Tomohisa Sakaue

BAZF, a novel component of cullin3-based E3 ligase complex, mediates VEGFR and Notch cross-signaling in angiogenesis

Angiogenic homeostasis is maintained by a balance between vascular endothelial growth factor (VEGF) and Notch signaling in endothelial cells (ECs). We screened for molecules that might mediate the coupling of VEGF signal transduction with down-regulation of Notch signaling, and identified B-cell chronic lymphocytic leukemia/lymphoma6-associated zinc finger protein (BAZF). BAZF was induced by VEGF-A in ECs to bind to the Notch signaling factor C-promoter binding factor 1 (CBF1), and to promote the degradation of CBF1 through polyubiquitination in a CBF1-cullin3 (CUL3) E3 ligase complex. BAZF disruption in vivo decreased endothelial tip cell number and filopodia protrusion, and markedly abrogated vascular plexus formation in the mouse retina, overlapping the retinal phenotype seen in response to Notch activation. Further, impaired angiogenesis and capillary remodeling were observed in skin-wounded BAZF(-/-) mice. We therefore propose that BAZF supports angiogenic sprouting via BAZF-CUL3-based polyubiquitination-dependent degradation of CBF1 to down-regulate Notch signaling.

Pathophysiology of Lung Injury Induced by Common Bile Duct Ligation in Mice

Background Liver dysfunction and cirrhosis affect vasculature in several organ systems and cause impairment of organ functions, thereby increasing morbidity and mortality. Establishment of a mouse model of hepatopulmonary syndrome (HPS) would provide greater insights into the genetic basis of the disease. Our objectives were to establish a mouse model of lung injury after common bile duct ligation (CBDL) and to investigate pulmonary pathogenesis for application in future therapeutic approaches. Methods Eight-week-old Balb/c mice were subjected to CBDL. Immunohistochemical analyses and real-time quantitative reverse transcriptional polymerase chain reaction were performed on pulmonary tissues. The presence of HPS markers was detected by western blot and microarray analyses. Results We observed extensive proliferation of CD31-positive pulmonary vascular endothelial cells at 2 weeks after CBDL and identified 10 upregulated and 9 down-regulated proteins that were associated with angiogenesis. TNF-α and MMP-9 were highly expressed at 3 weeks after CBDL and were less expressed in the lungs of the control group. Conclusions We constructed a mouse lung injury model by using CBDL. Contrary to our expectation, lung pathology in our mouse model exhibited differences from that of rat models, and the mechanisms responsible for these differences are unknown. This phenomenon may be explained by contrasting processes related to TNF induction of angiogenic signaling pathways in the inflammatory phase. Thus, we suggest that our mouse model can be applied to pulmonary pathological analyses in the inflammatory phase, i.e., to systemic inflammatory response syndrome, acute lung injury, and multiple organ dysfunction syndrome.

Protein kinase D2 and heat shock protein 90 beta are required for BCL6-associated zinc finger protein mRNA stabilization induced by vascular endothelial growth factor-A

Vascular endothelial growth factor (VEGF) is a major angiogenic factor that activates pro-angiogenic molecules to generate new vessels. Recently, we identified a VEGF-A-induced pro-angiogenic gene, BCL-6 associated zinc finger protein (BAZF), in endothelial cells. BAZF interacts with CBF1, a transcriptional regulator of Notch signaling, and downregulates Notch signaling by inducing the degradation of CBF1. A signal inhibition assay with a combination of chemical inhibitors and siRNA revealed that the protein kinase D (PRKD) family, mainly PRKD2, mediated BAZF gene expression by VEGF-A stimulation. A luciferase reporter assay showed that the promoter activity of the BAZF gene was unchanged by VEGF-A stimulation. However, we found that the stability of BAZF mRNA increased in a VEGF-A/PRKD2-dependent manner. In further studies to investigate the underlying mechanism, we successfully identified heat shock protein 90 beta (HSP90β) as a molecule that interacts with and stabilizes BAZF mRNA following VEGF-A/PRKD2 activation. These data suggest that HSP90β may positively regulate angiogenesis, not only as a protein chaperone, but also as an mRNA stabilizer for pro-angiogenic genes, such as BAZF, in a PRKD2 activity-dependent manner.

