THE ROLE OF THE ARP2/3 INHIBITORY PROTEIN ARPIN IN THE REGULATION OF ENDOTHELIAL BARRIER FUNCTIONS

The endothelium forms a semi‐permeable barrier in the vasculature that separates blood from underlying tissues. During inflammation, this barrier is altered usually leading to increased permeability and leukocyte recruitment. Such alterations include endothelial actin remodeling regulated by actin‐binding proteins (ABP) and junction architecture modulation. An ABP critical for actin remodeling is the Arp2/3 complex, which induces formation of branched actin networks. The Arp2/3 complex needs to be tightly regulated which is achieved by activation via nuclear promoting factors (NPF) and inhibition via proteins that locally antagonize the activity of NPFs such as PICK1, gadkin and arpin. Arpin contains a COOH‐terminal acidic motif that binds directly to the Arp2/3 complex in competition with NPF to inhibit its activity. Arpin has been shown to localize at the lamellipodial edge, where it colocalizes with the WAVE2 complex and antagonizes WAVE2 to regulate Arp2/3 activity in this specific subcellular location. However, nothing is known about the role of arpin in endothelial barrier regulation. First, we analyzed expression of arpin in different endothelial cell types and found that it is expressed at high levels in Human vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (HMEC‐1), brain‐derived Endothelial cells.3 (bEnd3) and primary murine lung endothelial cells (MLEC). To analyze arpin functions in endothelial barrier regulation, we generated arpin‐depleted HUVEC by transfecting them with lentiviruses encoding specific short hairpin RNA (shRNA) against arpin and a scrambled control sequence, and selecting stable clones using puromycin. Western blot analysis revealed arpin downregulation of more than 90%. Expression of the junctional proteins VE‐cadherin, claudin‐1, occludin, ZO‐1 and the apical adhesion molecule ICAM‐1 did not change in arpin‐depleted HUVECs. However, VE‐Cadherin staining revealed significant gaps at cell contacts and redistribution into the cytoplasm. Moreover, we consistently observed more actin filaments in arpin‐depleted HUVEC. As ROCK1 and ZIPK kinases were overexpressed in the absence of arpin, we conclude that these filaments are contractile stress fibers that destabilize junctions by generating pulling forces. In vitro filter‐based permeability assays showed that arpin‐depleted HUVEC are much more permeable compared to control cells transduced with lentivirus expressing scrambled shRNA. These data suggest that arpin regulates the endothelial barrier by two possible mechanisms: First, arpin may inhibit Arp2/3 to control junction protein internalization and vesicle trafficking; and second, arpin may inhibit Arp2/3 to control actin dynamics at junctions. Whether arpin additionally regulates Arp2/3‐independent processes is currently unknown, and will be investigated in the future. Our data reveal arpin as a new critical regulator of endothelial barrier integrity. Support or Funding Information This work was supported by a grant of the Mexican Council for Science and Technology (CONACyT, 284292 to MS).