Endothelial Notch signaling directly regulates the small GTPase RND1 to facilitate Notch suppression of endothelial migration

Swaminathan, Bhairavi; Youn, Seock Won; Naiche, L.A.; Du, Juan; Villa, Stephanie R.; Metz, Jordan B.; Feng, Huijuan; Zhang, Chaolin; Kopan, Raphael; Sims, Peter A.; Kitajewski, Jan

doi:10.1038/s41598-022-05666-1

Cited by 19 publications

(14 citation statements)

References 57 publications

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“…NOTCH1 activity in MCF10A cells has been mostly characterized using transfection of activated forms [61,62]. Here we have shown that it is possible to activate endogenous NOTCH1 using a short pulse of Ca 2+ chelation that destabilizes the NOTCH1 HD domain leading to S2 and S3 cleavage, as previously reported for a variety of different cell types [6,[63][64][65]. Interestingly, we find that MCF10A possess a low level basal S3 cleavage and that, upon S3 cleavage NOTCH1 is rapidly phosphorylated, an event that have been described to lead to proteasomal degradation of NICD.…”

Section: Discussionsupporting

confidence: 68%

Novel determinants of NOTCH1 trafficking and signaling in breast epithelial cells

Kobia

Almeida

Carminati

et al. 2023

Preprint

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The evolutionarily conserved Notch pathway controls cell–cell communication during development and in adult metazoans. It influences cell fate decisions, cell proliferation and cell differentiation, and contributes to the maintenance of normal tissue homeostasis. Consequently, misregulation of the Notch pathway is associated with a wide range of diseases, including congenital disorders and cancers with little to no cure. Signaling by Notch receptors is regulated by a complex set of cellular processes that include maturation and trafficking to the plasma membrane, endocytic uptake and sorting, lysosomal and proteasomal degradation, and ligand-dependent and independent proteolytic cleavages. We devised assays to follow quantitively the lifetime of endogenous human NOTCH1 receptor in breast epithelial cells in culture. Based on such analysis, we executed a high-content screen of 2749 human genes for which modulatory compounds exist, to identify new regulators of Notch signaling activation that might be amenable to pharmacologic intervention. We uncovered 39 new NOTCH1 genetic modulators that affect different steps of NOTCH1 cellular dynamics. In particular, we find thatPTPN23andHCN2act as positive NOTCH1 regulators by promoting endocytic trafficking and NOTCH1 maturation in the Golgi apparatus, respectively, whileSGK3serves as a negative regulator that can be modulated by pharmacologic inhibition. Our findings might be relevant in the search of new strategies to counteract pathologic Notch signaling.

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Section: Discussionsupporting

confidence: 68%

Novel determinants of NOTCH1 trafficking and signaling in breast epithelial cells

Kobia

Almeida

Carminati

et al. 2023

Preprint

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“…To further examine if O1 effectively inhibits RhoA in human cells, a RhoA activation assay in an S100 extract from HeLa cell was performed using G-LISA RhoA [ 66 ]. In this assay, RhoA-GTP, but not RhoA-GDP, can be detected.…”

Section: Resultsmentioning

confidence: 99%

Identification of a small RhoA GTPase inhibitor effective in fission yeast and human cells

Morishita

Nurse

2023

Open Biol.

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The Rho GTPase family proteins are key regulators of cytoskeletal dynamics. Deregulated activity of Rho GTPases is associated with cancers and neurodegenerative diseases, and their potential as drug targets has long been recognized. Using an economically effective drug screening workflow in fission yeast and human cells, we have identified a Rho GTPase inhibitor, O1. By a suppressor mutant screen in fission yeast, we find a point mutation in the rho1 gene that confers resistance to O1. Consistent with the idea that O1 is the direct inhibitor of Rho1, O1 reduced the cellular amount of activated, GTP-bound Rho1 in wild-type cells, but not in the O1-resistant mutant cells, in which the evolutionarily conserved Ala62 residue is mutated to Thr. Similarly, O1 inhibits activity of the human orthologue RhoA GTPase in tissue culture cells. Our studies illustrate the power of yeast phenotypic screens in the identification and characterization of drugs relevant to human cells and have identified a novel GTPase inhibitor for fission yeast and human cells.

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“…Sprout initiation is associated with changes in endothelial cell motility, and multiple genes involved in regulating these processes were upregulated. Although RND1 and SEMA7A have previously been shown to play a role in endothelial motility and angiogenesis, 61,62 few data are available on the role of NEDD9 , NUAK2 , RAPH1 , or RND3 . RND3 is an atypical Rho GTPase (also known as RhoE) that has previously been implicated in cell rounding, loss of stress fibers 63,64 and barrier function.…”

Section: Resultsmentioning

confidence: 99%

Identification of new markers of angiogenic sprouting using transcriptomics: New role for RND3

Abbey,

Duran,

Chen

et al. 2023

Preprint

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Background: New blood vessel formation requires endothelial cells to transition from a quiescent to an invasive phenotype. Transcriptional changes are vital for this switch, but a comprehensive genome-wide approach focused exclusively on endothelial cell sprout initiation has not been reported. Approach and Results: Using a model of human endothelial cell sprout initiation, we developed a protocol to physically separate cells that initiate the process of new blood vessel formation (invading cells) from non-invading cells. We used this model to perform multiple transcriptomics analyses from multiple donors to monitor endothelial gene expression changes. Single-cell Population Analyses, single-cell Cluster Analyses, and bulk RNA sequencing were used to delineate transcriptomic changes in invading cells. The results revealed a 39 gene signature that was consistent with activation of signal transduction, morphogenesis, and immune responses. Many of the genes were previously shown to regulate angiogenesis, and include multiple tip cell markers. Upregulation of SNAI1, PTGS2, and JUNB protein expression was confirmed in invading cells, and silencing JunB and SNAI1 significantly reduced invasion responses. Separate studies investigated Rounding 3 (RND3), also known as RhoE, which has not yet been implicated in angiogenesis. Silencing RND3 reduced endothelial invasion distance as well as filopodia length, fitting with a pathfinding role for RND3 via regulation of filopodial extensions. Analysis of in vivo retinal angiogenesis in Rnd3 heterozygous mice confirmed a decrease in filopodial length compared to wild type littermates. Conclusion: Validation of multiple genes, including RND3, revealed a functional role for this gene signature early in the angiogenic process. This study expands the list of genes that are associated with the acquisition of a tip cell phenotype during endothelial cell sprout initiation.

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Endothelial Notch signaling directly regulates the small GTPase RND1 to facilitate Notch suppression of endothelial migration

Cited by 19 publications

References 57 publications

Novel determinants of NOTCH1 trafficking and signaling in breast epithelial cells

Novel determinants of NOTCH1 trafficking and signaling in breast epithelial cells

Identification of a small RhoA GTPase inhibitor effective in fission yeast and human cells

Identification of new markers of angiogenic sprouting using transcriptomics: New role for RND3

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