Perivascular Adipose Tissue Angiotensin II Type 1 Receptor Promotes Vascular Inflammation and Aneurysm FormationNovelty and Significance

Perivascular adipose tissue exhibits characteristics of active local inflammation, which contributes to the development of atherosclerotic disease as a complication of obesity/metabolic syndrome. However, the precise role of perivascular adipose tissue in the progression of abdominal aortic aneurysm remains unclear. To test the hypothesis that genetic deletion of angiotensin II type 1a (AT1a) receptor in perivascular visceral adipose tissue (VAT) can attenuate aortic aneurysm formation in apolipoprotein E–deficient (ApoE−/−) mice, we performed adipose tissue transplantation experiments by using an angiotensin II–induced aneurysm murine model, in which we transplanted VAT from ApoE−/− or ApoE−/− AT1a−/− donor mice onto the abdominal aorta of ApoE−/− recipient mice. Compared with ApoE−/− VAT transplantation, ApoE−/− AT1a−/− VAT transplantation markedly attenuated aortic aneurysm formation, macrophage infiltration, and gelatinolytic activity in the abdominal aorta. AT1a receptor activation led to the polarization of macrophages in perivascular VAT toward the proinflammatory phenotype. Moreover, osteopontin expression and gelatinolytic activity were considerably lower in ApoE−/− AT1a−/− perivascular VAT than in ApoE−/− perivascular VAT, and angiotensin II–induced osteopontin secretion from adipocytes was eliminated after deletion of AT1a receptor in adipocytes. Notably, induction of macrophage migration by conditioned medium from angiotensin II–stimulated wild-type adipocytes was suppressed by treatment with an osteopontin-neutralizing antibody, and ApoE−/− OPN−/− VAT transplantation more potently attenuated aortic aneurysm formation than ApoE−/− VAT transplantation. Our findings indicate a previously unrecognized effect of AT1a receptor in perivascular VAT on the pathogenesis of abdominal aortic aneurysm.

The CUL3-SPOP-DAXX axis is a novel regulator of VEGFR2 expression in vascular endothelial cells

Vascular endothelial cell growth factor receptor 2 (VEGFR2) is an essential receptor for the homeostasis of endothelial cells. In this study, we showed that NEDD8-conjugated Cullin3 (CUL3)-based ubiquitin E3 (UbE3) ligase plays a crucial role in VEGFR2 mRNA expression. Human umbilical vein endothelial cells treated with MLN4924, an inhibitor of NEDD8-activating enzyme, or with CUL3 siRNA drastically lost their response to VEGF due to the intense decrease in VEGFR2 expression. Moreover, speckle-type POZ protein (SPOP) and death-domain associated protein (DAXX) were involved in the CUL3 UbE3 ligase complex as a substrate adaptor and a substrate, respectively. Knockdown of SPOP and CUL3 led to the upregulation of DAXX protein and downregulation of VEGFR2 levels. These levels were inversely correlated with one another. In addition, simultaneous knockdown of SPOP and DAXX completely reversed the downregulation of VEGFR2 levels. Moreover, the CUL3-SPOP-DAXX axis had the same effects on NOTCH1, DLL4 and NRP1 expression. Taken together, these findings suggest that the CUL3-SPOP-DAXX axis plays a very important role in endothelial cell function by targeting key angiogenic regulators.

Ectodomain Shedding of Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE-1) Is Induced by Vascular Endothelial Growth Factor A (VEGF-A).

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a type I transmembrane glycoprotein, is known as one of the most specific lymphatic vessel markers in the skin. In this study, we found that the ectodomain of LYVE-1 undergoes proteolytic cleavage, and this process produces soluble LYVE-1. We further identified the cleavage site for ectodomain shedding and generated an uncleavable mutant of LYVE-1. In lymphatic endothelial cells, ectodomain shedding of LYVE-1 was induced by vascular endothelial growth factor (VEGF)-A, an important factor for angiogenesis and lymphangiogenesis under pathological conditions. VEGF-A-induced LYVE-1 ectodomain shedding was mediated via the extracellular signal-regulated kinase (ERK) and a disintegrin and metalloproteinase (ADAM) 17. Wild-type LYVE-1, but not uncleavable LYVE-1, promoted migration of lymphatic endothelial cells in response to VEGF-A. Immunostaining analyses in human psoriasis skin lesions and VEGF-A transgenic mouse skin suggested that the ectodomain shedding of LYVE-1 occurred in lymphatic vessels undergoing chronic inflammation. These results indicate that the ectodomain shedding of LYVE-1 might be involved in promoting pathological lymphangiogenesis.

