Free Fatty Acid Induces Apoptosis via Retinoic Acid Receptor-Related Orphan Receptor Alpha (RORA)-Mediated Protein Tyrosine Phosphatase Non-Receptor Type 2 (PTPN2) Downregulation in Hepatic Cells

Background: Retinoic Acid Receptor-Related Orphan Receptor Alpha (RORA) is a transcription factor in addition to a nuclear orphan receptor. It was shown to regulate the expression of various proteins that regulate hepatic functions. RORA expression was downregulated under Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) conditions; however, there is limited data on the mechanisms by which RORA regulates MASLD. Protein Tyrosine Phosphatase Non-Receptor Type 2 (PTPN2) participates in the process of dephosphorylating various receptor and non-receptor tyrosine protein kinases, which play a significant role in the signalling of various cellular operations. PTPN2 was reported to be suppressed in obesity-associated metabolic dysfunction-associated steatohepatitis (MASH). Methods: HepG2 cells were treated with 300 µM palmitic acid (PA) for 72 h, and the expression of RORA, PTPN2, Bcl-xL (BCL2L1), BAX, and β-actin was determined using immunoblotting. Promoter analysis of PTPN2 was performed through JASPAR CORE database enrichment analysis. HepG2 cells were treated with either RORA natural agonist or synthetic antagonist, and the regulation of PTPN2 and apoptosis was studied. siRNAs targeting PTPN2 (siPTPN2) were transfected into HepG2 cells, and PTPN2 and apoptotic marker expression was assessed. Three sets of six C57BL/6 mice were given either CSAA, CDAA, or a normal diet for a total of 54 weeks. Liver tissues were separated after feeding, and immunoblotting was performed using total protein. Key Findings: RORA and PTPN2 expression were downregulated, and apoptosis was upregulated in PA-treated hepatic cells. JASPAR CORE database enrichment analysis of PTPN2 promoter analysis revealed the presence of ROR response element (RORE). PTPN2 expression was upregulated and downregulated in hepatic cells treated with RORA natural agonist and synthetic antagonist, respectively. Apoptosis was unregulated in siPTPN2-transfected hepatic cells. In CDAA diet-fed mice, RORA and PTPN2 expression were suppressed, and apoptotic markers were upregulated. Conclusions: According to the study’s findings, palmitic acid induced apoptosis by inhibiting RORA-mediated PTPN2 expression in hepatic cells. These findings can help in the development of novel therapeutic modalities using potent activators of RORA for the treatment of MASLD.