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1.
Development ; 148(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34383884

RESUMO

Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveals distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tricellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics the junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function in junction formation and stabilization during sprouting angiogenesis.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neovascularização Fisiológica/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Comunicação Celular/fisiologia , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Junções Intercelulares/metabolismo , Junções Intercelulares/fisiologia , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Peixe-Zebra/fisiologia
2.
Development ; 145(17)2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30042182

RESUMO

Although major progress in our understanding of the genes and mechanisms that regulate lymphatic vasculature development has been made, we still do not know how lumen formation and maintenance occurs. Here, we identify the Ras-interacting protein Rasip1 as a key player in this process. We show that lymphatic endothelial cell-specific Rasip1-deficient mouse embryos exhibit enlarged and blood-filled lymphatics at embryonic day 14.5. These vessels have patent lumens with disorganized junctions. Later on, as those vessels become fragmented and lumens collapse, cell junctions become irregular. In addition, Rasip1 deletion at later stages impairs lymphatic valve formation. We determined that Rasip1 is essential for lymphatic lumen maintenance during embryonic development by regulating junction integrity, as Rasip1 loss results in reduced levels of junction molecules and defective cytoskeleton organization in vitro and in vivo We determined that Rasip1 regulates Cdc42 activity, as deletion of Cdc42 results in similar phenotypes to those seen following the loss of Rasip1 Furthermore, ectopic Cdc42 expression rescues the phenotypes in Rasip1-deficient lymphatic endothelial cells, supporting the suggestion that Rasip1 regulates Cdc42 activity to regulate cell junctions and cytoskeleton organization, which are both activities required for lymphatic lumen maintenance.


Assuntos
Proteínas de Transporte/metabolismo , Citoesqueleto/metabolismo , Embrião de Mamíferos/embriologia , Células Endoteliais/metabolismo , Vasos Linfáticos/embriologia , Junções Íntimas/metabolismo , Animais , Proteínas de Transporte/genética , Citoesqueleto/genética , Embrião de Mamíferos/citologia , Células Endoteliais/citologia , Peptídeos e Proteínas de Sinalização Intracelular , Vasos Linfáticos/citologia , Camundongos , Camundongos Transgênicos , Junções Íntimas/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo
3.
Angiogenesis ; 19(2): 173-90, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26897025

RESUMO

Cardiovascular function depends on patent, continuous and stable blood vessel formation by endothelial cells (ECs). Blood vessel development initiates by vasculogenesis, as ECs coalesce into linear aggregates and organize to form central lumens that allow blood flow. Molecular mechanisms underlying in vivo vascular 'tubulogenesis' are only beginning to be unraveled. We previously showed that the GTPase-interacting protein called Rasip1 is required for the formation of continuous vascular lumens in the early embryo. Rasip1(-/-) ECs exhibit loss of proper cell polarity and cell shape, disrupted localization of EC-EC junctions and defects in adhesion of ECs to extracellular matrix. In vitro studies showed that Rasip1 depletion in cultured ECs blocked tubulogenesis. Whether Rasip1 is required in blood vessels after their initial formation remained unclear. Here, we show that Rasip1 is essential for vessel formation and maintenance in the embryo, but not in quiescent adult vessels. Rasip1 is also required for angiogenesis in three models of blood vessel growth: in vitro matrix invasion, retinal blood vessel growth and directed in vivo angiogenesis assays. Rasip1 is thus necessary in growing embryonic blood vessels, postnatal angiogenic sprouting and remodeling, but is dispensable for maintenance of established blood vessels, making it a potential anti-angiogenic therapeutic target.


Assuntos
Proteínas de Transporte/metabolismo , Neovascularização Fisiológica , Vasos Retinianos/embriologia , Vasos Retinianos/metabolismo , Envelhecimento/metabolismo , Animais , Aorta/crescimento & desenvolvimento , Feminino , Deleção de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Gravidez
4.
Cancer Biol Ther ; 24(1): 2193114, 2023 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-36967521

