Small ubiquitin-like modifier (SUMO) modification of proteins (SUMOylation) and deSUMOylation have emerged as important regulatory mechanisms for protein function. SENP1 (SUMO-specific protease) deconjugates SUMOs from modified proteins. We have created SENP1 knockout (KO) mice based on a Cre-loxP system. Global deletion of SENP1 (SENP1 KO) causes anemia and embryonic lethality between embryonic day 13.5 and postnatal day 1, correlating with erythropoiesis defects in the fetal liver. Bone marrow transplantation of SENP1 KO fetal liver cells to irradiated adult recipients confers erythropoiesis defects. Protein analyses show that the GATA1 and GATA1-dependent genes are down-regulated in fetal liver of SENP1 KO mice. This down-regulation correlates with accumulation of a SUMOylated form of GATA1. We further show that SENP1 can directly deSUMOylate GATA1, regulating GATA1-dependent gene expression and erythropoiesis by in vitro assays. Moreover, we demonstrate that GATA1 SUMOylation alters its DNA binding, reducing its recruitment to the GATA1-responsive gene promoter. Collectively, we conclude that SENP1 promotes GATA1 activation and subsequent erythropoiesis by deSUMOylating GATA1.

Epsin is a ubiquitin-binding endocytic adaptor, which is highly concentrated at clathrin-coated pits and coordinates acquisition of bilayer curvature with coat recruitment and cargo selection. Epsin is encoded by three distinct genes in mammals. Epsin 1 and 2 have broad tissue distribution with high-level expression in the brain. In contrast, epsin 3 was reported to be expressed primarily in immature keratinocytes. Here, we show that epsin 3 is selectively expressed at high levels in the stomach (including the majority of gastric cancers), where it is concentrated in parietal cells. In these cells, epsin 3 is enriched and colocalized with clathrin around apical canaliculi, the sites that control acidification of the stomach lumen via the exo-endocytosis of vesicles containing the H/K ATPase. Deletion of the epsin 3 gene in mice did not result in obvious pathological phenotypes in either the stomach or other organs, possibly because of overlapping functions of the other two epsins. However, levels of EHD1 and EHD2, two membrane tubulating proteins with a role in endocytic recycling, were elevated in epsin 3 knock-out stomachs, pointing to a functional interplay of epsin 3 with EHD proteins in the endocytic pathway of parietal cells. We suggest that epsin 3 cooperates with other bilayer binding proteins with curvature sensing/generating properties in the specialized traffic and membrane remodeling processes typical of gastric parietal cells.

OBJECTIVE: The goal of this study was to investigate the novel hypothesis that bone marrow kinase in the X chromosome (Bmx), an established inflammatory mediator of pathological angiogenesis, promotes lymphangiogenesis. METHODS AND RESULTS: We have recently demonstrated a critical role for Bmx in inflammatory angiogenesis. However, the role of Bmx in lymphangiogenesis has not been investigated. Here, we show that in wild-type mice, Bmx is upregulated in lymphatic vessels in response to vascular endothelial growth factor (VEGF). In comparison with wild-type mice, Bmx-deficient mice mount weaker lymphangiogenic responses to VEGF-A and VEGF-C in 2 mouse models. In vitro, Bmx is expressed in cultured human dermal microvascular lymphatic endothelial cells. Furthermore, pharmacological inhibition and short interfering RNA mediated silencing of Bmx reduces VEGF-A and VEGF-C-induced signaling and lymphatic endothelial cell tube formation. Mechanistically, we demonstrated that Bmx differentially regulates VEGFR-2 and VEGFR-3 receptor signaling pathways: Bmx associates with and directly regulates VEGFR-2 activation, whereas Bmx associates with VEGFR-3 and regulates downstream signaling without an effect on the receptor autophosphorylation. CONCLUSIONS: Our in vivo and in vitro results provide the first insight into the mechanism by which Bmx mediates VEGF-dependent lymphangiogenic signaling.

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: CCM1, CCM2, and CCM3. CCM3, also known as PDCD10 (programmed cell death 10), was initially identified as a messenger RNA whose abundance was induced by apoptotic stimuli in vitro. However, the in vivo function of CCM3 has not been determined. Here, we describe mice with a deletion of the CCM3 gene either ubiquitously or specifically in the vascular endothelium, smooth muscle cells, or neurons. Mice with global or endothelial cell-specific deletion of CCM3 exhibited defects in embryonic angiogenesis and died at an early embryonic stage. CCM3 deletion reduced vascular endothelial growth factor receptor 2 (VEGFR2) signaling in embryos and endothelial cells. In response to VEGF stimulation, CCM3 was recruited to and stabilized VEGFR2, and the carboxyl-terminal domain of CCM3 was required for the stabilization of VEGFR2. Indeed, the CCM3 mutants found in human patients lacking the carboxyl-terminal domain were labile and were unable to stabilize and activate VEGFR2. These results demonstrate that CCM3 promotes VEGFR2 signaling during vascular development.

