scholarly journals Modulation of neutral protease expression in human mesangial cells by hyperglycaemic culture

1996 ◽  
Vol 320 (3) ◽  
pp. 777-783 ◽  
Author(s):  
Nadia ABDEL WAHAB ◽  
Roger M. MASON
Keyword(s):  
Plasminogen Activator ◽  
Mesangial Cells ◽  
Plasminogen Activator Inhibitor ◽  
Culture Conditions ◽  
Plasminogen Activator Inhibitor 1 ◽  
Human Mesangial Cells ◽  
Cell Matrix ◽  
Tissue Plasminogen ◽  
Protease Expression ◽  
Activator Inhibitor

We report on the effect of prolonged hyperglycaemic (11 and 30 mM d-glucose) culture conditions on human mesangial cell matrix metalloproteinases (MMPs), plasminogen activators and their inhibitors. The results indicate that hyperglycaemic conditions modulate the potential proteolytic activity of the enzymes secreted by confluent cultures of these cells. Gelatinase A (MMP-2) activity was always higher in cultures maintained under hyperglycaemic than under normoglycaemic conditions (4 mM d-glucose). In contrast, gelatinase B (MMP-9) activity was decreased under the same conditions. Matrilysin (MMP-7) activity was decreased by up to 100% under hyperglycaemic conditions. Reverse transcriptase–PCR and Western-blotting analyses indicate that in all cases both the transcripts and the protein level were correlated with enzymic activity.One tissue inhibitor of metalloproteinases, TIMP-2, was barely detectable under hyperglycaemic conditions (30 mM d-glucose). In contrast, TIMP-1 increased during the initial 2 weeks of culture in hyperglycaemic conditions and remained elevated to the end of the experiment (4 weeks). Under normoglycaemic conditions TIMP-1 decreased after 2 weeks of culture. Hyperglycaemic conditions also decreased markedly the activity of tissue plasminogen activator (t-PA). This seemed to be due to increased synthesis of its inhibitor, plasminogen activator inhibitor 1, under these conditions rather than to decreased expression of the t-PA enzyme.

Decorin suppresses transforming growth factor-β-induced expression of plasminogen activator inhibitor-1 in human mesangial cells through a mechanism that involves Ca2+-dependent phosphorylation of Smad2 at serine-240

2002 ◽  
Vol 362 (3) ◽  
pp. 643-649 ◽  
Author(s):  
Nadia ABDEL-WAHAB ◽  
Stephen J. WICKS ◽  
Roger M. MASON ◽  
Andrew CHANTRY
Keyword(s):  
Growth Factor ◽  
Plasminogen Activator ◽  
Transforming Growth Factor ◽  
Mesangial Cells ◽  
Plasminogen Activator Inhibitor ◽  
Transforming Growth Factor Β ◽  
Receptor Activation ◽  
Plasminogen Activator Inhibitor 1 ◽  
Human Mesangial Cells ◽  
Activator Inhibitor

Transforming growth factor-β (TGFβ) is a key mediator of extracellular matrix (ECM) accumulation in sclerotic kidney diseases such as diabetic nephropathy. One of the main target cells for TGFβ in the kidney are glomerular mesangial cells, which respond by increasing expression of ECM proteins, such as collagens, laminin and fibronectin, while suppressing the expression of ECM-degrading proteases and increasing the synthesis of ECM protease inhibitors, including plasminogen activator inhibitor-1. Previous studies have shown that exposure of mesangial cells to chronic high-glucose conditions, such as those seen in diabetes, increases ECM deposition in a mechanism involving glucose-mediated up-regulation of TGFβ expression. Naturally occurring inhibitors of this TGFβ-dependent fibrotic response include decorin, a small leucine-rich proteoglycan. While the mechanism by which TGFβ stimulates gene expression via the Smad signal-transduction pathway is becoming clear, the precise mechanism by which decorin may impinge upon TGFβ activity remains to be established. In this study, for the first time we provide evidence that decorin can disrupt glucose- and TGFβ/Smad-dependent transcriptional events in human mesangial cells through a mechanism that involves an increase in Ca2+ signalling, the activation of Ca2+/calmodulin-dependent protein kinase II and ensuing phosphorylation of Smad2 at Ser-240. We show that decorin also induces Ser-240 phospho-Smad hetero-oligomerization with Smad4 and the nuclear localization of this complex independently of TGFβ receptor activation. Thus, in human mesangial cells, the mechanism of decorin-mediated inhibition of TGFβ signalling may involve activation of Ca2+ signalling, the subsequent phosphorylation of Smad2 at a key regulatory site, and the sequestration of Smad4 in the nucleus.

scholarly journals The mechanism of the reaction between human plasminogen-activator inhibitor 1 and tissue plasminogen activator

1990 ◽  
Vol 265 (1) ◽  
pp. 109-113 ◽  
Author(s):  
T L Lindahl ◽  
P I Ohlsson ◽  
B Wiman
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Peptide Bond ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Tissue Plasminogen ◽  
Activator Inhibitor ◽  
Pai 1

The structural events taking place during the reaction between PAI-1 (plasminogen-activator inhibitor 1) and the plasminogen activators sc-tPA (single-chain tissue plasminogen activator) and tc-tPA (two-chain tissue plasminogen activator) were studied. Complexes were formed by mixing sc-tPA or tc-tPA with PAI-1 in slight excess (on an activity basis). The complexes were purified from excess PAI-1 by affinity chromatography on fibrin-Sepharose. Examination of the purified complexes by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE) and N-terminal amino acid sequence analysis demonstrated that a stoichiometric 1:1 complex is formed between PAI-1 and both forms of tPA. Data obtained from both complexes revealed the amino acid sequences of the parent molecules and, in addition, a new sequence: Met-Ala-Pro-Glu-Glu-. This sequence is found in the C-terminal portion of the intact PAI-1 molecule and thus locates the reactive centre of PAI-1 to Arg346-Met347. The proteolytic activity of sc-tPA is demonstrated by its capacity to cleave the ‘bait’ peptide bond in PAI-1. The complexes were inactive and dissociated slowly at physiological pH and ionic strength, but rapidly in aq. NH3 (0.1 mol/l). Amidolytic tPA activity was generated on dissociation of the complexes, corresponding to 0.4 mol of tPA/mol of complex. SDS/PAGE of the dissociated complexes indicated a small decrease in the molecular mass of PAI-1, in agreement with proteolytic cleavage of the ‘bait’ peptide bond during complex-formation.

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