Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro
Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro
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DOI：
10.1042/0264-6021:3440061
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发表时间：
1999-11-15
影响因子：
4.1
通讯作者：
Caterson, B
中科院分区：
文献类型：
--
作者：
Little, CB;Flannery, CR;Caterson, B
研究了体外正常和骨关节炎（OA）关节软骨中聚集蛋白聚糖酶与基质金属蛋白酶（MMP）酶活性在聚集蛋白聚糖降解中的重要性，以进一步了解这两种酶活性在软骨退变发病机制中聚集蛋白聚糖催化剂的潜在作用。猪和牛的关节软骨保持在外植体培养长达20天的存在或不存在的分解代谢刺激视黄酸，白细胞介素-l或肿瘤坏死因子-a。使用比色测定法将蛋白聚糖从软骨的释放测量为糖胺聚糖（GAG）释放。蛋白聚糖降解产物（释放到培养基中和保留在软骨基质内）的分析通过蛋白质印迹法进行，所述蛋白质印迹法使用对由聚集蛋白聚糖核心蛋白（IGD）的球间结构域的聚集蛋白聚糖酶和MMP相关催化剂产生的N-和C-末端新表位特异性的抗体。进行了测定这些相同培养物中MMP-3和MMP-13的mRNA表达的研究。这些分析表明，所有三种分解代谢剂在4天内刺激> 80%的GAG从关节软骨释放。GAG释放的程度对应于聚集蛋白聚糖酶产生的聚集蛋白聚糖催化剂释放到介质中并保留在软骨内的增加。重要的是，没有证据表明MMP-产生的聚集蛋白聚糖代谢物释放到培养基中，也没有证据表明MMP-产生的催化剂在这些相同培养物的组织内积累。检测已知能够降解聚集蛋白聚糖IGD的两种MMP（MMP-3和MMP-13）的mRNA的表达。然而，这些MMPs的表达增加与聚集蛋白聚糖降解无关。分析使用猪软骨，培养或没有分解代谢刺激12小时至20天，表明IGD的聚集蛋白聚糖酶的主要裂解是负责释放聚集蛋白聚糖代谢产物在早期和晚期的时间点的文化。晚期OA人关节软骨样品的培养物表明，与正常猪或牛软骨相比，在不存在分解代谢刺激的情况下，聚集蛋白聚糖酶活性上调。此外，即使在这种晚期退行性软骨中，聚集蛋白聚糖酶而不是MMP活性负责从软骨中释放大部分聚集蛋白聚糖。这项研究表明，在体外对分解代谢刺激的反应中，聚集蛋白聚糖从正常和OA软骨中的释放涉及聚集蛋白聚糖酶而不是MMPs的初级切割。
The importance of aggrecanase versus matrix metalloproteinase (MMP) enzymic activities in the degradation of aggrecan in normal and osteoarthritic (OA) articular cartilage in vitro was studied in order to further our understanding of the potential role of these two enzyme activities in aggrecan catabolism during the pathogenesis of cartilage degeneration. Porcine and bovine articular cartilage was maintained in explant culture for up to 20 days in the presence or absence of the catabolic stimuli retinoic acid, interleukin-l or tumour necrosis factor-a. Release of proteoglycan from cartilage was measured as glycosaminoglycan (GAG) release using a colorimetric assay. Analysis of proteoglycan degradation products, both released into culture media and retained within the cartilage matrix, was performed by Western blotting using antibodies specific for the N- and C-terminal neoepitopes generated by aggrecanase- and MMP-related catabolism of the interglobular domain of the aggrecan core protein (IGD), In addition, studies determining the mRNA expression for MMP-3 and MMP-13 in these same cultures were undertaken. These analyses indicated that all three catabolic agents stimulated the release of > 80% of the GAG from the articular cartilage over 4 days. The degree of GAG release corresponded to an increase in aggrecanase-generated aggrecan catabolites released into the media and retained within the cartilage. Importantly, there was no evidence for the release of MMP-generated aggrecan metabolites into the medium, nor the accumulation of MMP-generated catabolites within the tissue in these same cultures. Expression of the mRNAs for two MMPs known to be capable of degrading the aggrecan IGD, MMP-3 and MMP-13, was detected. However, increased expression of these MMPs was not correlated with aggrecan degradation. Analyses using porcine cartilage, cultured with or without catabolic stimulation for 12 h to 20 days, indicated that primary cleavage of the IGD by aggrecanase was responsible for release of aggrecan metabolites at both the early and late time points of culture. Cultures of late-stage OA human articular cartilage samples indicated that aggrecanase activity was upregulated in the absence of catabolic Stimulation when compared with normal porcine or bovine cartilage. In addition, even in this late-stage degenerate cartilage, aggrecanase and not MMP activity was responsible for the release of the majority of aggrecan from the cartilage. This study demonstrates that the release of aggrecan from both normal and OA cartilage in, response to catabolic stimulation in vitro involves a primary cleavage by aggrecanase and not MMPs.