Transient receptor potential ankyrin 1 (TRPA1) as a factor and drug target in osteoarthritis: TRPA1 mediates fibroblast growth factor 2 expression in chondrocytes

Abstract

Purpose: Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, which is known to be involved in nociception and neurogenic inflammation. TRPA1 is widely expressed in neurons, and according to more recent findings, also in non-neuronal cells, including synoviocytes and keratinocytes. TRPA1 acts as a chemosensor of pungent environmental compounds, but it is also activated by agents formed endogenously in inflammatory and hypoxic conditions such as those found in osteoarthritic joints. We have recently shown in monosodium iodoacetate-induced experimental osteoarthritis that TRPA1 activation mediates inflammation, cartilage degradation and pain (Moilanen et al. Osteoarthritis Cartilage 2015). We have also shown that TRPA1 is functionally expressed in human chondrocytes, where it mediates inflammatory effects (Nummenmaa et al. Arthritis Res Ther 2016). Further, we found that anti-inflammatory drugs dexamethasone and aurothiomalate downregulate the expression of TRPA1 in human chondrocytes (Nummenmaa et al. RMD Open 2017), revealing TRPA1 as a potential mediator and drug target in osteoarthritis. Fibroblast growth factor 2 (FGF-2) belongs to the fibroblast growth factor family, which is a large group of molecules involved in the regulation of connective tissue development and metabolism. FGF-2 is found in the synovial fluid and cartilage of osteoarthritis patients, and has previously been associated with the pathogenesis of osteoarthritis. In the present study, we investigated the functional consequences of TRPA1 activation in chondrocytes focusing in particularly on the expression of FGF-2. Methods: Chondrocytes were isolated from cartilage tissue from wild type (WT) and TRPA1 deficient (knock-out, KO) mice and cultured in the absence or presence of IL-1β. Total RNA was isolated and genome-wide expression analysis was carried out with next generation RNA sequencing (NGS) method with Illumina HiSeq2500 at the Institute for Molecular Medicine Finland. Results from the RNA sequencing analysis were verified by quantitative RT-PCR. Primary human OA chondrocytes were isolated from cartilage samples obtained from patients undergoing knee replacement surgery. Chondrocytes were cultured in the presence of the TRPA1-inducing cytokine IL-1β alone and together with the selective TRPA1 antagonist HC-030031. For experiments investigating the effects of FGF-2 in chondrocytes, the cells were cultured in the presence of FGF-2. Expression of FGF-2 subsequent to TRPA1 activation was measured by quantitative RT-PCR, and aggrecan, collagen II and MMP-enzymes by quantitative RT-PCR/immunoassay. Results: The expression of FGF-2 was significantly downregulated in chondrocytes from TRPA KO mice compared to WT mice. The downregulation of FGF-2 was verified by performing quantitative RT-PCR analysis on the sequenced samples, and on a larger set of corresponding samples. The results were confirmed using primary human OA chondrocytes. Pharmacological inhibition of TRPA1 with the selective antagonist HC-030031 downregulated FGF-2 expression in these cells. We further investigated the effects of FGF-2 in human OA chondrocytes, and found that FGF-2 upregulated the production of cartilage matrix degrading enzymes MMP-1 and MMP-13, and downregulated the expression of cartilage matrix components aggrecan and collagen II. Conclusions: The results revealed a hitherto unknown role for TRPA1 in the regulation of FGF-2 expression in chondrocytes. Further, FGF-2 was confirmed to have catabolic and anti-anabolic effects in human OA chondrocytes. Inhibition of TRPA1 and subsequent downregulation of FGF-2 could therefore have beneficial effects on the balance of catabolic and anabolic mediators within OA cartilage. These results together with our recent data discussed above support the concept of TRPA1 as a potential mediator and drug target in osteoarthritis.