking down either IRF1 or SMARCC1 disrupted the pro-inflammatory effects induced by IL-1β in chondrocytes, leading to a mitigation of OA-like symptoms in rat models. These symptoms included inflammatory infiltration, cartilage degradation, and tissue injury. These findings suggest that targeting the IRF1-SMARCC1 regulatory axis, as well as the associated epigenetic modifications, could potentially be a novel approach in the development of OA therapies, offering new opportunities for disease management and improved patient outcomes.

Keywords: Epigenetics; GCN5; IRF1-SMARCC1 axis; Macrophages; Osteoarthritis; SETD2.

Graphical abstract

Figure 1

IRF1 is a regulon of SMARCC1 that potentially participates in the onset and development of OA. A-B, cell types differing in control and CCA groups in GSE152805 dataset; C, DEGs between OA- and non-OA-chondrocytes and the volcano plots generated by the findmarker function wilcoxon test; and to find differential genes and plot volcanoes; D, gene variation analysis to screen relevant signaling pathways in two types of macrophages based on the Hallmarker gene set; E-F, differential expression levels of IRF1 and SMARCC1 in OA; G, the regulatory role of IRF1 on SMARCC1 confirmed by ChIP-seq analysis.

Figure 2

Knockdown of SMARCC1 or IRF1 impairs the pro-inflammatory effect of IL-1β on chondrocytes. A-B, mRNA and protein expression of SMARCC1 and IRF1 in IL-1β-stimulated chondrocytes detected by RT-qPCR and WB analysis; C-D, mRNA and protein expression of SMARCC1 and IRF1 in IL-1β-stimulated chondrocytes after SMARCC1 or IRF1 treatment detected by RT-qPCR and WB analysis; E-F, viability and proliferation of the chondrocytes evaluated by CCK-8 and EdU labeling assays; G, number of apoptotic bodies in the chondrocytes determined by TUNEL assay; H–I, mRNA and protein levels of ECM production- or degradation-related factors in the chondrocytes determined by RT-qPCR and WB analysis; J, production of inflammatory factors IL-6 and TNF-α in the culture medium of chondrocytes detected by ELISA kits. Three biological replicates were performed. Differences were analyzed by the one- or two-way ANOVA followed by Tukey's multiple comparison test. **P < 0.01, ***P < 0.001.

Figure 3

IRF1 regulates M1-type polarization of macrophages via SMARCC1. A, a diagram for the co-culture system of M0 macrophages (PMA-treated THP-1 monocytes) and IL-1β-treated chondrocytes; B–C, mRNA and protein levels of M1 marker iNOS and M2 marker Arg1 in the macrophages determined by RT-qPCR or WB analysis; D, the proportions of M1 macrophages (CD86⁺) and M2 macrophages (CD206⁺) in the co-culture system analyzed by flow cytometry; E, chemotactic migration of macrophages in the co-culture system determined by Transwell assay; F, expression of M1-type cytokines (TNF-α and IL-23) and M2-type cytokines (IL-10 and TGF-β) in the culture medium analyzed by ELISA kits; G, proportion of M1/M2 macrophages (CD86/CD206) analyzed by dual-label immunofluorescence staining. Three biological replicates were performed. Differences were analyzed by the one- or two-way ANOVA followed by Tukey's multiple comparison test. **P < 0.01, ***P < 0.001.

Figure 4

Knockdown of SMARCC1 or IRF1 alleviates OA-like symptoms in rats. A, a diagram for rat model establishment and treatment; B, OA-like symptoms in rats determined by Safranin O staining and Mankin scoring; C, concentrations of TNF-α, IL-12, IL-10, MCP-1 and TGF-β in the synovial fluid examined by ELISA kits; D, cartilage degeneration in rats examined by toluidine blue staining; E ∼ F, pathological changes and inflammatory cell infiltration in the cartilage tissue examined by HE and safarin and fast green staining; G, expression of the M1-type protein iNOS and M2-type protein Arg1 in the cartilage tissue determined by immunofluorescence; H, apoptotic cells in the cartilage tissue examined by TUNEL assay; I, collagen content in the cartilage examined using a collagen deposition kit. In each group, n = 6. In the graphs, each spot refers to a rat. Differences were analyzed by the one- or two-way ANOVA followed by Tukey's multiple comparison test. **P < 0.01, ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Figure 5

