Adenoviral delivery of dominant-negative transforming growth factor beta type II receptor up-regulates transcriptional repressor SKI-like oncogene, decreases matrix metalloproteinase 2 in hepatic stellate cell and prevents liver fibrosis in rats.
BACKGROUND
Dominant-negative transforming growth factor beta type II receptor (TbetaRIIDeltacyt) is a protein that blocks transforming growth factor (TGF-beta) signaling. Because the consequences of blocking TGF-beta have not been completely elucidated in liver fibrosis, we analysed the effects of adenoviral delivery of TbetaRIIDeltacyt on profibrogenic genes and matrix metalloproteinase (MMP) proteins, as well as on TGF-beta signal repressor SKI-like oncogene (SnoN), in cultured hepatic stellate cells (HSCs) and in a rat model of liver fibrosis.


METHODS
To induce liver fibrosis, rats were treated with thioacetamide for 7 weeks and administrated once with Ad-TbetaRIIDeltacyt or Ad-betagal through the iliac vein. Fibrosis was measured by morphometric analysis. We evaluated SnoN by western blot, immunocytochemistry and immunohistochemistry; MMP activity was determined by zymography and profibrogenic gene expression by the real-time reverse transcriptase-polymerase chain reaction in cultured HSCs and liver tissue.


RESULTS
Profibrogenic gene expression of collagen alpha1 (I), TGF-beta1, platelet-derived growth factor-B, plasminogen activator inhibitor (PAI)-1, tissue inhibitor of matrix metalloproteinase-1 and MMP-2 was down-regulated; whereas MMP-3 was over-expressed in response to Ad-TbetaRIIDeltacyt in HSCs. Moreover, zymography assays corroborated MMP-2 and MMP-3 changes in activity. Surprisingly, anti-TGF-beta molecular intervention increased nuclear SnoN in HSCs. In vivo, Ad-TbetaRIIDeltacyt reduced liver fibrosis, increased nuclear SnoN in sinusoidal cells, and also produced significant suppression in collagen alpha1 (I), TGF-beta1, PAI-1, MMP-2 and over-expression in MMP-3 in thioacetamide-intoxicated animals.


CONCLUSIONS
The results obtained in the present study suggest that the molecular mechanism for the blocking effects of Ad-TbetaRIIDeltacyt in TGF-beta signaling acts via up-regulation of the transcriptional repressor SnoN, which antagonizes TGF-beta signaling (TGF-beta/Smad-pathway inhibitor). Consequently, profibrogenic genes are down-regulated.
Marquez-Aguirre
Ana
A
Institute for Molecular Biology in Medicine and Gene Therapy, University of Guadalajara, Department of Molecular Biology and Genomics, Jalisco, Mexico.
Sandoval-Rodriguez
Ana
A
Gonzalez-Cuevas
Jaime
J
Bueno-Topete
Miriam
M
Navarro-Partida
Jose
J
Arellano-Olivera
Immaculada
I
Lucano-Landeros
Silvia
S
Armendariz-Borunda
Juan
J
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001835:Body Weight; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D055166:Hepatic Stellate Cells / Q000166:cytology / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D008103:Liver Cirrhosis / Q000139:chemically induced / Q000473:pathology / Q000517:prevention & control*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013853:Thioacetamide / Q000494:pharmacology; D014161:Transduction, Genetic; D019076:Transgenes; D015854:Up-Regulation
D047908:Intracellular Signaling Peptides and Proteins; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D018125:Receptors, Transforming Growth Factor beta; D012097:Repressor Proteins; C060615:SKIL protein, human; C540291:Skil protein, rat; D013853:Thioacetamide; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2