Sema3d and Sema3e Direct Endothelial Motility through Distinct Molecular Signaling Pathways [Cell Biology]

May 13th, 2014 by Aghajanian, H., Choi, C., Ho, V. C., Gupta, M., Singh, M. K., Epstein, J. A.

Class 3 semaphorins were initially described as axonal growth cone guidance molecules that signal through plexin and neuropilin coreceptors, and since have been established to be regulators of vascular development. Semaphorin 3e (Sema3e) was previously shown to repel endothelial cells and is the only class 3 semaphorin known to be capable of signaling via a plexin receptor without a neuropilin coreceptor. Sema3e signals through plexin D1 (Plxnd1) to regulate vascular patterning by modulating the cytoskeleton and focal adhesion structures. Recently, we have shown that semaphorin 3d (Sema3d) mediates endothelial cell repulsion and pulmonary vein patterning during embryogenesis. Here we show that Sema3d and Sema3e similarly affect human umbilical vein endothelial cells (HUVECs) but through distinct molecular signaling pathways. Time-lapse imaging studies show that both Sema3d and Sema3e can inhibit cell motility and migration, and tube formation assays indicate that both can impede tubulogenesis. Endothelial cells incubated with either Sema3d or Sema3e demonstrate a loss of actin stress fibers and focal adhesions. However, the addition of neuropilin 1 (Nrp1) blocking antibody or siRNA knockdown of Nrp1 inhibits Sema3d-mediated but not Sema3e-mediated cytoskeletal reorganization, and siRNA knockdown of Nrp1 abrogates Sema3d but not Sema3e-mediated inhibition of tubulogenesis. On the other hand, endothelial cells deficient in Plxnd1 are resistant to endothelial repulsion mediated by Sema3e, but not Sema3d. Unlike Sema3e, Sema3d incubation results in phosphorylation of Akt in HUVECs, and inhibition of the Phosphoinositide 3-kinase (PI3K)/Akt pathway blocks the endothelial guidance and cytoskeletal reorganization functions of Sema3d but not Sema3e.