Kolling Institute of Medical Research, University of Sydney, and Royal North Shore Hospital, St. Leonards, New South Wales, Australia.
Arthritis Rheumatol. 2014 Jun;66(6):1525-36. doi: 10.1002/art.38401.
Levels of activated protein C (APC) are elevated in the synovial fluid of patients with osteoarthritis (OA), and increased APC levels are correlated with the levels of active matrix metalloproteinase 2 (MMP-2). This study sought to investigate whether APC is a relevant protein for activation of MMPs in the degradation of human OA cartilage, and to elucidate its mechanisms of action.
Human articular cartilage was cultured with or without interleukin-1α (IL-1α), in the presence or absence of APC or protein C, and an MMP or serine proteinase inhibitor. Aggrecan and collagen release and chondrocyte gene expression levels were quantified. Aggrecanase and MMP cleavage of aggrecan was examined with neoepitope-specific antibodies, and MMP activity was measured using gelatin zymography and fluorogenic peptide assay.
In human OA cartilage, APC induced aggrecan and collagen release, whereas in non-OA cartilage, costimulation with IL-1α was required. Inhibition of MMP activity reduced APC-induced cartilage proteolysis, and MMP-induced aggrecanolysis was confirmed by Western blotting. In cultures with APC alone, the activity of MMPs 2, 9, and 13 was significantly increased in OA cartilage, although APC could not directly activate MMPs 2 or 9. Expression of MMP1, MMP2, MMP9, MMP13, TIMP1, and TIMP3 was not altered by APC in OA cartilage. Human OA chondrocytes expressed messenger RNA for protein C, endothelial protein C receptor, thrombomodulin, and protease-activated receptor 1, but these were unaltered or down-regulated by APC. The induction of MMP activation and cartilage degradation by APC was dependent on its serine protease activity.
APC is a physiologically relevant activator of MMPs and cartilage breakdown in human OA. The effects of APC are dependent on its proteolytic activity and as-yet-undefined cell and/or cartilage matrix factors, and inhibition of this pathway may provide a novel therapeutic target to halt the progression of cartilage damage in OA.
在骨关节炎（OA）患者的滑液中，活化蛋白 C（APC）的水平升高，并且 APC 水平的升高与活性基质金属蛋白酶 2（MMP-2）的水平相关。本研究旨在探讨 APC 是否为 OA 软骨降解中 MMP 激活的相关蛋白，并阐明其作用机制。
用人白细胞介素-1α（IL-1α）培养关节软骨，有或无 APC 或蛋白 C，以及 MMP 或丝氨酸蛋白酶抑制剂。定量测定聚集蛋白聚糖和胶原的释放以及软骨细胞基因表达水平。用新表位特异性抗体检测聚集蛋白聚糖的聚集蛋白聚糖酶和 MMP 切割，并用明胶酶谱和荧光肽测定法测量 MMP 活性。
在人 OA 软骨中，APC 诱导聚集蛋白聚糖和胶原释放，而非 OA 软骨则需要 IL-1α 的共刺激。抑制 MMP 活性可减少 APC 诱导的软骨蛋白水解，并且通过 Western blot 证实了 MMP 诱导的聚集蛋白聚糖分解。在单独 APC 的培养物中，OA 软骨中 MMP2、9 和 13 的活性显著增加，尽管 APC 不能直接激活 MMP2 或 9。在 OA 软骨中，APC 未改变 MMP1、MMP2、MMP9、MMP13、TIMP1 和 TIMP3 的表达。人 OA 软骨细胞表达蛋白 C、内皮蛋白 C 受体、血栓调节蛋白和蛋白酶激活受体 1 的信使 RNA，但 APC 对其无改变或下调。APC 诱导 MMP 激活和软骨降解依赖于其丝氨酸蛋白酶活性。
APC 是人类 OA 中 MMP 和软骨降解的生理相关激活剂。APC 的作用取决于其蛋白水解活性以及尚未明确的细胞和/或软骨基质因素，抑制该途径可能为阻止 OA 中软骨损伤的进展提供新的治疗靶点。