A single nucleotide polymorphism in the DAB2IP gene is associated with risk of aggressive prostate cancer (PCa), and loss of DAB2IP expression is frequently detected in metastatic PCa. However, the functional role of DAB2IP in PCa remains unknown. Here, we show that the loss of DAB2IP expression initiates epithelial-to-mesenchymal transition (EMT), which is visualized by repression of E-cadherin and up-regulation of vimentin in both human normal prostate epithelial and prostate carcinoma cells as well as in clinical prostate-cancer specimens. Conversely, restoring DAB2IP in metastatic PCa cells reversed EMT. In DAB2IP knockout mice, prostate epithelial cells exhibited elevated mesenchymal markers, which is characteristic of EMT. Using a human prostate xenograft-mouse model, we observed that knocking down endogenous DAB2IP in human carcinoma cells led to the development of multiple lymph node and distant organ metastases. Moreover, we showed that DAB2IP functions as a scaffold protein in regulating EMT by modulating nuclear beta-catenin/T-cell factor activity. These results show the mechanism of DAB2IP in EMT and suggest that assessment of DAB2IP may provide a prognostic biomarker and potential therapeutic target for PCa metastasis.

Previously we have shown that tyrosine 718 of ASK1 when phosphorylated is critical for SOCS1 binding and SOCS1-mediated degradation of ASK1. However, the kinase and phosphatase responsible for phosphorylation and dephosphorylation of ASK1 at Tyr-718 are unknown. In this study, we identified JAK2 and SHP2 as a Tyr-718-specific kinase and phosphatase, respectively. Interferon-gamma (IFN-gamma) induced degradation of ASK1 in normal but not in SOCS1-KO endothelial cells (EC). IFN-gamma-induced tyrosine phosphorylation of ASK1 at Tyr-718 was blocked by a JAK2-specific inhibitor. IFN-gamma enhanced the association between JAK2 and ASK1, and the ASK1-JAK2 complex was labile and was stabilized by the proteasomal inhibitor MG132. Furthermore, JAK2, but not JAK1, directly bound to and phosphorylated ASK1 at Tyr-718, leading to an enhanced association of ASK1 with SOCS1 and subsequent ASK1 degradation. Next, we showed that overexpression of the SH2-containing protein-tyrosine phosphatase-2 (SHP2) augmented, whereas a phosphatase-inactive mutant of SHP2 inhibited, TNF-induced ASK1 dephosphorylation. SHP2 associated with ASK1 in response to tumor necrosis factor in EC. An SHP-2 substrate-trapping mutant formed a complex with tyrosine-phosphorylated ASK1, suggesting that ASK1 is a direct SHP2 substrate. Moreover, SHP2 wild type, but not a catalytically inactive mutant, dissociated SOCS1 from ASK1. IFN-gamma-induced ASK1 Tyr(P)-718 was enhanced in mouse EC deficient in SHP2 (SHP2-KO). In contrast, tumor necrosis factor-induced dephosphorylation of ASK1 at Tyr(P)-718 and activation of ASK1-JNK signaling, as well as EC apoptosis, are significantly reduced in SHP2-KO EC. Our data suggest that JAK2-SOCS1 and SHP2 reciprocally regulate ASK1 phosphorylation and stability in response to cytokines.

