TY - JOUR
T1 - ECM degradation by cultured human mesangial cells is mediated by a PA/plasmin/MMP-2 cascade
AU - Baricos, W. H.
AU - Cortez, S. L.
AU - El-Dahr, S. S.
AU - Schnaper, H. W.
N1 - Funding Information:
This work was funded by PHS grants ROl DK 45449-01 and DK 08712, and grant 91-942 from the American Heart Association (National Center). Portions of this work were presented at the Annual Meeting of the American Society of Nephrology, Boston, Massachusetts, November 14—17, 1993. The authors thank Dr. Hanna Abboud for supplying and characterizing the mesangial cells; Drs. Elliot Barnathan and D. Dichek for their gifts of cDNA probes; and Drs. Sudhir Shah, Vecihi Batuman, Suzanne Meleg-Smith, and Jeffrey Kopp for their critical review of the manuscript.
PY - 1995/4
Y1 - 1995/4
N2 - 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, α-2-antiplasmin (40 μg/ml) and aprotinin (216 KIU/ml), and partially reduced (-33 ± 1.8%, P < 0.01) by TIMP-1 (40 μg/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-l (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.
AB - 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, α-2-antiplasmin (40 μg/ml) and aprotinin (216 KIU/ml), and partially reduced (-33 ± 1.8%, P < 0.01) by TIMP-1 (40 μg/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-l (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.
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U2 - 10.1038/ki.1995.150
DO - 10.1038/ki.1995.150
M3 - Article
C2 - 7540230
AN - SCOPUS:0028945629
SN - 0085-2538
VL - 47
SP - 1039
EP - 1047
JO - Kidney international
JF - Kidney international
IS - 4
ER -
