Construction and characterization of peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1α over-expressing cell line derived from human hepatocyte carcinoma HepG2 cells).
OBJECTIVE
The aim was develop stable human cell line stable over-expressing transcription co-activator peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) with restored hepatospecific functions and increased expression of major xenobiotic metabolizing enzymes.


METHODS
Six clones of HepG2-PGC-1α 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 (HNF4α), 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-1α derived from HepG2 cells over-expressing PGC-1α displayed increased secretion of fibrinogen, but not albumin or alpha1-antitrypsin compared to parent HepG2 cells. We found increased levels of HNF4α, PXR and AhR proteins but not their mRNAs in HepG2-PGC1 cells. Basal expression of CYP3A4 protein in HepG2-PGC-1α 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-1α cells than in parent HepG2 cells. Induction of CYP1A1 mRNA by TCDD in HepG2-PGC-1α cells was comparable with that in parent HepG2 cells and ranged between 103 - 198 fold.


CONCLUSIONS
Stable expression of PGC-1α in HepG2 cells restores several hepatospecific functions, such as secretion of fibrinogen, expression of HNF4α1 and xenoreceptors PXR and AhR. However, the expression and induction of key drug-metabolizing enzymes (CYP1A1 and CYP3A4) were not improved.
Department of Cell Biology and Genetics, Faculty of Science, Palacky University Olomouc, Czech Republic.
Aneta
A
Novotna
Aneta
A
Doricakova
Petr
P
Pavek
Zdenek
Z
Dvorak
D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005839:Gentamicins / Q000494:pharmacology; D056945:Hep G2 Cells; D051557:Hepatocyte Nuclear Factor 4 / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D047495:PPAR gamma / Q000378:metabolism*; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D000077297:Pregnane X Receptor; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism; D013723:Teratogens / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D051792:Basic Helix-Loop-Helix Proteins; D014408:Biomarkers, Tumor; D019363:Cytochrome P-450 CYP1A1; D051544:Cytochrome P-450 CYP3A; D005340:Fibrinogen; D005839:Gentamicins; D051557:Hepatocyte Nuclear Factor 4; D047495:PPAR gamma; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D000072317:Polychlorinated Dibenzodioxins; D000077297:Pregnane X Receptor; D018336:Receptors, Aryl Hydrocarbon; D011987:Receptors, Steroid; D013723:Teratogens; D014157:Transcription Factors; C111235:AHR protein, human; C496592:HNF4A protein, human; C486497:PPARGC1A protein, human; C010680:antibiotic G 418; C569463:CYP1A1 protein, human; C510163:CYP3A4 protein, human