Reduced NR2F6 expression reshapes the VPA-induced transcriptome in human hepatocytes and increases lipid accumulation

Valproic acid (VPA) is an antiepileptic drug associated with hepatic steatosis, yet the transcriptional regulators determining cellular susceptibility to VPA remain incompletely defined. In a time-series RNA-sequencing analysis of primary human liver spheroids, NR2F6 emerged as one of the nuclear regulators predicted to shape the hepatocellular response to VPA. In parallel, a shRNA screen targeting 42 nuclear receptors in HepG2 cells independently identified NR2F6 as a sensitizer of VPA toxicity. Functional validation in HepG2 and HepaRG models demonstrated that NR2F6 knockdown significantly increased VPA-induced lipid accumulation, whereas lipid accumulation triggered by oleic and palmitic acid remained unaffected, indicating a VPA-specific steatogenic vulnerability. To characterize NR2F6-dependent transcriptional programs, we performed RNA-sequencing in shNR2F6 and shGFP HepaRG cells exposed to VPA for 72 h. Although VPA was the principal driver of transcriptional variance, reduced NR2F6 expression markedly amplified the VPA-induced transcriptomic response. shNR2F6 cells exhibited coordinated upregulation of nuclear-encoded oxidative phosphorylation genes across Complexes I, III, IV, and V, while mitochondrial genome-encoded subunits remained unchanged, suggesting nuclear-driven mitochondrial compensation. NR2F6 knockdown also altered key lipid-associated pathways, including reduced induction of CPT1A and exaggerated induction of PLIN2, linking NR2F6 deficiency to impaired fatty-acid import and enhanced lipid-droplet accumulation. Together, these results identify NR2F6 as a key modulator of hepatocellular adaptation to VPA, linking nuclear receptor signaling to mitochondrial and lipid-metabolic remodeling and revealing a previously unrecognized regulatory node in drug-induced steatosis.