RESUMO

A GTPase binding protein, Ras interacting protein 1 (RASIP1), has been reported with a tumor-promoting role in lung cancer cells, and its role in lymphoma remains unknown. The analysis of medical databank shows that RASIP1 is upregulated in diffuse large B-cell lymphoma (DLBCL) specimens. In this article, we demonstrated that RASIP1 is highly expressed in DLBCL cell lines, compared with primary B cells. The gain- and loss-of-function experiments were performed to investigate the effects of RASIP1 on DLBCL cells. CCK-8, flow cytometry, western blot, and transwell assays demonstrated that silence of RASIP1 inhibited proliferation, cell cycle transition, and invasion and induced significant apoptosis in DLBCL cells, and ectopic expression of RASIP1 played opposite roles. Xenograft results revealed that RASIP1 facilitated the growth of DLBCL cells in vivo. These findings suggest that RASIP1 may be required for malignancy of DLBCL cells. In addition, we also found that the expression of RASIP1 was negatively regulated by forkhead box O3 (FOXO3), which has been reported to suppress the proliferation of DLBCL cells. Our results indicate that FOXO3 is bound to the promoter sequence of RASIP1 and inhibits its transcription. The suppressive effects of FOXO3 on proliferation and invasion of DLBCL cells were neutralized by RASIP1. In conclusion, we demonstrate that FOXO3 negatively regulated RASIP1 facilitates growth and invasion of DLBCL cells, provides novel diagnostic markers and therapeutic targets for DLBCL in clinic.


Assuntos
Apoptose , Peptídeos e Proteínas de Sinalização Intracelular , Linfoma Difuso de Grandes Células B , Humanos , Proteínas de Transporte/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo
5.
Inflamm Regen ; 43(1): 41, 2023 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-37553580

RESUMO

BACKGROUND: During mouse embryonic development, definitive hematopoiesis is first detected around embryonic day (E) 10.5 in the aorta-gonad-mesonephros (AGM) region. Hematopoietic stem cells (HSCs) arise in the dorsal aorta's intra-aortic hematopoietic cell clusters (IAHCs). We have previously reported that a transcription factor Sox17 is expressed in IAHCs, and that, among them, CD45lowc-Kithigh cells have high hematopoietic activity. Furthermore, forced expression of Sox17 in this population of cells can maintain the formation of hematopoietic cell clusters. However, how Sox17 does so, particularly downstream signaling involved, remains poorly understood. The purpose of this study is to search for new Sox17 targets which contribute to cluster formation with hematopoietic activity. METHODS: RNA-sequencing (RNA-seq) analysis was done to identify genes that are upregulated in Sox17-expressing IAHCs as compared with Sox17-negative ones. Among the top 7 highly expressed genes, Rasip1 which had been reported to be a vascular-specific regulator was focused on in this study, and firstly, the whole-mount immunostaining was done. We conducted luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay to examine whether Sox17 regulates Rasip1 gene expression via binding to its enhancer element. We also analyzed the cluster formation and the multilineage colony-forming ability of Rasip1-transduced cells and Rasip1-knockdown Sox17-transduced cells. RESULTS: The increase of the Rasip1 expression level was observed in Sox17-positive CD45lowc-Kithigh cells as compared with the Sox17-nonexpressing control. Also, the expression level of the Rasip1 gene was increased by the Sox17-nuclear translocation. Rasip1 was expressed on the membrane of IAHCs, overlapping with the endothelial cell marker, CD31, and hematopoietic stem/progenitor marker (HSPC), c-Kit. Rasip1 expression was observed in most part of c-Kit+Sox17+ cells in IAHCs. Luciferase reporter assay and ChIP assay indicated that one of the five putative Sox17-binding sites in the Rasip1 enhancer region was important for Rasip1 expression via Sox17 binding. Rasip1 knockdown in Sox17-transduced cells decreased the cluster formation and diminished the colony-forming ability, while overexpression of Rasip1 in CD45lowc-Kithigh cells led to a significant but transient increase in hematopoietic activity. CONCLUSIONS: Rasip1 knockdown in Sox17-transduced CD45lowc-Kithigh cells displayed a significant decrease in the multilineage colony-forming ability and the cluster size. Rasip1 overexpression in Sox17-untransduced CD45lowc-Kithigh cells led to a significant but transient increase in the multilineage colony-forming ability, suggesting the presence of a cooperating factor for sustained hematopoietic activity.

