Abstract
Purpose :
The tetraspan transmembrane (TM) tight junction protein occludin is located in centrosomes during endothelial division. Retinal neovascularization induced by vascular endothelial growth factor (VEGF) can be blocked by delivery of a phospho-inhibitory point mutant (S490A) of occludin. However, the molecular mechanisms by which occludin regulates cell growth and neovascularization remain unclear. We hypothesized that occludin regulates VEGF-driven endothelial proliferation through its centrosomal localization. The current study utilized mutational analysis to identify regions of occludin that regulate targeting to centrosomes and VEGF-driven endothelial proliferation.
Methods :
Centrosomal localization of occludin mutants was tested by transfecting U2OS cells, which have well-characterized, easily identifiable centrosomes. VEGF-induced proliferation was examined after nucleofection of occludin mutants in bovine retinal endothelial cells (BREC) using Click-iT EdU cell proliferation assay in 3D culture. Occludin localization was examined by immunofluorescence confocal and super-resolution microscopy.
Results :
Occludin mutational analysis revealed a requirement for TM region 4, the carboxy terminal tail including the coiled-coil region and Ser490 phosphorylation for centrosomal targeting and endothelial proliferation in response to VEGF. In particular, a construct mimicking a natural splice variant, possessing TM4 and the carboxy terminal tail of occludin, was sufficient for targeting to centrosomes but the carboxy terminal tail without TM4 failed to do so, suggesting a membrane requirement. The amine-terminus/first extracellular loop was not required for centrosomal localization. Surprisingly, this region was required for endothelial proliferation in response to VEGF revealing a second region of occludin that controls proliferation. BODIPY staining revealed that phospho-occludin co-localizes with lipid markers at centrosomes during mitosis and super-resolution microscopy suggests the fusion of occludin containing vesicles at centrosomes during mitosis.
Conclusions :
These results reveal that occludin contributes to vascular endothelial growth and differentiation. The studies suggest that VEGF-driven mitosis requires occludin localization to centrosomes that is dependent on Ser490 phosphorylation and transmembrane insertion.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.