Stable overexpression of pregnane X receptor in HepG2 cells increases its potential for bioartificial liver application.
To bridge patients with acute liver failure to transplantation or liver regeneration, a bioartificial liver (BAL) is urgently needed. A BAL consists of an extracorporeal bioreactor loaded with a bioactive mass that would preferably be of human origin and display high hepatic functionality, including detoxification. The human hepatoma cell line HepG2 exhibits many hepatic functions, but its detoxification function is low. In this study, we investigated whether stable overexpression of pregnane X receptor (PXR), a master regulator of diverse detoxification functions in the liver [eg, cytochrome P450 3A (CYP3A) activity], would increase the potential of HepG2 for BAL application. Stable overexpression was achieved by lentiviral expression of the human PXR gene, which yielded cell line cBAL119. In monolayer cultures of cBAL119 cells, PXR transcript levels increased 29-fold versus HepG2 cells. Upon activation of PXR by rifampicin, the messenger RNA levels of CYP3A4, CYP3A5, and CYP3A7 increased 49- to 213-fold versus HepG2 cells. According to reporter gene assays with different inducers, the highest increase in CYP3A4 promoter activity (131-fold) was observed upon induction with rifampicin. Inside BALs, the proliferation rates, as measured by the DNA content, were comparable between the 2 cell lines. The rate of testosterone 6beta-hydroxylation, a measure of CYP3A function inside BALs, increased 4-fold in cBAL119 BALs versus HepG2 BALs. Other functions, such as apolipoprotein A1 synthesis, urea synthesis, glucose consumption, and lactate production, remained unchanged or increased. Thus, stable PXR overexpression markedly increases the potential of HepG2 for BAL application.
Nibourg
Geert A A
GA
Department of Experimental Surgery and University of Amsterdam, Amsterdam, the Netherlands.
Huisman
Maarten T
MT
van der Hoeven
Tessa V
TV
van Gulik
Thomas M
TM
Chamuleau
Robert A F M
RA
Hoekstra
Ruurdtje
R
D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000235:genetics; D019149:Bioreactors; D049109:Cell Proliferation; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis / Q000235:genetics; D004790:Enzyme Induction; D017930:Genes, Reporter; D005822:Genetic Vectors; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics; D019164:Liver, Artificial; D000077297:Pregnane X Receptor; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011987:Receptors, Steroid / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012293:Rifampin / Q000494:pharmacology; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D011987:Receptors, Steroid; D013739:Testosterone; D001189:Aryl Hydrocarbon Hydroxylases; C510164:CYP3A5 protein, human; C103123:CYP3A7 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D012293:Rifampin