Single-cell RNA sequencing identifies endothelial-derived HBEGF as promoting pancreatic beta cell proliferation in mice via the EGFR–Kmt5a–H4K20me pathway

Pancreatic beta cell mass is dynamically regulated in response to increased physiological and pathological demands. Understanding the mechanisms that control physiological beta cell proliferation could provide valuable insights into novel therapeutic approaches to diabetes. Here, we aimed to analyse the intracellular and extracellular signalling pathways involved in regulating the physiological proliferation of beta cells using single-cell RNA-seq (scRNA-seq) and in vitro functional assays. Islets isolated from nulliparous mice, mice at different time points of gestation and mice at day 4 after delivery were analysed using scRNA-seq. Bioinformatics analyses of scRNA-seq data were performed to determine the heterogeneous transcriptomic characteristics of beta cells and to identify the proliferating subpopulation. CellChat was used to analyse cell–cell communication and identify the ligand–receptor pairs between beta cell subclusters as well as between non-beta cells and proliferating beta cells. In vitro functional assays were conducted in mouse and rat beta cell lines and isolated mouse primary islets to validate the role of Kmt5a– mono-methylation of histone H4 at lysine 20 (H4K20me) signalling and endothelial-derived heparin-binding EGF-like growth factor (HBEGF) in beta cell proliferation. Of 43,724 endocrine and non-endocrine cells within islets analysed by scRNA-seq, 15,569 beta cells were clustered into eight distinct populations, each exhibiting unique heterogeneity. A proliferating beta cell subcluster was identified that highly expressed the histone methyltransferase Kmt5a. Activation of Kmt5a–H4K20me signalling upregulated the expression of Cdk1 and promoted beta cell proliferation. The crosstalk between endothelial cells and the proliferating beta cell subcluster, mediated by the HBEGF–EGF receptor (EGFR) ligand–receptor interaction, increased as beta cell mass expanded. HBEGF increased the expression levels of genes involved in the cell cycle and promoted beta cell proliferation by regulating the Kmt5a–H4K20me signalling pathway. Our study demonstrates that, under physiological conditions, endothelial-derived HBEGF regulates beta cell proliferation through the Kmt5a–H4K20me signalling pathway, which may serve as a potential target to promote beta cell expansion and treat diabetes. The scRNA-seq and RNA-seq datasets are available from the Gene Expression Omnibus (GEO) using the accession numbers GSE278860 and GSE278861, respectively.