ARHGEF12 activates Rap1A and not RhoA in human capillary endothelial cells to reduce tumor necrosis factor-induced leak
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PROVIDER: PRJNA786218 | ENA |
REPOSITORIES: ENA
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Project description:In acute critical illness the capillary barrier may break down resulting in hypovolemia, extracellular edema, tissue dysoxia and even death. Restoration of the tight junction dependent capillary barrier is regulated by small GTPases, the specific regulatory molecules most active in this setting are not well described. Transcriptional profiling of tight junction forming human dermal microvascular ECs (HDMECs) and adherens junctions forming human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is significantly differentially regulated in HDMECs before and after stimulation with tumor necrosis factor (TNF). HDMEC depleted of ArhGEF12 demonstrate significantly exacerbated TNF-induced decrease in trans-endothelial electrical resistance and disruption of tight junction proteins claudin-5 and ZO-1. ArhGEF12 is a RhoA-GEF, an established finding which is inconsistent with our observed results. Pulldown activation assays from of HDMECs depleted of ArhGEF12 and treated with TNF show decreased Rap1A activity after four hours and paradoxically increased RhoA activity after 12 hours. In cell-free pulldown assays, immunoprecipitated ArhGEF12 effectively activates Rap1A and RhoA and not Rap2A-C, RhoB-C or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in tight junction forming EC, ArhGEF12 selectively activates Rap1A to promote capillary barrier restoration in a mechanism independent of traditionally described cAMP-mediated Epac1 activation. The unique dual in vitro specificity of ArhGEF12 for RhoA, Rap1A and not Rap1B cannot be explained by sequence identity and requires additional investigation.
2021-12-07 | GSE190181 | GEO
Project description:Endothelial glycocalyx dysfunction in monoclonal gammopathy-associated systemic capillary leak syndrome (Clarkson disease)
| PRJNA842668 | ENA
Project description:Rho family small GTPases serve as molecular switches in the regulation of diverse cellular functions including actin cytoskeleton remodeling, cell migration, gene transcription, and cell proliferation. Importantly, Rho overexpression is frequently seen in many carcinomas. However, published studies have almost invariably utilized immortal or tumorigenic cell lines to study Rho GTPase functions and there are no studies on the potential of Rho small GTPase to overcome senescence checkpoints and induce preneoplastic transformation of human mammary epithelial cells (hMECs). We found that ectopic expression of wild-type RhoA as well as a constitutively-active RhoA mutant (G14V) in primary hMEC strains led to their immortalization and preneoplastic transformation. Significantly, RhoA-T37A mutant, known to be incapable of interacting with many well known Rho-effectors ,was also capable of immortalizing hMECs.Our results demonstrate that RhoA can induce the preneoplastic transformation of hMECs by altering multiple pathways linked cellular transformation and breast cancer. Through microarray analysis, we want to identify genes and pathways linked to RhoA induced hMECs immortalization. Experiment Overall Design: 4 samples, in triplicate analyses per sample.
2008-10-26 | E-GEOD-12917 | biostudies-arrayexpress
Project description:The small GTPase RhoA regulates a variety of cellular processes, including cell motility, proliferation, survival and permeability. In addition, there are reports suggesting that the RhoA-ROCK axis plays a role in VEGF-mediated angiogenesis, whereas other work has shown opposite effects. To elucidate this conflicting data, we examined HUVEC and HCAEC after stable overexpression (lentiviral transduction) of constitutively active (G14V/Q63L), dominant-negative (T19N), or wild-type RhoA using a variety of in vitro angiogenesis assays (proliferation, migration, tube formation, angiogenic sprouting, endothelial cell viability) and a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. We observed that expression of active as well as wild-type RhoA but not expression of dominant-negative RhoA significantly increased endothelial cell death as well as inhibited endothelial cell proliferation, migration, tube formation and angiogenic sprouting of endothelial cells in vitro and reduced HUVEC-related angiogenesis in vivo. Inhibition of RhoA by C3 transferase antagonized inhibitory RhoA effects and strongly enhanced VEGF-induced angiogenic sprouting in control-treated cells. In contrast, inhibition of RhoA effectors ROCK1/2 and LIMK1/2 had no significant effect on RhoA-related effects, but again increased angiogenic sprouting and migration of control-treated cells. In line with these data, VEGF did not activate RhoA in HUVEC as measured by a FRET-based biosensor. Furthermore, global transcriptome and subsequent bioinformatic gene ontology (GO) enrichment analyses revealed that constitutively active RhoA induces a differentially expressed gene pattern that is enriched for GO biological process terms such as mitotic nuclear division, cell proliferation, cell motility and cell adhesion and includes a significant decrease in VEGFR-2 and NOS3 expression. Thus, our data demonstrate that increased RhoA activity has the potential to trigger endothelial dysfunction and anti-angiogenic effects independently of its well-characterized downstream effectors ROCK and LIMK.
2022-07-06 | GSE182806 | GEO
Project description:Using mice with a keratinocyte-restricted deletion of the RhoA gene, we explored its role in the regulation of gene expression in vivo and in vitro RNA was isolated from RhoA-null or wildtype keratinocytes, in two different conditions, freshly isolated (in vivo) or cultured (in vitro), using the RNeasy kit (Qiagen, Germany). Hybridization to GeneChip Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA) was carried out at the Copenhagen University Hospital Microarray Center using standard protocols. 2 biological replicates were analyzed for each in vivo genotype (2 samples from RhoA-null, 2 from wildtype), while 3 replicates for each in vitro genotype.
