Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.

The recruitment model for gene activation presumes that DNA is a platform on which the requisite components of the transcriptional machinery are assembled. In contrast to this idea, we show here that Rap1͞Gcr1͞Gcr2 transcriptional activation in yeast cells occurs through a large anchored protein platform, the Nup84 nuclear pore subcomplex. Surprisingly, Nup84 and associated subcomplex components activate transcription themselves in vivo when fused to a heterologous DNA-binding domain. The Rap1 coactivators Gcr1 and Gcr2 form an important bridge between the yeast nuclear pore complex and the transcriptional machinery. Nucleoporin activation may be a widespread eukaryotic phenomenon, because it was first detected as a consequence of oncogenic rearrangements in acute myeloid leukemia and related syndromes in humans. These chromosomal translocations fuse a homeobox DNA-binding domain to the human homolog (hNup98) of a transcriptionally active component of the yeast Nup84 subcomplex. We conclude that Rap1 target genes are activated by moving to contact compartmentalized nuclear assemblages, rather than through recruitment of the requisite factors to chromatin by means of diffusion. We term this previously undescribed mechanism ''reverse recruitment'' and discuss the possibility that it is a central feature of eukaryotic gene regulation. Reverse recruitment stipulates that activators work by bringing the DNA to an nuclear pore complex-tethered platform of assembled transcriptional machine components.chromatin boundaries ͉ leukemia ͉ silencing ͉ synthetic genetic array ͉ gene regulation A n underlying assumption of both the stepwise and preassembly alternatives (1) of the recruitment model of in vivo gene activation (2-6) is that activators work by bringing the transcriptional machinery to the DNA, i.e., that the machinery itself diffuses relatively freely within the nuclear compartment. We have been studying the repressor͞activator protein Rap1 of Saccharomyces cerevisiae, which recognizes identical motifs in mediating either transcriptional activation (of glycolytic genes and ribosomal protein genes; refs. 7-9) or repression (of silent mating type loci and telomeres; refs. 10-15) and with its coactivators Gcr1 and Gcr2 participates in coordination of growth with cell-cycle progression (16,17). Numerous aspects of Rap1 activation have conformed poorly with the ''free diffusion'' aspect of the recruitment model for transcriptional activation. One such aspect is the presence of an unusually large activation domain that is easily inactivated by means of mutations throughout the N-terminal 280 residues of Gcr1, spanning four distinct hypomutable regions (8,17,18); two of these hypomutable regions overlap with putative transmembrane domains.We report here independent approaches demonstrating that the Rap1͞Gcr1͞Gcr2 activation assemblage (7-9, 19), like its silencing counterpart, is anchored at the nuclear periphery. For example, synthetic genetic array (SGA) analysis identified a robust genetic network that connects the Ra...

Objective We previously showed that endothelial epsin deficiency causes elevated VEGFR2 and enhanced VEGF signaling, resulting in aberrant tumor angiogenesis and tumor growth in adult mice. However, direct evidence demonstrating that endothelial epsins regulate angiogenesis specifically through VEGFR2 downregulation is still lacking. In addition, whether the lack of epsins causes abnormal angiogenesis during embryonic development remains unclear. Approach and Results A novel strain of endothelial epsin-deleted mice that are heterozygous for VEGFR2 (Epn1fl/fl; Epn2−/−; Flkfl/+; iCDH5 Cre mice) was created. Analysis of embryos at different developmental stages shows that deletion of epsins causes defective embryonic angiogenesis and retards embryo development. In vitro angiogenesis assays using isolated primary endothelial cells (EC) from Epn1fl/fl; Epn2−/−; iCDH5 Cre (EC-iDKO) and Epn1fl/fl; Epn2−/−; Flkfl/+; iCDH5 Cre (EC-iDKO-Flkfl/+) mice demonstrated that VEGFR2 reduction in epsin depleted cells is sufficient to restore normal VEGF signaling, EC proliferation, EC migration and EC network formation. These findings were complemented by in vivo wound healing, inflammatory angiogenesis, and tumor angiogenesis assays in which reduction of VEGFR2 was sufficient to rescue abnormal angiogenesis in endothelial epsin-deleted mice. Conclusions Our results provide the first genetic demonstration that epsins function specifically to downregulate VEGFR2 by mediating activated VEGFR2 internalization and degradation and that genetic reduction of VEGFR2 level protects against excessive angiogenesis caused by epsin loss. Our findings indicate epsins may be a potential therapeutic target in conditions where tightly regulated angiogenesis is crucial, such as in diabetic wound healing and tumors.