Background Recent studies have demonstrated that hyaluronan (HA) suppresses inflammatory cytokine-induced catabolic proteases such as member of a disintegrin and metalloproteinase with thrombospondin type1 motifs (ADAMTS), or matrix metalloproteinases (MMPs)1). However little is known their mechanical stress-induced change and/or effects of HA on these protease expressions. We previously reported that runt-related transcription factor 2 (RUNX-2) has an important role in regulation of mechanical stress-induced expression of ADAMTS aggrecanases2). In addition, it has been suggested that inflammatory cytokines such as IL-1β also play an important role on the expression of ADAMTSs or MMPs3).
Objectives To investigate the mechanism that HA inhibits mechanical stress-induced expression of catabolic proteases by chondrocytes.
Methods Normal Human Articular Chondrocytes from knee joint were purchased from Lonza (Walkersville, MD, USA). High molecular weight HA (2700kDa) was purchased from Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). Cells were cultured at 37°C, and were seeded onto type II collagen coated stretch chambers. They were transferred to serum-free medium with or without HA (1.0mg/ml) for 12h before cyclic tensile strain (CTS), and a uni-axial CTS (0.5 Hz, 10% elongation) was applied for 30 min using the ST-140 mechanical stretch system(STREX, Osaka, Japan). RNA was isolated at 1, 6, 12, 24h after CTS, reverse transcribed, and the expression of ADAMTS-4, -5, -9, MMP-3, -13, and RUNX-2 were analyzed by real-time PCR. The concentration of IL-1βin the supernatant was measured using ELISA.
Results CTS induced the expression of ADAMTS-4, -9, MMP-13 at 24h, and MMP-3 at 6h after CTS in control samples. In HA treated samples, the expression of ADAMTS-4, -9, MMP-13, MMP-3 were significantly downregulated. ADAMTS-5 gene expression was biphasic (1h and 12h after CTS) in control samples, and only the second peak (cytokine dependent peak) was reduced in HA treated samples. There was no significant difference in the expression of RUNX-2 between both samples. The concentration of IL-1β in the supernatant increased in control samples at 6h after CTS, but decreased in HA treated samples.
Conclusions HA treatment suppressed the mechanical stress-induced expression of ADAMTS-4, -9, MMP-3, -13 almost completely, and the expression of ADAMTS-5 partially, presumably by inhibition of IL-1β expression but not through RUNX-2 pathways. These data suggest the clinical chondro-protective effects of HA against mechanical stress-induced cartilage degeneration.
Yatabe T, Mochizuki S, Takizawa M, Chijiiwa M, Okada A, et al. Hyaluronan inhibits expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic chondrocytes. Ann Rheum Dis 2009;68:1051-1058
Tetsunaga T, Nishida K, Furumatsu T, Naruse K, Hirohata S, et al. Regulation of mechanical stress-induced MMP-13 and ADAMTS-5 expression by RUNX-2 transcriptional factor in SW1353 chondrocyte-like cells. Osteoarthritis Cartilage 2011;19:222-232
Julovi SM, Yasuda T, Shimizu M, Hiramitsu T, Nakamura T, et al. Inhibition of interleukin-1β-stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage. Arthritis Rheum 2004;50:516-525
Disclosure of Interest None Declared
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