2015-01-07 | E-GEOD-64714 | biostudies-arrayexpress
Project description:Rationale: Endothelial cells (ECs) line the inner surface of continuous capillaries and form a permselective barrier between blood and tissues to limit paracellular fluid and solute flux. The capillary barrier is maintained by intercellular tight junctions (TJs) which are regulated, in part, by differential activity of small GTPases. Tumor necrosis factor (TNF) causes TJ disassembly in a process dependent upon NF-κB-mediated gene expression. Activated (GTP-bound) RhoB has been identified as a downstream effector of TNF-induced leak both through Rho-associated coiled-coil kinase-mediated phosphorylation of RelA, enhancing TNF-induced NF-κB-mediated gene activation, and through sequestration of Rac1 away from the cell junction, preventing barrier restoration. To date, no TNF-induced, RhoB-selective GTP exchange factor (GEF) has been identified in ECs. Objective: Identify a TNF-induced RhoB specific GEF in capillary ECs. Method and Results: human dermal microvascular ECs (HDMECs) were used because they form TJs. Candidate GEFs were identified by whole transcriptome profiling of HDMECs with or without TNF stimulation. Fifty-seven of 73 known GEFs were expressed in HDMECs and 17 were significantly upregulated by TNF. Functional impact of each of the latter group was assessed by measuring the effects of depletion by short interfering RNA knockdown on TNF-induced reduction of trans-endothelial electrical resistance (TEER) of HDMEC monolayers. Depletions of ArhGEF10 or RhoB or double depletion of ArhGEF10/RhoB similarly decreased TNF-mediated reduction of TEER. ArhGEF10 knockdown prevented TNF-induced disruption of TJ proteins assessed by confocal microscopy. TNF-induced increases in the levels of activated RhoB in HDMEC lysates were markedly reduced with only minimal alterations in RhoA or RhoC activation. Finally, immunoisolated ArhGEF10 selectively catalyzed GTP exchange for GDP in RhoB, but not RhoA or RhoC, in vitro. Conclusion: ArhGEF10 is a TNF-induced RhoB-selective GEF for RhoB in cultured human capillary ECs that contributes to TJ disruption.
2021-05-10 | GSE161021 | GEO
Project description:Rho family small GTPases serve as molecular switches in the regulation of diverse cellular functions including actin cytoskeleton remodeling, cell migration, gene transcription, and cell proliferation. Importantly, Rho overexpression is frequently seen in many carcinomas. However, published studies have almost invariably utilized immortal or tumorigenic cell lines to study Rho GTPase functions and there are no studies on the potential of Rho small GTPase to overcome senescence checkpoints and induce preneoplastic transformation of human mammary epithelial cells (hMECs). We found that ectopic expression of wild-type RhoA as well as a constitutively-active RhoA mutant (G14V) in primary hMEC strains led to their immortalization and preneoplastic transformation. Significantly, RhoA-T37A mutant, known to be incapable of interacting with many well known Rho-effectors ,was also capable of immortalizing hMECs.Our results demonstrate that RhoA can induce the preneoplastic transformation of hMECs by altering multiple pathways linked cellular transformation and breast cancer. Through microarray analysis, we want to identify genes and pathways linked to RhoA induced hMECs immortalization.
2008-09-26 | GSE12917 | GEO
Project description:RHOA is a recurrently mutated gene in diffuse-type gastric cancer, whose molecular influence in gastric cancer has not been well uncovered. We aimed to clarify the contribution of RHOA to pro-oncogenesis to improve the understanding of the role of RHOA as a therapeutic target. We performed microarray analysis after RHOA knockdown for HGC-27, AGS, CCK-81, and SW948.
2021-02-01 | GSE110237 | GEO
Project description:The goal of this study is to determine the feasibility of CEA assessments at home using (automated) capillary sampling in patients in the follow-up after treatment for colorectal cancer.
The main questions it aims to answer are:
* To determine the success rate of capillary sampling at home by the patient
* To assess reliability and satisfaction of (automated) capillary CEA measurements Participants will be asked to perform automated capillary sampling and lancet capillary sampling at home twice after regular check-up visits in the hospital, with an interval of 3-6 months in between. During this hospital visit, a CEA measurement in blood sampled by venipuncture will be performed to act as a reference for the CEA measurements in (automated) capillary blood to be sampled at home.
Reliability of CEA measurements will be assessed for automated capillary and lancet capillary sampling compared to venipuncture.
Satisfaction in terms of patient reported outcomes (pain, burden, ease of use, and preference) will be evaluated.
| 2031 | ecrin-mdr-crc
Project description:The goal of this study is to identify transcriptomic effects of RhoA G17V expression in mouse CD4+ T lymphocytes. To this end, mice conditionally expressing RhoA G17V were generated by replacing exon 3 of RhoA with a minigene containing RhoA exons 3 to 6 flanked by loxP sites followed by a neomycin selection cassette and a mutant Rhoa exon 3 encoding the G17V substitution. These mice were then crossed with the CD4CreERT2 line (Tg(Cd4-cre/ERT2)11Gnri, Jackson Laboratories), which expresses a 4-hydroxi-tamoxifen-inducible form of the Cre recombinase under the control of the mouse Cd4 promoter to generate conditional inducible CD4 specific RhoA G17V mice (Rhoaco-G17V/+ CD4CreERT2). Mice were treated with vehicle only or Tamoxifen to induce expression of the RhoA G17V in CD4+ T cells. CD4CreERT2 mice were included as a negative control. CD4+ T cells were isolated from single cell suspensions of spleen by negative selection using the mouse naive CD4+ T Cell Isolation Kit (Milteny#130-104-453) according to the manufacturer’s protocol. Total RNA was extracted and standard Illumina poly-A RNA seq was performed. Naive mouse CD4+ T cells expressing RhoA G17V displayed an enrichment in a Tfh gene expression program.
2017-09-01 | GSE101340 | GEO