Desert Hedgehog promotes endothelium integrity through ADAM17 degradation

Introduction Endothelium integrity is critical to vascular homeostasis, as failure of this system results in serious consequences such as hemorrhage, edema, inflammation and tissue ischemia. We recently identified Desert Hedgehog (DHH) as a critical regulator of endothelium integrity, downstream of KLF2. Objective The purpose of the present study was to characterize signaling pathways mediating Dhh-regulation of endothelium integrity. Methods We used BioGRID database to identify possible interactors of the DHH receptor, Patched-1 (PTCH1). Among these we chose to test the role of Disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) starting with in vitro assays including co-immunoprecipitation, western blot, immunofluorescence and endothelium permeability assays. Results First, we confirmed ADAM17 interaction with PTCH1 via co-immunoprecipitation assays in HeLa cells. Then, we showed that either DHH overexpression in HeLa cells or treatment of cultured endothelial cells (ECs) with soluble DHH induces ADAM17 protein degradation. By using either lysosome (Bafilomycin A1 and chloroquine) or proteasome inhibitors (MG132), we found that DHH induces ADAM17 degradation via the lysosome. To test whether DHH promotes adherens junction integrity by degrading ADAM17, cultured EC were either transfected with DHH siRNA alone, or with both DHH and ADAM17 siRNAs. We found that while DHH siRNA alone induces Cdh5 junction destabilization and increases endothelial permeability, these effects were prevented in the presence of ADAM17 siRNA, demonstrating DHH knockdown promotes Cdh5 junction destabilization and increased endothelial permeability via ADAM17. Conclusion Altogether, these results demonstrate that DHH promotes endothelium integrity by inducing ADAM17 degradation. We will soon verify these results in vivo in Dhh endothelial specific KO mice treated or not with the ADAM17 inhibitor Aderbasib.