6.
Cancer Manag Res ; 10: 4537-4552, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30349386

RESUMO

BACKGROUND: Runt-related transcription factor 1 (RUNX1), an essential regulator of hematopoiesis, is overexpressed in patients with nonsmall-cell lung cancer (NSCLC) and is correlated with enhanced metastatic ability. Ras-interacting protein 1 (Rasip1), a potential oncogene, is required for blood vessel formation, and recently, it has been shown that Rasip1 is widely expressed in NSCLC patients. We noticed that Rasip1 promoter contains several potential RUNX1-binding sequences. However, the relationship between Rasip1 and RUNX1 in NSCLC is still unknown. In this study, the potential function of RUNX1 involving in Rasip1 expression and the potential role of Rasip1 in lung cancer cells were investigated. MATERIALS AND METHODS: Rasip1 and RUNX1 expressions were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting in NSCLC cells lines. A549 and H1299 cells were transfected with plasmids or interfering RNA (siRNA) to upregulate or downregulate the expression of Rasip1 and RUNX1. Cell motility was assessed by transwell and wound-healing assay. Location of Rasip1 and RUNX1 was detected via immunofluorescence. Meanwhile, chromatin immunoprecipitation was done using an anti-RUNX1 antibody. Rasip1 promoter was constructed, and cells were lysed for the analysis of luciferase activity. RESULTS: In this study, we showed that ectopic expression or knockdown of RUNX1 resulted in a significant increase or reduction in Rasip1 expression, respectively. RUNX1 bound directly to a specific DNA sequence within Rasip1 promoter and modulated its transcription. Furthermore, silencing of Rasip1 inhibited the migration of RUNX1-overexpressing NSCLC cells through inactivation of Rac1 pathway. Moreover, we found that Rasip1 was expressed ubiquitously in NSCLC cells lines and enhanced cell migration. In addition, EGFR signaling was involved both in the expression and the subcellular localization of Rasip1. CONCLUSION: Our data indicated that Rasip1 is regulated in part by the transcription factor RUNX1 and might be developed as a therapeutic target for NSCLC.

7.
Elife ; 5: e11394, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26780829

RESUMO

Heart of Glass (HEG1), a transmembrane receptor, and Rasip1, an endothelial-specific Rap1-binding protein, are both essential for cardiovascular development. Here we performed a proteomic screen for novel HEG1 interactors and report that HEG1 binds directly to Rasip1. Rasip1 localizes to forming endothelial cell (EC) cell-cell junctions and silencing HEG1 prevents this localization. Conversely, mitochondria-targeted HEG1 relocalizes Rasip1 to mitochondria in cells. The Rasip1-binding site in HEG1 contains a 9 residue sequence, deletion of which abrogates HEG1's ability to recruit Rasip1. HEG1 binds to a central region of Rasip1 and deletion of this domain eliminates Rasip1's ability to bind HEG1, to translocate to EC junctions, to inhibit ROCK activity, and to maintain EC junctional integrity. These studies establish that the binding of HEG1 to Rasip1 mediates Rap1-dependent recruitment of Rasip1 to and stabilization of EC cell-cell junctions.


Assuntos
Células Endoteliais/fisiologia , Junções Intercelulares/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Linhagem Celular , Humanos , Ligação Proteica
8.
Structure ; 24(12): 2152-2162, 2016 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-27839947

RESUMO

Ras-interacting protein 1 (Rasip1) is an endothelial-specific Rap1 and Ras effector, important for vascular development and angiogenesis. Here, we report the crystal structure of the Rasip1 RA domain (RRA) alone, revealing the basis of dimerization, and in complex with Rap1 at 2.8 Å resolution. In contrast to most RA domains, RRA formed a dimer that can bind two Rap1 (KD = 0.9 µM) or Ras (KD = 2.2 µM) molecules. We solved the Rap1-RRA complex and found that Rasip1 binds Rap1 in the Switch I region, and Rap1 binding induces few conformation changes to Rasip1 stabilizing a ß strand and an unstructured loop. Our data explain how Rasip1 can act as a Rap1 and Ras effector and show that Rasip1 defines a subgroup of dimeric RA domains that could mediate cooperative binding to membrane-associated Ras superfamily members.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Sítios de Ligação , Dimerização , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas rap1 de Ligação ao GTP/química , Proteínas ras/química
9.
Cell Adh Migr ; 8(2): 76-83, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24622510

