Funato Sakie, Yasuhara Rika, Yoshimura Kentaro, Miyamoto Yoichi, Kaneko Kotaro, Suzawa Tetsuo, Chikazu Daichi, Mishima Kenji, Baba Kazuyoshi, Kamijo Ryutaro
Department of Biochemistry, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
Department of Prosthodontics, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
Cell Tissue Res. 2017 Apr;368(1):135-144. doi: 10.1007/s00441-016-2551-2. Epub 2017 Jan 9.
Osteoarthritis is a degenerative joint disease caused by excessive death of chondrocytes and loss of the extracellular matrix (ECM) in articular cartilage. We previously reported that reactive oxygen species (ROS) generated by the NADPH oxidase (NOX) isoform NOX-2 are involved in chondrocyte death induced by interleukin-1β (IL-1β). In this study, we investigate the role of NOX-2 in the production and degradation of ECM by chondrocytes. Although IL-1β lowered the mRNA expression of type II collagen (Col2a1) and aggrecan (Acan) in mouse chondrocyte-like ATDC5 cells, RNA silencing of Nox2 did not change the mRNA expression of these major components of the ECM of cartilage. Hence, NOX-2 is not involved in the IL-1β-induced suppression of ECM production. On the other hand, the NOX inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), the ROS scavenger N-acetylcysteine and an antisense oligodeoxynucleotide for Nox2 prevented the loss of proteoglycan induced by IL-1β in highly differentiated ATDC5 cells. Furthermore, AEBSF did not affect the expression of hyaluronidase-1 and -2, whereas it suppressed hyaluronidase activity in culture medium. IL-1β-induced intra- and extracellular acidification was also suppressed by AEBSF, as was the antisense oligodeoxynucleotide for Nox2. Since hyaluronidase activity is known to be higher under acidic conditions, NOX-2 probably contributes to ECM loss by the activation of hyaluronidase through acidification.
骨关节炎是一种退行性关节疾病，由软骨细胞过度死亡和关节软骨中细胞外基质（ECM）丧失所致。我们之前报道过，烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶（NOX）亚型NOX-2产生的活性氧（ROS）参与白细胞介素-1β（IL-1β）诱导的软骨细胞死亡。在本研究中，我们调查了NOX-2在软骨细胞产生和降解ECM中的作用。虽然IL-1β降低了小鼠软骨样ATDC5细胞中II型胶原（Col2a1）和聚集蛋白聚糖（Acan）的mRNA表达，但Nox2的RNA沉默并未改变软骨ECM这些主要成分的mRNA表达。因此，NOX-2不参与IL-1β诱导的ECM产生抑制。另一方面，NOX抑制剂4-（2-氨基乙基）苯磺酰氟（AEBSF）、ROS清除剂N-乙酰半胱氨酸和针对Nox2的反义寡脱氧核苷酸可防止高分化ATDC5细胞中IL-1β诱导的蛋白聚糖丢失。此外，AEBSF不影响透明质酸酶-1和-2的表达，却抑制了培养基中的透明质酸酶活性。AEBSF还抑制了IL-1β诱导的细胞内和细胞外酸化，针对Nox2的反义寡脱氧核苷酸也有同样作用。由于已知酸性条件下透明质酸酶活性更高，NOX-2可能通过酸化激活透明质酸酶，从而导致ECM丢失。
