对ADAMTS4和MT4-MMP的分析表明，二者均参与白细胞介素-1处理的牛软骨中的蛋白聚糖分解。
Analysis of ADAMTS4 and MT4-MMP indicates that both are involved in aggrecanolysis in interleukin-1-treated bovine cartilage.
作者信息
Patwari P, Gao G, Lee J H, Grodzinsky A J, Sandy J D
机构信息
Massachusetts Institute of Technology, Department of Electrical Engineering, Cambridge, MA 02139, USA.
出版信息
Osteoarthritis Cartilage. 2005 Apr;13(4):269-77. doi: 10.1016/j.joca.2004.10.023.
OBJECTIVE
To investigate the mechanism of aggrecanolysis in interleukin-1 (IL-1)-treated cartilage tissue by examining the time course of aggrecan cleavages and the tissue and medium content of membrane type 4-matrix metalloproteinases (MT4-MMP) and a disintegrin and metalloproteinase with thrombospondin type I motifs (ADAMTS)4.
METHODS
Articular cartilage explants were harvested from newborn bovine femoropatellar groove. The effects of IL-1 treatment with or without aggrecanase blockade were investigated by Western analysis of aggrecan fragment generation, ADAMTS4 species (p68 and p53), and MT4-MMP, as well as by realtime PCR (polymerase chain reaction) for ADAMTS4 and 5. Aggrecanase was blocked with mannosamine (ManN), an inhibitor of glycosylphosphatidylinositol anchor synthesis, and esculetin (EST), an inhibitor of MMP-1, MMP-3, and MMP-13 gene expression.
RESULTS
IL-1 treatment caused a major increase in MT4-MMP abundance in the tissue and medium. ADAMTS4 (p68) was abundant in fresh cartilage and this was retained in the tissue in untreated cartilage. IL-1 treatment for 6 days caused a marked loss of p68 from the cartilage and the appearance of p53 in the medium. Addition of either 1.35 mM ManN or 31-500 microM EST blocked IL-1-mediated aggrecanolysis and this was accompanied by nearly complete inhibition of the MT4-MMP increase, the p68 loss and the formation of p53. IL-1 treatment increased mRNA abundance for ADAMTS4 ( approximately 3-fold) and ADAMTS5 ( approximately 10-fold) but this was not accompanied by a marked change in enzyme protein abundance.
CONCLUSION
These studies support a central role for MT4-MMP in IL-1-induced cartilage aggrecanolysis and are consistent with the identification of p68 as the aggrecanase that cleaves within the CS2 domain, and of p53 as the aggrecanase that generates G1-NITEGE. Since the induction by IL-1 was not accompanied by marked changes in total ADAMTS4 protein, but rather in partial conversion of p68 to p53 and release of both from the tissue, we conclude that aggrecanolysis in this model system results from MT4-MMP-mediated processing of a resident pool of ADAMTS4 and release of the p68 and p53 from their normal association with the cell surface.
目的
通过检测聚集蛋白聚糖裂解的时间进程以及膜型4-基质金属蛋白酶（MT4-MMP）和含血小板反应蛋白基序的解聚素和金属蛋白酶（ADAMTS）4在组织及培养基中的含量，研究白细胞介素-1（IL-1）处理的软骨组织中聚集蛋白聚糖降解的机制。
方法
从新生牛股骨髌沟获取关节软骨外植体。通过对聚集蛋白聚糖片段生成、ADAMTS4种类（p68和p53）以及MT4-MMP进行蛋白质印迹分析，以及对ADAMTS4和5进行实时聚合酶链反应（PCR），研究有无聚集蛋白聚糖酶阻断情况下IL-1处理的效果。用甘露糖胺（ManN，糖基磷脂酰肌醇锚合成抑制剂）和七叶亭（EST，MMP-1、MMP-3和MMP-13基因表达抑制剂）阻断聚集蛋白聚糖酶。
结果
IL-1处理导致组织和培养基中MT4-MMP丰度显著增加。ADAMTS4（p68）在新鲜软骨中含量丰富，在未处理的软骨组织中也保持这种状态。IL-1处理6天导致软骨中p68显著丢失，培养基中出现p53。添加1.35 mM ManN或31 - 500 microM EST均可阻断IL-1介导的聚集蛋白聚糖降解，同时几乎完全抑制MT4-MMP增加、p68丢失以及p53形成。IL-1处理使ADAMTS4（约3倍）和ADAMTS5（约10倍）的mRNA丰度增加，但酶蛋白丰度无明显变化。
结论
这些研究支持MT4-MMP在IL-1诱导的软骨聚集蛋白聚糖降解中起核心作用，与将p68鉴定为在CS2结构域内裂解的聚集蛋白聚糖酶以及将p53鉴定为产生G1-NITEGE的聚集蛋白聚糖酶一致。由于IL-1诱导并未伴随ADAMTS4总蛋白的明显变化，而是p68部分转化为p53并二者均从组织中释放，我们得出结论，该模型系统中的聚集蛋白聚糖降解是由MT4-MMP介导处理驻留的ADAMTS4池以及p68和p53从其与细胞表面的正常结合中释放所致。
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