Suramin enhances chondrogenic properties by regulating the p67^phox/PI3K/AKT/SOX9 signalling pathway

Abstract

Aims Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism. Methods Porcine chondrocytes were treated with vehicle or various doses of suramin. The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN); COL1A1; COL10A1; SRY-box transcription factor 9 (SOX9); nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX); interleukin (IL)-1β; tumour necrosis factor alpha (TNFα); IL-8; and matrix metallopeptidase 13 (MMP-13) in chondrocytes at both messenger RNA (mRNA) and protein levels was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. In addition, the supplementation of suramin to redifferentiation medium for the culture of expanded chondrocytes in 3D pellets was evaluated. Glycosaminoglycan (GAG) and collagen production were evaluated by biochemical analyses and immunofluorescence, as well as by immunohistochemistry. The expression of reactive oxygen species (ROS) and NOX activity were assessed by luciferase reporter gene assay, immunofluorescence analysis, and flow cytometry. Mutagenesis analysis, Alcian blue staining, reverse transcriptase polymerase chain reaction (RT-PCR), and western blot assay were used to determine whether p67phox was involved in suramin-enhanced chondrocyte phenotype maintenance. Results Suramin enhanced the COL2A1 and ACAN expression and lowered COL1A1 synthesis. Also, in 3D pellet culture GAG and COL2A1 production was significantly higher in pellets consisting of chondrocytes expanded with suramin compared to controls. Surprisingly, suramin also increased ROS generation, which is largely caused by enhanced NOX (p67phox) activity and membrane translocation. Overexpression of p67phox but not p67phoxAD (deleting amino acid (a.a) 199 to 212) mutant, which does not support ROS production in chondrocytes, significantly enhanced chondrocyte phenotype maintenance, SOX9 expression, and AKT (S473) phosphorylation. Knockdown of p67phox with its specific short hairpin (sh) RNA (shRNA) abolished the suramin-induced effects. Moreover, when these cells were treated with the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) inhibitor LY294002 or shRNA of AKT1, p67phox-induced COL2A1 and ACAN expression was significantly inhibited. Conclusion Suramin could redifferentiate dedifferentiated chondrocytes dependent on p67phox activation, which is mediated by the PI3K/AKT/SOX9 signalling pathway. Cite this article: Bone Joint Res 2022;11(10):693–708.

Dihydrocaffeic acid improves IL-1β-induced inflammation and cartilage degradation via inhibiting NF-κB and MAPK signalling pathways

“…The most crucial mediators of chondrocytes metabolism are collagen II, aggrecan, and SOX9. 23 , 24 Therefore, we investigated whether DHCA can restore the expression of aforementioned mediators in chondrocytes activated with IL-1β. As shown in Figures 3a and 3b , IL-1β stimulation drastically reduced the expression of aggrecan, collagen II, and SOX9 at both mRNA and protein levels in mouse chondrocytes.…”

Identification of cell-specific epigenetic patterns associated with chondroitin sulfate treatment response in an endemic arthritis, Kashin-Beck disease

“…Next, 500 ng of the extracted RNA was reverse transcribed into complementary DNA according to the manufacturer’s protocol of Maxima H Minus cDNA Synthesis Master Mix with dsDNase (Thermo Fisher Scientific). 36 Then, 25 μl of reaction volume was used for the qRT-PCR on CFX96 Real-Time PCR System (Bio-Rad Laboratories, USA) using SYBR Green qPCR Master Mix (Bimake.com, USA) to detect expression levels of DMR-related genes. 37 All reactions were carried out at an annealing temperature of 60°C; cycling conditions were: 95°C-30 s (one cycle), 95°C-5 s, followed by 60°C-30 s (40 cycles).…”

Identification and experimental validation of key extracellular proteins as potential targets in intervertebral disc degeneration

“…15 Furthermore, the aberrant expression of various proteases in degenerated disc tissue and their roles in ECM proteolysis have been reported, representing potential biomarkers or therapeutic targets for IDD. 12,16 Bioinformatics analysis of microarray transcription profiles is a novel approach for exploring disease pathogenesis, identifying disease biomarkers, and discovering therapeutic targets. 4,16 The Gene Expression Omnibus (GEO) database contains microarray expression profiling data for many diseases.…”

“…12,16 Bioinformatics analysis of microarray transcription profiles is a novel approach for exploring disease pathogenesis, identifying disease biomarkers, and discovering therapeutic targets. 4,16 The Gene Expression Omnibus (GEO) database contains microarray expression profiling data for many diseases. 17 We downloaded the IDD patient IVD tissue microarray gene expression profiling dataset (GSE23130) from the GEO database and used R software to screen for differentially expressed genes (DEGs) between IVD tissue samples of early and late IDD.…”

“… 15 Furthermore, the aberrant expression of various proteases in degenerated disc tissue and their roles in ECM proteolysis have been reported, representing potential biomarkers or therapeutic targets for IDD. 12 , 16 …”