Takeda Yuki, Niki Yasuo, Fukuhara Yusuke, Fukuda Yoshitsugu, Udagawa Kazuhiko, Shimoda Masayuki, Kikuchi Toshiyuki, Kobayashi Shu, Harato Kengo, Miyamoto Takeshi, Matsumoto Morio, Nakamura Masaya
Department of Orthopaedic Surgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.
Department of Pathology, School of Medicine, Keio University, Tokyo, Japan.
BMC Musculoskelet Disord. 2021 Mar 1;22(1):238. doi: 10.1186/s12891-021-04095-x.
Mechanical overload applied on the articular cartilage may play an important role in the pathogenesis of osteoarthritis. However, the mechanism of chondrocyte mechanotransduction is not fully understood. The purpose of this study was to assess the effects of compressive mechanical stress on interleukin-1 receptor (IL-1R) and matrix-degrading enzyme expression by three-dimensional (3D) cultured ATDC5 cells. In addition, the implications of transient receptor potential vanilloid 4 (TRPV4) channel regulation in promoting effects of compressive mechanical loading were elucidated.
ATDC5 cells were cultured in alginate beads with the growth medium containing insulin-transferrin-selenium and BMP-2 for 6 days. The cultured cell pellet was seeded in collagen scaffolds to produce 3D-cultured constructs. Cyclic compressive loading was applied on the 3D-cultured constructs at 0.5 Hz for 3 h. The mRNA expressions of a disintegrin and metalloproteinases with thrombospondin motifs 4 (ADAMTS4) and IL-1R were determined with or without compressive loading, and effects of TRPV4 agonist/antagonist on mRNA expressions were examined. Immunoreactivities of reactive oxygen species (ROS), TRPV4 and IL-1R were assessed in 3D-cultured ATDC5 cells.
In 3D-cultured ATDC5 cells, ROS was induced by cyclic compressive loading stress. The mRNA expression levels of ADAMTS4 and IL-1R were increased by cyclic compressive loading, which was mostly prevented by pyrollidine dithiocarbamate. Small amounts of IL-1β upregulated ADAMTS4 and IL-1R mRNA expressions only when combined with compressive loading. TRPV4 agonist suppressed ADAMTS4 and IL-1R mRNA levels induced by the compressive loading, whereas TRPV4 antagonist enhanced these levels. Immunoreactivities to TRPV4 and IL-1R significantly increased in constructs with cyclic compressive loading.
Cyclic compressive loading induced mRNA expressions of ADAMTS4 and IL-1R through reactive oxygen species. TRPV4 regulated these mRNA expressions, but excessive compressive loading may impair TRPV4 regulation. These findings suggested that TRPV4 regulates the expression level of IL-1R and subsequent IL-1 signaling induced by cyclic compressive loading and participates in cartilage homeostasis.
施加于关节软骨的机械性过载可能在骨关节炎的发病机制中起重要作用。然而，软骨细胞机械转导的机制尚未完全明确。本研究的目的是评估压缩机械应力对三维（3D）培养的ATDC5细胞白细胞介素-1受体（IL-1R）和基质降解酶表达的影响。此外，还阐明了瞬时受体电位香草酸受体4（TRPV4）通道调节在压缩机械负荷促进作用中的意义。
将ATDC5细胞培养于含有胰岛素-转铁蛋白-硒和骨形态发生蛋白-2的生长培养基的藻酸盐珠中6天。将培养的细胞团接种于胶原支架上以制备3D培养构建体。对3D培养构建体施加0.5 Hz的循环压缩负荷3小时。测定有无压缩负荷时具有血小板反应蛋白基序的解聚素和金属蛋白酶4（ADAMTS4）及IL-1R的mRNA表达，并检测TRPV4激动剂/拮抗剂对mRNA表达的影响。评估3D培养的ATDC5细胞中活性氧（ROS）、TRPV4和IL-1R的免疫反应性。
在3D培养的ATDC5细胞中，循环压缩负荷应力诱导ROS产生。循环压缩负荷增加了ADAMTS4和IL-1R的mRNA表达水平，而吡咯烷二硫代氨基甲酸盐大多可阻止这种增加。仅在与压缩负荷联合时，少量白细胞介素-1β上调ADAMTS4和IL-1R的mRNA表达。TRPV4激动剂抑制压缩负荷诱导的ADAMTS4和IL-1R mRNA水平，而TRPV4拮抗剂则增强这些水平。在施加循环压缩负荷的构建体中，TRPV4和IL-1R的免疫反应性显著增加。
循环压缩负荷通过活性氧诱导ADAMTS4和IL-1R的mRNA表达。TRPV4调节这些mRNA表达，但过度的压缩负荷可能损害TRPV4调节。这些发现提示TRPV4调节循环压缩负荷诱导的IL-1R表达水平及随后的IL-1信号传导，并参与软骨内环境稳定。
