V-ATPase-Driven Lysosomal Activation Orchestrates MEK2-Induced Endothelial Reprogramming

Direct lineage reprogramming holds therapeutic promise but often depends on transcription factor overexpression, resulting in unstable phenotypes. Here, we describe a novel strategy to convert fibroblasts into endothelial-like cells by activating lysosomal activity. Constitutively active MEK2 induces an endothelial gene program via sustained MAPK/ERK signaling, leading to enhanced vacuolar ATPase (V-ATPase) activity, lysosomal acidification, extracellular matrix degradation, and angiogenic behavior. V-ATPase inhibition impairs these effects, whereas pharmacologic activation with EN6 recapitulates key features of reprogramming and promotes nuclear translocation of TFEB, a master lysosomal regulator. Consistently, TFEB overexpression-particularly a phospho-deficient mutant-boosts lysosomal function and endothelial gene expression. These findings define a MAPK-V-ATPase-TFEB axis that drives endothelial reprogramming and highlight the lysosome as a central hub for cell fate transitions, offering an organelle-centric framework for regenerative medicine. HighlightsO_LISustained MEK2 activation reprograms fibroblasts into endothelial-like cells C_LIO_LIMEK2 enhances lysosomal acidification by upregulating V-ATPase subunits C_LIO_LIV-ATPase drives acidification and ECM remodeling for endothelial reprogramming C_LIO_LIV-ATPase activation promotes TFEB nuclear entry and endothelial gene expression C_LI