Investigation of Orai1 inhibition in HepG2 cells reveals cholesterol pathways remodelling with implications for cardiovascular disease

Abstract Introduction ORAI1-mediated calcium signalling plays a central role in vascular smooth muscle cell (VSMC) remodelling. A recent UK Biobank study has identified an ORAI1 missense variant associated with dyslipidaemia, atherosclerosis and hospitalisation for peripheral arterial disease, suggesting a potential systemic role for ORAI1 in lipid metabolism (1). Previous transcriptomic profiling in PDGF-stimulated human aortic VSMC showed that pharmacological ORAI1 inhibition with the small molecule inhibitor, JPIII affects inflammatory, proliferative and cholesterol-related pathways (2). The effects of JPIII on hepatic lipid-regulatory pathways remain unclear. Purpose To investigate the transcriptional responses to ORAI1 inhibition using JPIII in HepG2 hepatocytes and compare with previous human VSMC RNA-seq findings to identify shared and/or unique cell-type-specific pathways relevant to lipid metabolism and cardiovascular biology. Methods HepG2 cells were treated with JPIII (30 µM, n=4) or vehicle (n=4) for 24 h. Total RNA-seq was performed and aligned to GRCh38 using HISAT2. Differentially expressed genes (DEGs) were identified using a 5% FDR threshold. Functional enrichment (KEGG, Reactome, GO, DisGeNET) was performed on significant DEGs. A comparative analysis with previously VSMC RNA-seq under the same JPIII treatment was conducted using Venn analysis and direct gene-level matching. Results PCA demonstrated clear separation between JPIII-treated and vehicle samples (Fig1), indicating a strong transcriptional shift. ORAI1 inhibition in HepG2 resulted in 2171 DEGs (888 upregulated; 1248 downregulated; FDR ≤ 0.05, |log2FC| ≥ 1). Comparison with VSMC data (1470 DEGs, 668 up; 802 down) identified 183 shared genes (84 up; 104 down in HepG2), representing an 8% overlap. Pathway enrichment analysis showed that JPIII strongly affected lipid-regulatory pathways, including PPAR signalling, fatty-acid and cholesterol metabolism, folate/one-carbon pathways, and ER/oxidative-stress responses (ATF3, DDIT3). Where pathways related to the cell cycle, DNA replication, and mitotic control were suppressed. Similarly to VSMC, HepG2 cells showed changes in TGF-β, inflammatory signalling and ECM remodelling pathways (MMP2, MMP3, LOX, PLAU). HepG2-specific effects centred on lipid homeostasis, whereas VSMC showed dominant suppression of proliferative remodelling. DisGeNET enrichment linked to atherosclerosis, dyslipidaemia, and cardiometabolic disorders aligning with UK Biobank ORAI1 variant associations. Conclusion ORAI1 inhibition using JPIII induces transcriptional remodelling in hepatocytes, including strong modulation of lipid-metabolic, inflammatory and stress-response pathways. A partial overlap with VSMC transcriptional profiles, alongside clear hepatocyte-specific pathways highlight tissue-dependent roles for ORAI1 signalling. These findings support ORAI1 as a potential regulator of systemic lipid metabolism with relevance to cardiovascular disease.HepG2 transcriptomic effects of JPIIIFor image description, please refer to the figure legend and surrounding text.