Construction and characterization of peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1 alpha over-expressing cell line derived from human hepatocyte carcinoma HepG2 cells)
Abstrakt
Aims. The aim was develop stable human cell line stable over-expressing transcription co-activator peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1 alpha) with restored hepatospecific functions and increased expression of major xenobiotic metabolizing enzymes.
Methods. Six clones of HepG2-PGC-1 alpha and one control clone HepG2-pcDNA3 were isolated and analyzed for secretion of hepatospecific markers, fibrinogen, albumin and alpha1-antitrypsin.
Expression levels of protein and mRNA of hepatocyte nuclear factor (HNF4 alpha), pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) were determined. We measured basal and ligand inducible expression of CYP1A1 and CYP3A4.
Results. Stably transfected cell line HepG2-PGC-1 alpha derived from HepG2 cells over-expressing PGC-1 alpha displayed increased secretion of fibrinogen, but not albumin or alpha1-antitrypsin compared to parent HepG2 cells.
We found increased levels of HNF4 alpha, PXR and AhR proteins but not their mRNAs in HepG2-PGC1 cells. Basal expression of CYP3A4 protein in HepG2-PGC-1 alpha cells was increased but rifampicin-inducible expression of CYP3A4 protein was lowered in comparison with parent HepG2 cells.
Induction of CYP3A4 mRNA varied between 1.3 - 1.9 fold in individual clones. Expression of TCDD-inducible CYP1A1 protein was lower in HepG2-PGC-1 alpha cells than in parent HepG2 cells.
Induction of CYP1A1 mRNA by TCDD in HepG2-PGC-1 alpha cells was comparable with that in parent HepG2 cells and ranged between 103 - 198 fold. Conclusion.
Stable expression of PGC-1 alpha in HepG2 cells restores several hepatospecific functions, such as secretion of fibrinogen, expression of HNF4 alpha 1 and xenoreceptors PXR and AhR. However, the expression and induction of key drug-metabolizing enzymes (CYP1A1 and CYP3A4) were not improved.