The Role of Formins in Endothelial Adherens Junction Regulation

Abstract

Adherens junctions are cadherin-dependent structures that mediate intercellular signaling and structural integrity of the endothelial barrier. Formins are a highly conserved family of cytoskeletal remodeling proteins whose activity has been implicated in regulating adherens junction formation in other cell-types. Therefore, we tested the hypothesis that formin activity is essential for adherens junction assembly in endothelial cells. A small-molecule formin inhibitor (smiFH2) was used to determine the effect of formin inhibition on junction formation using an in vitro vascular permeability assay. We determined that smiFH2 treatment caused a dose-dependent inhibition of junction formation. We used siRNAs to knockdown expression of the seven formins shown to be expressed in TIME cells and determined that individual knockdown of FHOD1, FHOD3 and Dia1 significantly increased the permeability of the endothelial monolayer. Interestingly, FMNL2 knockdown actually potentiated barrier function. Knockdown of the remaining formins had little or no effect on junction formation. Knockdown of FHOD3 had the greatest inhibitory effect on junction assembly; VE-cadherin protein levels were decreased in FHOD3-depleted cells. The FHOD3 knockdown cells were also elongated in comparison to controls and formed thin linear adherens junctions and few focal adherens junctions. In contrast, the morphology of FMNL2-depleted cells did not appear obviously different from controls. In conclusion, our results suggest that multiple formins play diverse roles in adherens junction formation and maintenance in endothelial cells.