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PV1 is an endothelial-specific integral membrane glycoprotein associated with the stomatal diaphragms of caveolae, transendothelial channels, and vesiculo-vacuolar organelles and the diaphragms of endothelial fenestrae. Multiple PV1 homodimers are found within each stomatal and fenestral diaphragm. We investigated the function of PV1 within these diaphragms and their regulation and found that treatment of endothelial cells in culture with phorbol myristate acetate (PMA) led to upregulation of PV1. This correlated with de novo formation of stomatal diaphragms of caveolae and transendothelial channels as well as fenestrae upon PMA treatment. The newly formed diaphragms could be labeled with anti-PV1 antibodies. The upregulation of PV1 and formation of stomatal and fenestral diaphragms by PMA was endothelium specific and was the highest in microvascular endothelial cells compared with their large vessel counterparts. By using a siRNA approach, PV1 mRNA silencing prevented the de novo formation of the diaphragms of caveolae as well as fenestrae and transendothelial channels. Overexpression of PV1 in endothelial cells as well as in cell types that do not harbor caveolar diaphragms in situ induced de novo formation of caveolar stomatal diaphragms. Lastly, PV1 upregulation by PMA required the activation of Erk1/2 MAP kinase pathway and was protein kinase C independent. Taken together, these data show that PV1 is a key structural component, necessary for the biogenesis of the stomatal and fenestral diaphragms.