RNA sequencing in human HepG2 hepatocytes reveals PPAR-α mediates transcriptome responsiveness of bilirubin

Abstract

Bilirubin is a potent antioxidant that reduces inflammation and the accumulation of fat. There have been reports of gene responses to bilirubin, which was mostly attributed to its antioxidant function. Using RNA sequencing, we found that biliverdin, which is rapidly reduced to bilirubin, induced transcriptome responses in human HepG2 hepatocytes in a peroxisome proliferator-activated receptor (PPAR)-α-dependent fashion (398 genes with >2-fold change; false discovery rate P < 0.05). For comparison, a much narrower set of genes demonstrated differential expression when PPAR-α was suppressed via lentiviral shRNA knockdown (23 genes). Gene set enrichment analysis revealed the bilirubin-PPAR-α transcriptome mediates pathways for oxidation-reduction processes, mitochondrial function, response to nutrients, fatty acid oxidation, and lipid homeostasis. Together, these findings suggest that transcriptome responses from the generation of bilirubin are mostly PPAR-α dependent, and its antioxidant function regulates a smaller set of genes.

Keywords: PPAR; biliverdin; gene; liver; transcription.

Fig. 1.

RNA sequencing of the transcriptome responsiveness in PPAR-α-shRNA and Scramble-shRNA HepG2 hepatocytes. PPAR-α knockdown (KD) and Scramble-shRNA (Scramble) hepatocytes were treated with biliverdin (BV) [50 μM] or vehicle (Veh) in dialyzed serum for 24 h. A: heat map for genes changed with BV and Veh in PPAR-α KD and Scramble hepatocytes with a fold >2 and <−1.6. B: volcano plots for Scramble (BV versus Veh) and PPAR-α KD (BV versus Veh). C: Venn diagram with a minimum absolute fold change of 2 and false discovery rate (FDR) P value < 0.05. Yellow, genes changed with BV treatments in Scramble; light blue, genes changed in PPAR-α KD cells with BV treatment. The overlap is genes that were changed by BV in both cell types (n = 3).

Fig. 2.

Real-time PCR of mRNA to confirm RNA-sequencing responses. Real-time PCR of genes changed in RNA sequencing for PPAR-α knockdown (KD) and Scramble hepatocytes treated biliverdin (BV) [50 μM] or vehicle (Veh) in dialyzed fatty-acid free serum for 24 h. A: PPAR-α target genes. B: genes changed in PPAR-α KD and Scramble HepG2 cells. C: genes changed only in the PPAR-α KD, and not Scramble HepG2 cells. ns, Not significant; *P < 0.05, **P < 0.01, ***P < 0.001; ± SE; n = 3.

Fig. 3.

Pathway analysis from RNA sequencing for molecular (A), cellular (B), and biological functions (C). Gene set enrichment analysis of the bilirubin-PPAR-α transcriptome for genes that exhibited differential expression (FDR P value < 0.05) between BV versus vehicle treatments for Scramble versus PPAR-α KD (n = 8,501 genes).

Fig. 4.

Schematic of pathways affected by the bilirubin-PPAR-α transcriptome. Bilirubin (BR) is rapidly generated from biliverdin (BV) by biliverdin reductase A (BVRA). BR has antioxidant and ligand functions and, primarily through PPAR-α, regulates transcriptome responses for pathways that improve mitochondrial function and fat burning. A smaller set of genes may be induced by BV activation of BVRA’s transcriptional function or BR’s antioxidant action.