OBJECTIVE: Thioredoxin-2 (Trx2), a major antioxidant protein in mitochondria, enhances nitric oxide bioavailability and inhibits ASK1-dependent apoptosis in endothelial cells (ECs). However, the in vivo role of Trx2 in angiogenesis has not been defined. Here we used EC-specific transgenesis of Trx2 (Trx2-TG) in mice to determine the in vivo function of Trx2 in arteriogenesis and angiogenesis. METHODS AND RESULTS: In a femoral artery ligation model, Trx2-TG mice had enhanced capacity in limb perfusion recovery and ischemic reserve capacity compared to the nontransgenic littermates. Ischemia-initiated arteriogenesis in the upper limb was augmented in Trx2-TG mice. Trx2-TG mice also showed significantly enhanced capillary formation and maturation in the lower limb. In nontransgenic limb, ischemia specifically induced a downregulation of Trx2 protein, leading to increased oxidative stress, ASK1 activation, and EC apoptosis. In contrast, Trx2-TG maintained a constitutive level of Trx2, reducing the ischemia-induced deleterious responses. We then defined the mechanism by which Trx2 increases angiogenesis using ECs isolated from Trx2-TG mice. Trx2-TG ECs showed increased NO and NO-dependent migration. In addition, these cells were more resistant to oxidative stress-induced activation of ASK1 signaling and apoptosis. Moreover, Trx2-augmented EC survival is NO-independent. To define the relative contributions of Trx2-increased NO and Trx2-reduced ASK1 apoptotic activity to angiogenesis in vivo, we examined Trx2 effects on ischemia-induced angiogenesis in eNOS-deficient mice. The eNOS deletion caused severe impairment in the functional flow recovery in response to ischemia. Trx2 expression in eNOS-KO mice still dramatically inhibited ischemia-induced ASK1 and EC apoptosis, leading to an enhanced functional flow recovery. CONCLUSIONS: These in vivo and in vitro data support that Trx2 maintains EC function by two parallel pathways-scavenging ROS to increase NO bioavailability and inhibiting ASK1 activity to enhance EC survival, facilitating ischemia-mediated arteriogenesis and angiogenesis.

Epsins are endocytic adaptors with putative functions in general aspects of clathrin-mediated endocytosis as well as in the internalization of specific membrane proteins. We have now tested the role of the ubiquitously expressed epsin genes, Epn1 and Epn2, by a genetic approach in mice. While either gene is dispensable for life, their combined inactivation results in embryonic lethality at E9.5-E10, i.e., at the beginning of organogenesis. Consistent with studies in Drosophila, where epsin endocytic function was linked to Notch activation, developmental defects observed in epsin 1/2 double knockout (DKO) embryos recapitulated those produced by a global impairment of Notch signaling. Accordingly, expression of Notch primary target genes was severely reduced in DKO embryos. However, housekeeping forms of clathrin-mediated endocytosis were not impaired in cells derived from these embryos. These findings support a role of epsin as a specialized endocytic adaptor, with a critical role in the activation of Notch signaling in mammals.

ASK1-interacting protein-1 (AIP1), a recently identified member of the Ras GTPase-activating protein family, is highly expressed in vascular ECs and regulates EC apoptosis in vitro. However, its function in vivo has not been established. To study this, we generated AIP1-deficient mice (KO mice). Although these mice showed no obvious defects in vascular development, they exhibited dramatically enhanced angiogenesis in 2 models of inflammatory angiogenesis. In one of these models, the enhanced angiogenesis observed in the KO mice was associated with increased VEGF-VEGFR2 signaling. Consistent with this, VEGF-induced ear, cornea, and retina neovascularization were greatly augmented in KO mice and the enhanced retinal angiogenesis was markedly diminished by overexpression of AIP1. In vitro, VEGF-induced EC migration was inhibited by AIP1 overexpression, whereas it was augmented by both AIP1 knockout and knockdown, with the enhanced EC migration caused by AIP1 knockdown being associated with increased VEGFR2 signaling. We present mechanistic data that suggest AIP1 is recruited to the VEGFR2-PI3K complex, binding to both VEGFR2 and PI3K p85, at a late phase of the VEGF response, and that this leads to inhibition of VEGFR2 signaling. Taken together, our data demonstrate that AIP1 functions as an endogenous inhibitor in VEGFR2-mediated adaptive angiogenesis in mice.

We have previously shown that ASK1-interacting protein 1 (AIP1) transduces tumor necrosis factor-induced ASK1-JNK signaling. Because endoplasmic reticulum (ER) stress activates ASK1-JNK signaling cascade, we investigated the role of AIP1 in ER stress-induced signaling. We created AIP1-deficient mice (AIP1-KO) from which mouse embryonic fibroblasts and vascular endothelial cells were isolated. AIP1-KO cells show dramatic reductions in ER stress-induced, but not oxidative stress-induced, ASK1-JNK activation and cell apoptosis. The ER stress-induced IRE1-JNK/XBP-1 axis, but not the PERK-CHOP1 axis, is blunted in AIP1-KO cells. ER stress induced formation of an AIP1-IRE1 complex, and the PH domain of AIP1 is critical for the IRE1 interaction. Furthermore, reconstitution of AIP1-KO cells with AIP1 wild type, not an AIP1 mutant with a deletion of the PH domain (AIP1-DeltaPH), restores ER stress-induced IRE1-JNK/XBP-1 signaling. AIP1-IRE1 association facilitates IRE1 dimerization, a critical step for activation of IRE1 signaling. More importantly, AIP1-KO mice show impaired ER stress-induced IRE1-dependent signaling in vivo. We conclude that AIP1 is essential for transducing the IRE1-mediated ER stress response.