Xie D L, Hui F, Meyers R, Homandberg G A
Department of Biochemistry, Rush Medical College at Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612-3864.
Arch Biochem Biophys. 1994 Jun;311(2):205-12. doi: 10.1006/abbi.1994.1228.
We have reported that three different Fn fragments (Fn-f) added to bovine articular cartilage cultured in serum-free DMEM cause marked elevation of proteoglycan (PG) degradation and release into the culture media. We report here that the PG release required the continual presence of Fn-f, that PG release still occurred when serum-free cultures were switched to bovine synovial fluid media, and that addition of recombinant IGF-1, TGF-beta, and recombinant interferon gamma to cultures did not affect Fn-f-mediated PG release. The Fn-f caused a 25-fold enhanced release of stromelysin-1 protein from cartilage by Day 1 and up to 120-fold by Day 3. The stromelysin form released was 43 kDa, the activated form of pro-stromelysin-1. This stromelysin form apparently played a major role in Fn-f-mediated PG release, since addition of Sepharose-bound anti-stromelysin-1 to cartilage cultures greatly slowed rates of PG release. Potential activators of pro-stromelysin-1, plasmin, and u-PA (urinary plasminogen activator), were also detected in conditioned media of Fn-f-treated cartilage. u-PA levels were increased in the presence of the Fn-f but by only a few fold. Addition of alpha-1-antiproteinase inhibitor, which can block enzymatic activity of u-PA, was found to inhibit about half the PG-releasing activity of the Fn-f. Levels of TIMP-1, the 30-kDa tissue inhibitor of metalloproteinases, which can inhibit stromelysin, doubled within 24 h when a Fn-f was added to culture. These data suggest that stromelysin-1 may be a major mediator of Fn-f-mediated PG release from cartilage.
我们曾报道，将三种不同的纤连蛋白片段（Fn-f）添加到在无血清DMEM中培养的牛关节软骨中，会导致蛋白聚糖（PG）降解显著增加，并释放到培养基中。我们在此报告，PG的释放需要Fn-f持续存在，当无血清培养物换成牛滑液培养基时PG仍会释放，并且向培养物中添加重组胰岛素样生长因子-1（IGF-1）、转化生长因子-β（TGF-β）和重组干扰素-γ不会影响Fn-f介导的PG释放。到第1天，Fn-f使软骨中基质金属蛋白酶-1（stromelysin-1）蛋白的释放增强了25倍，到第3天增强了120倍。释放的stromelysin形式为43 kDa，即前基质金属蛋白酶-1的活化形式。这种stromelysin形式显然在Fn-f介导的PG释放中起主要作用，因为向软骨培养物中添加琼脂糖结合的抗基质金属蛋白酶-1可大大减缓PG的释放速率。在Fn-f处理的软骨的条件培养基中还检测到了前基质金属蛋白酶-1、纤溶酶和尿激酶型纤溶酶原激活剂（u-PA）的潜在激活剂。在Fn-f存在的情况下，u-PA水平有所升高，但仅升高了几倍。发现添加可阻断u-PA酶活性的α-1抗蛋白酶抑制剂可抑制约一半的Fn-f介导的PG释放活性。金属蛋白酶组织抑制剂-1（TIMP-1），一种30 kDa的金属蛋白酶组织抑制剂，当向培养物中添加Fn-f时，其水平在24小时内翻倍，而TIMP-1可抑制基质金属蛋白酶。这些数据表明，基质金属蛋白酶-1可能是Fn-f介导的软骨中PG释放的主要介质。
