Urokinase plasminogen activator stimulates function of active forms of stromelysin and gelatinases (MMP-2 and MMP-9) in cirrhotic tissue.
BACKGROUND
The authors' previous data support the notion that adenoviral-driven urokinase plasminogen activator (u-PA) expression results in reversion of experimental liver cirrhosis. The specific aim of the present study was to decipher the mechanisms involved in the regulation by endogenous/gene-delivered u-PA of matrix metalloproteinases (MMP) and related proteins engaged in degradation of excessive hepatic connective tissue.


METHODS
Tissue slices from cirrhotic rat livers were incubated with u-PA-rich supernatants from 24-h-cultured hepatic stellate cells (HSC). Matrix metalloproteinase-2, -9 and tissue inhibitor of metalloproteinases-1 (TIMP-1) were detected by western blot and biologic activity. The HSC that discontinued u-PA production were transfected with the adenovector Adu-PA and serum-free supernatants evaluated for proteolytic activity by MMP-3, MMP-2 and MMP-9. Collagen I, transforming growth factor-beta1 (TGF-beta1), plasminogen activator inhibitor-1 (PAI-1) and TIMP-1 mRNA levels were also evaluated.


CONCLUSIONS
Endogenous u-PA from cultured HSC significantly induced the active forms of MMP-2 (68 kDa) and MMP-9 (78 kDa) in cirrhotic tissue slices. The TIMP-1 molecular forms demonstrated that u-PA pushed the presence of 'free' TIMP-1 (not complexed with MMP; 71%) in cirrhotic tissue. When non-producing u-PA-HSC were transfected with adenoviral vector coding for the functional human protein u-PA (Adhu-PA), an overactivation of MMP-3, MMP-2 and MMP-9 (800%, 48% and 100%, respectively) was found as compared with HSC transfected with control adenovirus encoding green fluorescent protein (Ad-GFP). Finally, gene expression of collagen I, TGF-beta1, PAI-1 and TIMP-1 were downregulated by Adhu-PA action as well.
González-Cuevas
Jaime
J
Institute for Molecular Biology in Medicine and Gene Therapy, CUCS, University of Guadalajara, OPD Civil Hospital of Guadalajara, Jalisco, Mexico.
Bueno-Topete
Miriam
M
Armendariz-Borunda
Juan
J
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002908:Chronic Disease; D005500:Follow-Up Studies; D008106:Liver Cirrhosis, Experimental / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000187:drug effects / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3 / Q000187:drug effects / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology*
D019715:Tissue Inhibitor of Metalloproteinase-1; D014568:Urokinase-Type Plasminogen Activator; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9