Effect of sulphur mustard on the expression of urokinase in cultured 3T3 fibroblasts.
The expression of plasminogen activator (PA), a serine proteinase involved in the degradation of extracellular matrix proteins, has been investigated in 3T3 fibroblasts after in vitro exposure to sulphur mustard (SM). Expression of the cell-associated enzyme has been assessed with a synthetic substrate assay and at the mRNA level. Twenty-four hours after 100 microM SM, cell viability (monitored by MTT assay) was not significantly affected, but protein synthesis (tritiated leucine incorporation) was reduced to < 20% of the control value. PA activity was significantly increased compared to control cells with a 20-fold increase after 24 h. This up-regulation was independent of the cell density, occurred maximally between days 1 and 4 and persisted for at least 6 days after exposure. Lower concentrations of SM (< or = 10 microM) did not significantly affect PA activity. Northern blotting experiments revealed an increased expression of urokinase (u-PA) transcripts in cells treated with 100 microM SM, with a peak at 10 h after exposure. Conditioned culture medium from cell cultures treated with 100 microM SM did not affect the expression of PA activity in naive or SM-treated cultures. Thiodiglycol (100 microM), the main metabolite of SM, did not influence the expression of PA in the same system. Different compounds were tested for modulation of the PA upregulation after SM exposure. Nicotinamide (5 mM), vitamin D3 (10(-10)M), extracellular calcium (2 mM) or EGTA (5 mM) had no effect. Ryanodine (10 microM) amplified the PA up-regulation by a factor of 2 and vanadate (500 microM) reduced it by approximately 50%. Dexamethasone (1 microM) added directly after SM treatment almost completely prevented the induction of PA at both the protein and mRNA levels. Overall these results demonstrate an up-regulation of urokinase in 3T3 fibroblasts after treatment with SM, which is possibly mediated by intracellular calcium mobilization. Further studies are needed to identify the significance of this proteolytic response in the pathogenesis of blistering and/or DNA repair mechanisms.
Detheux
M
M
Laboratory of Medical Toxicology, Catholic University of Louvain, Brussels, Belgium.
Jijakli
H
H
Lison
D
D
D016475:3T3 Cells / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002470:Cell Survival; D002619:Chemical Warfare Agents / Q000494:pharmacology*; D003879:Dermatologic Agents / Q000494:pharmacology*; D003907:Dexamethasone / Q000494:pharmacology; D051379:Mice; D009151:Mustard Gas / Q000494:pharmacology*; D010960:Plasminogen Activators / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D002619:Chemical Warfare Agents; D003879:Dermatologic Agents; D012333:RNA, Messenger; D003907:Dexamethasone; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator; D009151:Mustard Gas