IRF1 recruits H3K27ac and H3K4me3 modifications to regulate SMARCC1 expression. A-B, enrichment of H3K27ac, H3K36me3, H3K4me1, and H3K4me3 modifications in the genome of 11 OA patients according to MACS2 and call 4 peaks analysis of the GSE112655 ChIP-seq dataset; C-D, binding relationships between H3K27ac and H3K4me3 and the promoter of SMARCC1 and its upstream 10 kb in rat cartilage or in the extracted chondrocyte determined by ChIP-qPCR; E-F, mRNA and protein levels of SMARCC1 and IRF1 in chondrocytes after MG149 or BRD9359 treatment detected by RT-qPCR and WB analysis. For cellular experiments, three biological replicates were performed. For animal studies, n = 6 in each group. Differences were analyzed by the one- or two-way ANOVA followed by Tukey's multiple comparison test. **P < 0.01, ***P < 0.001.

Figure 6

GCN5-SETD2 is involved in epigenetic regulation of SMARCC1 expression. A, RT-qPCR for SMARCC1 mRNA expression in chondrocytes after treatment of a library of commercial histone modification compounds; B–C, mRNA and protein levels of CBP, PCAF, GCN5, G9a, SETD2 in rat cartilage tissues and in the isolated chondrocytes examined by RT-qPCR and WB analysis; D, binding relationship of GCN5 and SETD2 with the SMARCC1 promoter and its upstream 10 kb in rat cartilage tissues and chondrocytes detected by ChIP-qPCR assay; E, construction of pGL3-E-Luc luciferase reporter vector containing the 10 kb sequence upstream of the promoter of SMARCC1 to examine the effect of GCN5 and SETD2 overexpression on the luciferase activity in 293T cells; F-G, mRNA and protein expression of SMARCC1 in chondrocytes after GCN5 and SETD2 overexpression determined by RT-qPCR and WB analysis. H, Fluorescence co-localization experiments confirmed the binding relationship between IRF1 and Gcn5 as well as SETD2. I, ChIP experiments validated the binding relationship of Gcn5 and Setd2 with Smarcc1 in chondrocytes following the knockdown of Irf1. For cellular experiments, three biological replicates were performed. For animal studies, n = 6 in each group. Differences were analyzed by the one- or two-way ANOVA followed by Tukey's multiple comparison test. **P < 0.01, ***P < 0.001.

Figure 7

Overexpression of GCN5 or SETD2 impairs the attenuating effect of sh-SMARCC1 on OA symptoms. A, a diagram for animal treatment; B, OA-like symptoms in rats determined by Safranin O staining and Mankin scoring; C, cartilage degeneration in rats examined by toluidine blue staining; D, pathological changes and inflammatory cell infiltration in the cartilage tissue examined by HE staining; E, expression of the M1-type protein iNOS and M2-type protein Arg1 in the cartilage tissue determined by immunofluorescence; F, apoptotic cells in the cartilage tissue examined by TUNEL assay; G, concentrations of TNF-α, IL-12, IL-10, MCP-1 and TGF-β in the synovial fluid examined by ELISA kits; H ∼ I, mRNA and protein levels of GCN5, SETD2, and SMARCC1 in rat cartilage tissues analyzed by RT-qPCR and WB analysis. In each group, n = 6. In the graphs, each spot refers to a rat. Differences were analyzed by the one- or two-way ANOVA followed by Tukey's multiple comparison test. **P < 0.01, ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Figure 8

SMARCC1 expression is significantly increased in cartilage tissue of clinical OA patients. A, expression of IRF1, GCN5, SETD2 and SMARCC1 in cartilage tissues from 13 patients with OA and another 7 patients without detected by RT-qPCR; B, correlations of the Mankin's score of OA patients with the detected IRF1, GCN5, SETD2 and SMARCC1 expression levels; C, correlation between IRF1, GCN5, SETD2 and SMARCC1 expression levels analyzed by Spearman correlation analysis. In panels A–C each point represents one subject. Differences were analyzed by the one- or two-way ANOVA followed by Tukey's multiple comparison test. **P < 0.01, ***P < 0.001.

Figure 9

The transcriptional activity of SMARCC1 can be influenced by IRF1 through the recruitment of GCN5 and SETD2, consequently regulating the H3K27ac and H3K4me3 modifications in close proximity to the SMARCC1 promoter.