RESUMO

The ability of blood vessels to sense and respond to stimuli such as fluid flow, shear stress, and trafficking of immune cells is critical to the proper function of the vascular system. Endothelial cells constantly remodel their cell-cell junctions and the underlying cytoskeletal network in response to these exogenous signals. This remodeling, which depends on regulation of the linkage between actin and integral junction proteins, is controlled by a complex signaling network consisting of small G proteins and their various downstream effectors. In this commentary, we summarize recent developments in understanding the small G protein RAP1 and its effector RASIP1 as critical mediators of endothelial junction stabilization, and the relationship between RAP1 effectors and modulation of different subsets of endothelial junctions.   The vasculature is a dynamic organ that is constantly exposed to a variety of signaling stimuli and mechanical stresses. In embryogenesis, nascent blood vessels form via a process termed vasculogenesis, wherein mesodermally derived endothelial precursor cells aggregate into cords, which subsequently form a lumen that permits trafficking of plasma and erythrocytes. (1)(,) (2) Angiogenesis occurs after establishment of this primitive vascular network, where new vessels sprout from existing vessels, migrate into newly expanded tissues, and anastomose to form a functional and complex circulatory network. (1)(,) (2) In the mouse, this process occurs through the second half of embryogenesis and into postnatal development in some tissues, such as the developing retinal vasculature. (3) Further, angiogenesis occurs in a variety of pathological conditions, such as diabetic retinopathy, age-related macular degeneration, inflammatory diseases such as rheumatoid arthritis, wound healing, and tumor growth. (1)(,) (2)(,) (4) Both vasculogenesis and angiogenesis are driven through signaling by vascular endothelial growth factor (VEGF), and therapeutic agents targeting this pathway have shown efficacy in a number of diseases. (5)(-) (9) Blood vessels must have a sufficient degree of integrity so as to not allow indiscriminate leak of plasma proteins and blood cells into the underlying tissue. However, vessels must be able to sense their environment, respond to local conditions, and mediate the regulated passage of protein, fluid, and cells. For example, endothelial cells are the primary point of attachment for immune cells leaving the blood stream and entering tissue, and leukocytes subsequently migrate either through the endothelial cell body itself (the transcellular route), or through transient disassembly of cell-cell junctions (the paracellular route). (10) Precise regulation of endothelial junctions is critical to the proper maintenance of vascular integrity and related processes, and disruption of vascular cell-cell contacts is an underlying cause or contributor to numerous pathologies such as cerebral cavernous malformations (CCM) and hereditary hemorrhagic telangiectasia (HHT). (11)(-) (13) Understanding the basic mechanisms of endothelial junction formation and maintenance will therefore lead to a greater chance of success of therapeutic intervention in these pathologic conditions, especially in instances where targeting of VEGF signaling is insufficient to resolve vascular abnormalities.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Transporte/metabolismo , Junções Intercelulares/genética , Neovascularização Fisiológica/genética , Proteínas rap1 de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Proteínas de Transporte/genética , Diferenciação Celular/genética , Células Endoteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Junções Intercelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas rap1 de Ligação ao GTP/genética
10.
Cell Adh Migr ; 8(2): 100-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24714377

RESUMO

The small G-protein Rap1 plays an important role in the regulation of endothelial barrier function, a process controlled largely by cellâ€"cell adhesions and their connection to the actin cytoskeleton. During the various stages of barrier dynamics, different guanine nucleotide exchange factors (GEFs) control Rap1 activity, indicating that Rap1 integrates multiple input signals. Once activated, Rap1 induces numerous signaling cascades, together responsible for the increased endothelial barrier function. Most notably, Rap1 activation results in the inhibition of Rho to decrease radial stress fibers and the activation of Cdc42 to increase junctional actin. This implies that Rap regulates endothelial barrier function by dual control of cytoskeletal tension. The molecular details of the signaling pathways are becoming to be elucidated.


Assuntos
Citoesqueleto de Actina/metabolismo , Adesão Celular/genética , Células Endoteliais/metabolismo , Proteínas de Ligação a Telômeros/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína KRIT1 , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Complexo Shelterina , Transdução de Sinais/genética , Fibras de Estresse/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
11.
Artigo em Chinês | WPRIM | ID: wpr-450808

RESUMO

Objective To explore the expression levels of RAS-interacting protein 1 (RASIP1) mRNA and protein in hepatocellular carcinoma (HCC) tissues and its cell lines,and to analyze the relationship between RASIP1 and tumorigenesis and progression of hepatocellular carcinoma.Methods The expression levels of RASIP1 mRNA and protein in 29 hepatocellular carcinoma tissues and the corresponding adjacent non-cancer liver tissues (ANLTs),as well as those in the HCC cell lines such as LO2,HEPG2,MHCC97-H and HCCLM3 were detected using real-time PCR and western blot.Results The RASIP1 expression levels decreased significantly in HCC tissues when compared with the corresponding ANLTs; The expression levels of RASIP1 mRNA and protein in LO2 were significantly higher than those in other HCC cell lines (P < 0.05) ; The expression levels of RASIP1 mRNA and protein in MHCC97-H and HCCLM3 were significantly lower than those in HepG2 (P < 0.05).Conclusions HCC tissues had lower expression than those in ANLTs.On analyzing the RASIP1 levels of HCC tissues and its cell lines,we speculated that RASIP1 might suppress recurrence and metastasis of HCC.

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