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)

Aneta Novotna,Aneta Doricakova,Petr Pavek,Zdenek Dvorak

doi:10.5507/bp.2012.075

Aneta Novotna, Aneta Doricakova + Show 2 more

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https://doi.org/10.5507/bp.2012.075

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Abstract

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. 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. 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. 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.

Highlights

Altered phenotype and loss of hepatocyte-specific function of human cancer cell lines derived from liver cells is probably the consequence of diminished expression of key transcriptional factors and their co-activators, such as peroxisome proliferator-activated receptor gamma co-activator (PGC-1α)
We found no significant difference in the expression levels of proliferator-activated receptor gamma co-activator 1α (PGC-1α) mRNA in HepG2-PGC-1α cells compared to HepG2-pcDNA3 and parent HepG2 cells
The loss of hepatic phenotype of cancer cells is at least partly caused by the under-expression of transcription factors or co-activators such as (PGC-1α, SRC-1, SRC2, c/EBP, HNF4α) and others

Summary

Introduction

Altered phenotype and loss of hepatocyte-specific function of human cancer cell lines derived from liver cells is probably the consequence of diminished expression of key transcriptional factors and their co-activators, such as peroxisome proliferator-activated receptor gamma co-activator (PGC-1α). The major mechanism of PGC-1α induction is activation of the cAMP signaling pathways through cAMP binding to CREB binding site in proximal promoter of PGC1α (ref.[10]). Another mechanism of PGC-1α induction involves stimulation of activating transcription factor 2 (ATF2) in brown adipose tissue by cold, or calcium signaling in exercised skeletal muscles and heart[11,12]. PGC-1α plays an important role in adaptive thermogenesis It is involved in glucose metabolism and maintenance of lipid and energy homeostasis. Fasting induces PGC-1α expression in adult liver, which leads to activation of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase through interactions with the transcription factor HNF4α and forkhead box O1 (FOXO-1)

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