Podosome formation impairs endothelial barrier function by sequestering zonula occludens proteins

Podosomes and tight junctions (TJs) are subcellular compartments that both exist in endothelial cells and localize at cell surfaces. In contrast to the well‐characterized role of TJs in maintaining cerebrovascular integrity, the specific function of endothelial podosomes remains unknown. Intriguingly, we discovered cross‐talk between podosomes and TJs in human brain endothelial cells. Tight junction scaffold proteins ZO‐1 and ZO‐2 localize at podosomes in response to phorbol‐12‐myristate‐13‐acetate treatment. We found that both ZO proteins are essential for podosome formation and function. Rather than being derived from new protein synthesis, podosomal ZO‐1 and ZO‐2 are relocated from a pre‐existing pool found at the peripheral plasma membrane with enhanced physical interaction with cortactin, a known protein marker for podosomes. Sequestration of ZO proteins in podosomes weakens tight junction complex formation, leading to increased endothelial cell permeability. This effect can be further attenuated by podosome inhibitor PP2. Altogether, our data revealed a novel cellular function of podosomes, specifically, their ability to negatively regulate tight junction and endothelial barrier integrity, which have been linked to a variety of cerebrovascular diseases. Podosome formation in human brain endothelial cells sequesters tight junction proteins ZO‐1 and ZO‐2, which leads to dysfunctional endothelial barrier. Inhibition of podosome formation can rescue such defects, highlighting anti‐podosome treatment as a promising therapy for blood brain barrier‐related cerebrovascular diseases.