Ubiquitin-conjugating enzyme V variant 1 enables cellular responses toward fibroblast growth factor signaling in endothelium.

Ubiquitination has been shown to provide an essential regulatory role in modulating the duration and amplitude of the signaling activity in angiogenesis. While successive enzymatic reactions mediated by three distinct types of enzymes commonly known as E1, E2, and E3 are required for ubiquitination, the role of E3s which govern the final step of ubiquitination has been extensively analyzed in angiogenesis. In contrast, the role of E2s, which determine the context and functional consequences of ubiquitination, remains largely unknown with respect to angiogenesis. To better elucidate the role of E2s in modulating endothelial behaviors during angiogenesis, we first systematically analyze the expression pattern of E2s in endothelial cells (ECs) using previously published scRNA-seq data and identify ubiquitin-conjugating enzyme variant 1 (UBE2V1), an unconventional E2 without innate catalytic activity, as one of the most abundantly expressed E2s in ECs. While ubiquitously expressed in diverse cell types, abrogation of UBE2V1 significantly impairs proliferation and viability of human umbilical vein endothelial cells (HUVECs) without affecting other cell types, suggesting that UBE2V1 is likely to possess nonredundant functions in ECs. Consistent with this idea, UBE2V1 appears to be critical for morphogenesis and migration of ECs during angiogenesis. Interestingly, we find that UBE2V1 is essential for fibroblast growth factor 2 (FGF2)-induced angiogenesis, but appears to have minor effects on vascular endothelial growth factor-A-induced angiogenesis in vitro as well as in vivo. Therefore, it seems that UBE2V1 could enable ECs to distinguish two related yet distinct angiogenic cues. Mechanistically, we show that UBE2V1 promotes ubiquitination of MEK kinase 1, a key mediator of FGF2 signaling, to enhance phosphorylation of extracellular signal-regulated kinase 1/2 in HUVECs. Taken together, our results illustrate the unique role of UBE2V1 as a key modulator for angiogenic behaviors in ECs.

Pax9 is essential for granulopoiesis but dispensable for erythropoiesis in zebrafish.

Paired Box (Pax) gene family, a group of transcription regulators have been implicated in diverse physiological processes. However, their role during hematopoiesis which generate a plethora of blood cells remains largely unknown. Using a previously reported single cell transcriptomics data, we analyzed the expression of individual Pax family members in hematopoietic cells in zebrafish. We have identified that Pax9, which is an essential regulator for odontogenesis and palatogenesis, is selectively localized within a single cluster of the hematopoietic lineage. To further analyze the function of Pax9 in hematopoiesis, we generated two independent pax9 knock-out mutants using the CRISPR-Cas9 technique. We found that Pax9 appears to be an essential regulator for granulopoiesis but dispensable for erythropoiesis during development, as lack of pax9 selectively decreased the number of neutrophils with a concomitant decrease in the expression level of neutrophil markers. In addition, embryos, where pax9 was functionally disrupted by injecting morpholinos, failed to increase the number of neutrophils in response to pathogenic bacteria, suggesting that Pax9 is not only essential for developmental granulopoiesis but also emergency granulopoiesis. Due to the inability to initiate emergency granulopoiesis, innate immune responses were severely compromised in pax9 morpholino-mediated embryos, increasing their susceptibility and mortality. Taken together, our data indicate that Pax9 is essential for granulopoiesis and promotes innate immunity in zebrafish larvae.

Antiatherogenic Effect of Resveratrol Attributed to Decreased Expression of ICAM-1 (Intercellular Adhesion Molecule-1).

Objective- Increasing evidence shows that resveratrol has antiatherogenic effects, but its underlying mechanisms are unknown. Thus, we evaluated the molecular mechanisms underlying the antiatherogenic effect of resveratrol. Approach and Results- Using the previously established mouse atherosclerosis model of partial ligation of the left carotid artery, we evaluated the role of resveratrol in antiatherosclerosis. We attempted to determine the mechanisms associated with focal adhesions using vascular endothelial cells. The results showed that resveratrol stimulated focal adhesion kinase cleavage via resveratrol-increased expression of lactoferrin in endothelial cells. Furthermore, we found that an N-terminal focal adhesion kinase fragment cleaved by resveratrol contained the FERM (band 4.1, ezrin, radixin, and moesin)-kinase domain. Furthermore, resveratrol inhibited lipopolysaccharide-stimulated adhesion of THP-1 human monocytes by decreased expression of ICAM-1 (intercellular adhesion molecule-1). A decreased ICAM-1 level was also observed in the left carotid artery of mice treated with resveratrol. To understand the relationship between resveratrol-induced antiinflammation and focal adhesion disruption, endothelial cells were transfected with FERM-kinase. Ectopically expressed FERM-kinase, the resveratrol-cleaved focal adhesion kinase fragment, was found in the nuclear fraction and inhibited the transcription level of icam-1 via the Nrf2 (nuclear factor erythroid 2-related factor 2)-antioxidant response element complex. Finally, ectopically expressed FERM-kinase blocked tumor necrosis factor-α- or IL- (interleukin) stimulated monocytic binding to endothelial cells. Conclusions- Our results show that resveratrol inhibits the expression of ICAM-1 via transcriptional regulation of the FERM-kinase and Nrf2 interaction, thereby blocking monocyte adhesion. These suppressive effects on the inflammatory mechanism suggest that resveratrol delayed the onset of atherosclerosis.