ECM degradation by cultured human mesangial cells is mediated by a PA/plasmin/MMP-2 cascade

Abstract

We examined the role of the plasminogen activator/plasmin system in extracellular matrix (ECM) degradation by human mesangial cells cultured on thin films of 125I-labeled ECM (Matrigel). ECM degradation (release of 125I 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 micrograms/ml) and aprotinin (216 KIU/ml), and partially reduced (-33 +/- 1.8%, P < 0.01) by TIMP-1 (40 micrograms/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).(ABSTRACT TRUNCATED AT 250 WORDS)