Purification, molecular cloning and functional characterization of swine phosphatidylethanolamine-binding protein 4 from seminal plasma

Phosphatidylethanolamine-binding proteins (PEBPs) are found in various species and have multiple functions. In this study, we purified the swine homolog of human PEBP4 (sPEBP4) from swine seminal plasma, cloned the sPEBP4 cDNA and functionally characterized this protein. The molecular mass of the purified protein was calculated to be 25 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. The full-length cDNA of sPEBP4 contains 815 bp with an open reading frame of 669 bp that encodes a protein 222 residues in length. sPEBP4 contains a putative phosphatidylethanolamine-binding domain between residues 79 and 195; however, this domain did not show lipid binding activity. The overall amino acid sequence identity of PEBP4s from swine, human, mouse, bovine and canine ranges between 56.1% and 82.4%. Immunohistochemical staining and western blotting analysis showed that sPEBP4 is secreted from epithelial cells in the epididymis to the seminal plasma. To explore the role of sPEBP4 in the seminal plasma, we tested the effect of sPEBP4 on swine sperm motility. Sperms suspended in phosphate-buffered saline began to swim after the addition of purified sPEBP4, but not when swine serum albumin was added, indicating that sPEBP4 promotes sperm motility.

Neddylated Cullin 3 is required for vascular endothelial‐cadherin‐mediated endothelial barrier function

Vascular endothelial (VE)‐cadherin, a major endothelial adhesion molecule, regulates vascular permeability, and increased vascular permeability has been observed in several cancers. The aim of this study was to elucidate the role of the NEDD8‐Cullin E3 ligase, in maintaining barrier permeability. To this end, we investigated the effects of the inhibition of Cullin E3 ligases, by using inhibitors and knockdown techniques in HUVECs. Furthermore, we analyzed the mRNA and protein levels of the ligases by quantitative RT‐PCR and Western blotting, respectively. The results revealed that NEDD8‐conjugated Cullin 3 is required for VE‐cadherin‐mediated endothelial barrier functions. Treatment of HUVECs with MLN4924, a chemical inhibitor of the NEDD8‐activating enzyme, led to high vascular permeability due to impaired cell–cell contact. Similar results were obtained when HUVECs were treated with siRNA directed against Cullin 3, one of the target substrates of NEDD8. Immunocytochemical staining showed that both treatments equally depleted VE‐cadherin protein localized at the cell–cell borders. However, quantitative RT‐PCR showed that there was no significant difference in the VE‐cadherin mRNA levels between the treatment and control groups. In addition, cycloheximide chase assay revealed that the half‐life of VE‐cadherin protein was dramatically reduced by Cullin 3 depletion. Together, these findings suggest that neddylated Cullin 3 plays a crucial role in endothelial cell barrier function by regulating VE‐cadherin.

Spatiotemporal visualization of proHB-EGF ectodomain shedding in living cells.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family, each of which is produced as a type I transmembrane precursor. The juxtamembrane domain of proHB-EGF, a precursor of HB-EGF, is cleaved by a disintegrin and metalloproteases. HB-EGF is released into the extracellular space and strongly activates EGF receptor. The relevance of better understanding proHB-EGF shedding relates to the importance of the process in the proliferation, differentiation and survival of various types of cells. Shedding of proHB-EGF is normally evaluated using an alkaline phosphatase-tagged proHB-EGF assay or a western blotting assay that involves multiple cells, which makes it difficult to observe spatiotemporal differences in the activities of the individual cells. In this study, we developed a fluorescent proHB-EGF-based metalloprotease biosensor, named Fluhemb, to visualize spatiotemporal regulation of proHB-EGF shedding in individual cells using a simple method that measures changes in fluorescence ratios. Fluhemb might be very useful for detecting the activity of proHB-EGF shedding in various types of cells under different conditions in vitro and in vivo.

The Cullin-3–Rbx1–KCTD10 complex controls endothelial barrier function via K63 ubiquitination of RhoB

RhoGTPases control endothelial cell (EC) migration, adhesion, and barrier formation. Whereas the relevance of RhoA for endothelial barrier function is widely accepted, the role of the RhoA homologue RhoB is poorly defined. RhoB and RhoA are 85% identical, but RhoB’s subcellular localization and half-life are uniquely different. Here, we studied the role of ubiquitination for the function and stability of RhoB in primary human ECs. We show that the K63 polyubiquitination at lysine 162 and 181 of RhoB targets the protein to lysosomes. Moreover, we identified the RING E3 ligase complex Cullin-3–Rbx1–KCTD10 as key modulator of endothelial barrier integrity via its regulation of the ubiquitination, localization, and activity of RhoB. In conclusion, our data show that ubiquitination controls the subcellular localization and lysosomal degradation of RhoB and thereby regulates the stability of the endothelial barrier through control of RhoB-mediated EC contraction.