Suramin enhances chondrogenic properties by regulating the p67phox/PI3K/AKT/SOX9 signalling pathway

Bone & Joint Research

article

Zi-Miao Liu¹  Po-Chih Shen¹  Cheng-Chang Lu¹  Shih-Hsiang Chou¹  Yin-Chun Tien¹ 

[1] Department of Orthopedics, Kaohsiung Medical University Hospital, Kaohsiung Medical University

关键词: Suramin;    Reactive oxygen species;    NADPH oxidase;    p67phox;    Chondrocyte redifferentiation;    chondrocytes;    Col2a1;    western blot;    reverse transcriptase-polymerase chain reaction;    mRNA;    RNA;    staining;    phosphate;    collagen;    Alcian blue;   

DOI  :  10.1302/2046-3758.1110.BJR-2022-0013.R2

学科分类：骨科学

来源: British Editorial Society Of Bone And Joint Surgery

【 摘 要 】

AimsAutologous 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.MethodsPorcine 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.ResultsSuramin 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.ConclusionSuramin could redifferentiate dedifferentiated chondrocytes dependent on p67phox activation, which is mediated by the PI3K/AKT/SOX9 signalling pathway.

【 授权许可】

CC BY-NC