Protein Kinase C Promotes Notch1 Cleavage Through Both Ligand-Dependent and Hyaluronic Acid–ADAM10-Dependent Mechanisms

Notch1 signaling is essential for endothelial cell fate determination and vascular development. While PKC has been implicated as an upstream activator of Notch1 signaling, the molecular mechanisms by which PKC promotes Notch1 proteolytic cleavage remain poorly defined. Here, using murine endothelial MS1 cells and mouse aortic endothelial cells (MAECs), we identify two cooperative pathways through which PKC drives generation of the Notch1 intracellular domain (NICD). First, PKC activation increases mRNA and protein levels of the Notch1 ligands Jag1 and Dll4. A NICD-Dll4 positive feedback loop further amplifies Notch signaling. Second, PKC upregulates hyaluronic acid synthase 2 (HAS2) mRNA and protein expression, promoting hyaluronan (HA) synthesis. Various genetic (HAS2 knockdown, CD44 knockdown, Hyal2 overexpression) and pharmacological (4-methylumbelliferone, 4-MU) treatments confirmed that HA is required for PKC to activate Notch1. Inhibition of HA production or signaling is associated with downregulation of ADAM10 on the cell surface. Direct phosphorylation of Notch1 at S1791 is dispensable for this effect. Together, these findings reveal a dual pathway model in which PKC coordinates ligand upregulation and HA signaling to promote Notch1 cleavage, identifying the extracellular matrix as a previously unrecognized regulator of Notch1 signal strength in endothelial cells.