ECM DEGRADATION BY CULTURED HUMAN MESANGIAL CELLS IS MEDIATED BY A PAPLASMIN/MMP-2 CASCADE/

We examined the role of the plasminogen activator/plasmin system in ex tracellular matrix (ECM) degradation by human mesangial cells cultured on thin films of I-125-labeled ECM (Matrigel). ECM degradation (relea se of I-125 into the medium) was dependent on exogenous plasminogen, p roportional to the number of mesangial cells and amount of plasminogen added, and coincident with the appearance of plasmin in the medium. E CM degradation was completely blocked (P < 0.001) by two plasmin inhib itors, alpha-2-antiplasmin (40 mu g/ml) and aprotinin (216 KIU/ml), an d 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 t he presence of latent matrix metalloproteinase-2 (MMP-2) which was con verted to a lower molecular weight, active form in the presence of mes angial cells and plasminogen. Northern analysis of poly A + RNA prepar ed from cultured human mesangial cells revealed mRNA for tissue-type p lasminogen 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 mes angial cells was demonstrated by Western blotting and ELISA which reve aled 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 degra dation was reduced by a monoclonal antibody (MAb) against human tPA (- 54 +/- 8.6%) or human uPA (-39 +/- 5.2%) compared to cells treated wit h identical amounts of non-specific monoclonal IgG (P < 0.01). In cont rast, MAb against human PAI-1 increased ECM degradation four-fold (P < 0.001). A MAb against human uPAR had no significant effect on ECM deg radation. Taken together, our results indicate that ECM degradation by cultured human mesangial cells is mediated by a proteinase cascade. T his cascade is initiated by tPA and generates plasmin and active MMP-2 , which together carry out the degradation of the ECM. We postulate th at decreased activity of this cascade may represent a final common pat hway contributing to glomerular ECM accumulation in progressive renal disease.