HAPLN1 Exhibits Dual Effects: Facilitating Extracellular Matrix Restoration while Enhancing Inflammatory Mediator...

Hyaluronan and proteoglycan link protein 1 (HAPLN1) secreted by fibroblast-like synoviocytes (FLSs) plays a critical role in the pathological process of inflammatory arthritis. This study aimed to investigate the impact and underlying mechanisms of HAPLN1 in an inflamed chondrocyte model. IL-1β-treated SW1353 chondrocytes were exposed to recombinant HAPLN1 (rHAPLN1). A transwell coculture system was used to assess interactions between the chondrocytes and FLSs transfected with the HAPLN1 overexpression plasmid. Cell viability and proliferation were evaluated using cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay. RNA sequencing and bioinformatic analyses, including the identification of differentially expressed genes (DEGs), gene functional enrichment and protein-protein interaction analyses were conducted to explore the potential mechanisms. Our fundings revealed that HAPLN also increased the levels of inflammatory mediators (TNF-α, IL-6, MMP1, MMP3, MMP9, and ADAMTS-5) in the inflamed chondrocytes, suggesting its pro-inflammatory effect. However, HAPLN1 enhanced cell viability, and the expression of Collagen II and TGF-β, the restoration effect of extracellular matrix (ECM). Bioinformatics analysis suggested the upregulated DEGs were enriched in the inflammatory processes, and the pathways related to ECM, TNF and IL-6 were activated in inflamed chondrocytes following rHAPLN1 exposure. Additionally, the protein-protein interaction networks indicated that HAPLN1 interacts with the molecules related to ECM, inflammation, and PI3K/AKT/mTOR pathway. Western blot assay further verified that the phosphorylation of PI3K, AKT, and mTOR was elevated in the rHAPLN1-treated chondrocytes. Our findings highlight the dual effects of HAPLN1 on the arthritic chondrocytes, which may be associated with the activation of the PI3K/AKT/mTOR pathway.