Bcl-2 overexpression in hepatic stellate cell line CFSC-2G, induces a pro-fibrotic state.
OBJECTIVE
Development of hepatic fibrosis is a complex process that involves oxidative stress (OS) and an altered balance between pro- and anti-apoptotic molecules. Since Bcl-2 overexpression preserves viability against OS, our objective was to address the effect of Bcl-2 overexpression in the hepatic stellate cells (HSC) cell-line CFSC-2G under acetaldehyde and H(2)O(2) challenge, and explore if it protects these cells against OS, induces replicative senescence and/or modify extracellular matrix (ECM) remodeling potential.


METHODS
To induce Bcl-2 overexpression, HSC cell line CFSC-2G was transfected by lipofection technique. Green fluorescent protein-only CFSC-2G cells were used as a control. Cell survival after H(2)O(2) treatment and total protein oxidation were assessed. To determine cell cycle arrest, proliferation-rate, DNA synthesis and senescence were assessed. Matrix metalloproteinases (MMP), tissue-inhibitor of MMP (TIMP), transglutaminases (TG) and smooth muscle a-actin (alpha-SMA) were evaluated by western blot in response to acetaldehyde treatment as markers of ECM remodeling capacity in addition to transforming growth factor-beta (TGF-beta) mRNA.


RESULTS
Cells overexpressing Bcl-2 survived approximately 20% more than control cells when exposed to H(2)O(2) and approximately 35% proteins were protected from oxidation, but Bcl-2 did not slow proliferation or induced senescence. Bcl-2 overexpression did not change alpha-SMA levels, but it increased TIMP-1 (55%), tissue transglutaminases (tTG) (25%) and TGF-beta mRNA (49%), when exposed to acetaldehyde, while MMP-13 content decreased (47%).


CONCLUSIONS
Bcl-2 overexpression protected HSC against oxidative stress but it did not induce replicative senescence. It increased TIMP-1, tTG and TGF-beta mRNA levels and decreased MMP-13 content, suggesting that Bcl-2 overexpression may play a key role in the progression of fibrosis in chronic liver diseases.
González-Puertos
Viridiana Y
VY
Health and Sciences Department, Biological and Health Sciences Division, Metropolitan Independent University, México, DF, México.
Hernández-Pérez
Elizabeth
E
Nuño-Lámbarri
Natalia
N
Ventura-Gallegos
José L
JL
López-Diázguerrero
Norma E
NE
Robles-Díaz
Guillermo
G
Gutiérrez-Ruiz
María C
MC
Konigsberg
Mina
M
D000079:Acetaldehyde / Q000494:pharmacology; D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D016922:Cellular Senescence; D004261:DNA Replication; D004305:Dose-Response Relationship, Drug; D005109:Extracellular Matrix / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D055166:Hepatic Stellate Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008103:Liver Cirrhosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics; D011503:Transglutaminases / Q000378:metabolism; D015854:Up-Regulation
D000079:Acetaldehyde; D000199:Actins; D019204:GTP-Binding Proteins; D006861:Hydrogen Peroxide; D053509:Matrix Metalloproteinase 13; D016877:Oxidants; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D016212:Transforming Growth Factor beta; D011503:Transglutaminases; C498862:Acta2 protein, rat; C507083:Mmp13 protein, rat