ECM DEGRADATION BY CULTURED HUMAN MESANGIAL CELLS IS MEDIATED BY A PA/PLASMIN/MMP-2 CASCADE
ECM DEGRADATION BY CULTURED HUMAN MESANGIAL CELLS IS MEDIATED BY A PA/PLASMIN/MMP-2 CASCADE
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DOI：
10.1038/ki.1995.150
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发表时间：
1995-04-01
影响因子：
19.6
通讯作者：
SCHNAPER, HW
中科院分区：
文献类型：
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作者：
BARICOS, WH;CORTEZ, SL;SCHNAPER, HW
我们研究了纤溶酶原激活物/纤溶酶系统在细胞外基质(ECM)降解中的作用。ECM的降解(I-125释放到培养液中)依赖于外源纤溶酶原，与系膜细胞数量和加入的纤溶酶原数量成正比，并与培养液中纤溶酶的出现相一致。两种纤溶酶抑制剂α-2-抗纤溶酶(40mU/ml)和抑肽酶(216kIU/ml)可完全阻断细胞外基质的降解(P&0.001)，而基质金属蛋白酶的特异性抑制剂TIMP-1(40mU/ml)可部分抑制ECM的降解(-33+/-1.8%，P<；0.01)。在无纤溶酶原条件下培养的细胞培养上清液的酶谱分析显示，在系膜细胞和纤溶酶原存在的情况下，存在潜伏的基质金属蛋白酶-2(MMP2)，该酶在系膜细胞和纤溶酶原存在下转化为低分子量的活性形式。Northern分析显示，培养的人肾小球系膜细胞中存在组织型纤溶酶原激活物(TPA)、尿激酶型纤溶酶原激活物(UPA)、纤溶酶原激活物抑制物-1(PAI-1)和uPA受体(UPAR)的mRNA。体外培养的人肾小球系膜细胞培养上清液中存在uPA蛋白，显示PAI-1(1.2+/-0.1×10(-9)M)明显高于uPA(1.2+/-0.1×10(-12)M)和tPA(0.19+/-0.04×10(-9)M)。抗人tPA(-54+/-8.6%)或人uPA(-39+/-5.2%)的单抗(MAb)与同等数量的非特异性单抗(P<；0.01)处理的细胞相比，ECM的降解率降低。相反，抗人PAI-1的单抗使细胞外基质的降解增加四倍(P<；0.001)。抗人uPAR单抗对ECM降解无明显影响。综上所述，我们的结果表明，培养的人肾小球系膜细胞对ECM的降解是由蛋白水解酶级联介导的。这个级联反应是由tPA启动的，并产生纤溶酶和活性的基质金属蛋白酶-2，它们共同完成细胞外基质的降解。我们推测，在进展性肾脏疾病中，这种级联反应的活性降低可能是导致肾小球ECM积聚的最终共同途径。
We examined the role of the plasminogen activator/plasmin system in extracellular matrix (ECM) degradation by human mesangial cells cultured on thin films of I-125-labeled ECM (Matrigel). ECM degradation (release of I-125 into the medium) was dependent on exogenous plasminogen, proportional to the number of mesangial cells and amount of plasminogen added, and coincident with the appearance of plasmin in the medium. ECM degradation was completely blocked (P < 0.001) by two plasmin inhibitors, alpha-2-antiplasmin (40 mu g/ml) and aprotinin (216 KIU/ml), and partially reduced (-33 +/- 1.8%, P < 0.01) by TIMP-1 (40 mu g/ml), a specific inhibitor of matrix metalloproteinases. Zymography of medium obtained from cells cultured in the absence of plasminogen revealed the presence of latent matrix metalloproteinase-2 (MMP-2) which was converted to a lower molecular weight, active form in the presence of mesangial cells and plasminogen. Northern analysis of poly A + RNA prepared from cultured human mesangial cells revealed mRNA for tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA receptor (uPAR). The presence of uPA protein in medium obtained from cultured human mesangial cells was demonstrated by Western blotting and ELISA which revealed a large molar excess of PAI-1 (1.2 +/- 0.1 x 10(-9) M) over uPA (1.2 +/- 0.1 x 10(-12) M) and tPA (0.19 +/- 0.04 x 10(-9) M). ECM degradation was reduced by a monoclonal antibody (MAb) against human tPA (-54 +/- 8.6%) or human uPA (-39 +/- 5.2%) compared to cells treated with identical amounts of non-specific monoclonal IgG (P < 0.01). In contrast, MAb against human PAI-1 increased ECM degradation four-fold (P < 0.001). A MAb against human uPAR had no significant effect on ECM degradation. Taken together, our results indicate that ECM degradation by cultured human mesangial cells is mediated by a proteinase cascade. This cascade is initiated by tPA and generates plasmin and active MMP-2, which together carry out the degradation of the ECM. We postulate that decreased activity of this cascade may represent a final common pathway contributing to glomerular ECM accumulation in progressive renal disease.