Fibrinolytic activity of human mesothelial cells is counteracted by rapid uptake of tissue-type plasminogen activator.
BACKGROUND
Human mesothelial cells (HMCs) have an important role in maintaining an adequately functioning fibrinolytic system in the peritoneal cavity by secreting the fibrinolytic enzymes tissue-type and urokinase-type plasminogen activator (t-PA and u-PA), as well as a specific PA inhibitor, PA inhibitor type 1 (PAI-1). In this study, we investigated whether the fibrinolytic capacity of HMCs is further counterbalanced by rapid uptake of t-PA and u-PA from the medium.


METHODS
Cultured HMCs were used to study the uptake and degradation of radiolabeled t-PA and u-PA in the absence or presence of an inhibitor of cellular protein degradation, chloroquine, and of specific receptor antagonists. Northern blotting and ligand-blotting techniques were applied to demonstrate the presence of specific receptors for binding of t-PA and u-PA.


RESULTS
At 37 degreesC, HMCs rapidly internalized and degraded 125I-t-PA and 125I-u-PA, which could be inhibited by an excess of unlabeled t-PA and u-PA, respectively, and by the lysosomotropic agent chloroquine. Northern blot analysis showed the expression of low-density lipoprotein (LDL) receptor-related protein (LRP), very low density lipoprotein (VLDL) receptor, and u-PA receptor. The addition of recombinant 39 kDa receptor-associated protein (RAP; an inhibitor of LRP and VLDL receptor) almost completely blocked the degradation of t-PA and partly that of u-PA. RAP ligand blotting demonstrated predominantly the presence of LRP, suggesting a major role for the LRP in mediating uptake and degradation of t-PA in HMCs. Endocytosis of u-PA occurs via two different pathways. After binding to u-PA receptor, a RAP-inhibitable and a non-RAP-inhibitable route for u-PA degradation was demonstrated. Tumor necrosis factor alpha (TNFalpha) diminished the fibrinolytic activity of HMCs by decreasing t-PA and increasing PAI-1 synthesis. The fall in t-PA levels could be counteracted by inhibiting t-PA degradation by either RAP or chloroquine. Interestingly, chloroquine also quenched the TNFalpha-induced changes in t-PA and PAI-1 mRNA levels. Using TNFalpha mutants and agonistic or blocking monoclonal antibodies specific for the TNF receptors p55 and p75, we found evidence that chloroquine interfered with the activation of the TNF receptor p55 and/or its intracellular signaling route.


CONCLUSIONS
Receptor-mediated endocytosis plays a crucial role in regulating the fibrinolytic capacity of HMCs by its participation in the degradation of t-PA and u-PA, and in the TNFalpha-induced decrease in t-PA and the increase in PAI-1 expression.
Sitter
T
T
Gaubius Laboratory, TNO Prevention and Health, Leiden, The Netherlands.
Toet
K
K
Quax
P
P
Kooistra
T
T
D000906:Antibodies / Q000494:pharmacology; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000494:pharmacology; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D017077:Culture Media, Conditioned; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005342:Fibrinolysis / Q000502:physiology*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes; D037181:Lectins, C-Type; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D000906:Antibodies; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D007457:Iodine Radioisotopes; D037181:Lectins, C-Type; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; C524898:PLAUR protein, human; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D011973:Receptors, LDL; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C036053:VLDL receptor; D002738:Chloroquine; C103332:GST-RAP protein, recombinant; D005982:Glutathione Transferase; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator